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Dysfunctional AMPA receptor trafficking in traumatic brain injury
by
Joshua David Bell
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Institute of Medical Science
University of Toronto
© Copyright by Joshua David Bell, 2010
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Dysfunctional AMPA receptor trafficking
in traumatic brain injury
Joshua D Bell, Institute of Medical Science University of Toronto: Doctor of Philosophy; 2010
Abstract Traumatic brain injury (TBI) is a devastating public health problem for patients
and their families. The neurodegeneration that follows TBI is complex, but can be
broadly subdivided into primary and secondary damage. Though primary damage is
irreversible and therefore unsalvageable, extensive literature aimed at understanding the
tissue, cellular, inflammatory and subcellular processes following the injury have proven
unequivocally that secondary pathophysiological events are delayed and progressive in
nature. Understanding these secondary events at the cellular levels is critical in the
eventual establishment of targeted therapeutics aimed at limiting progressive injury after
an initial trauma.
One such secondary event is referred to in the literature as excitotoxicity; a
sustained and de-regulated activation of glutamate receptors that leads to rapid cytotoxic
edema and calcium overload. Our understanding of excitotoxicity has evolved to include
not only a role for elevated extracellular glutamate in mediating neuronal damage, but
also post-synaptic receptor modifications that render glutamate profoundly more toxic to
injured neurons than healthy tissue.
In this thesis, we explored the hypothesis that glutamate excitotoxicity can be
perpetuated by trauma-induced post-synaptic modification of the AMPA receptor.
Specifically, we used a cortical culture model of TBI as well as the fluid percussion
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injury device to test the hypothesis that TBI confers a reduction of surface GluR2 protein,
an AMPA receptor subunit that limits neuronal calcium permeability. We conjectured
that this decrease in the expression of surface GluR2 would increase the expression of
calcium-permeable AMPA receptors, thereby rendering neurons vulnerable to secondary
excitotoxic injury. We further investigated the subcellular mechanisms responsible for
the internalization of surface GluR2, and the phenotypic consequences of GluR2
endocytosis in both models.
Our data revealed that both models of TBI resulted in a regulated signaling
cascade leading to the phosphorylation and internalization of GluR2. By exogenously
interrupting the trafficking of GluR2 protein with an inhibitory peptide, we further
observed that GluR2 internalization was mediated by a protein interaction involving
protein interacting with C kinase 1 (PICK1) and protein kinase C alpha (PKCα), two PDZ
domain-containing proteins that mediate GluR2 trafficking during constitutive synaptic
plasticity. We observed that GluR2 endocytosis was NMDA receptor dependent, and
resulted in increased neuronal calcium permeability, augmented AMPA receptor
mediated electrophysiological activity and increased susceptibility to delayed cell death.
Finally, we demonstrated that the interruption of GluR2 trafficking is cytoprotective,
suggesting that sustaining surface GluR2 protein protects neurons against secondary
injury. Overall, our findings suggest that experimental TBI promotes the expression of
injurious GluR2-lacking AMPA receptors, thereby enhancing cellular vulnerability to
secondary excitotoxicity.
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Acknowledgements
I want to thank a number of people who’ve helped me throughout my PhD
program. Firstly, I want to thank my supervisor, Andrew Baker. Dr Baker has been the
ideal supervisor, allowing me to pursue my own intellectual curiosities, while shaping my
interests into scientifically testable hypotheses. He has supported my ideas, challenged
my thinking, and helped me grow tremendously as a person and a scientist. He is a credit
to the IMS graduate program and the university. Outside of the lab, Dr Baker has also
fostered collaboration and connectivity with other labs, taking me to numerous
conferences where I’ve been able to share my data with audiences well beyond U of T. I
cannot possibly thank him enough for all that he has taught me about research and about
life.
I also want to acknowledge my fellow lab members. I want to thank Dr Eugene
Park for his guidance and willingness to discuss ideas, but more importantly for his
friendship. Dr Park’s experience in the lab made my program infinitely easier, as he
shared with me all that he knew about completing a successful doctoral program in the
IMS, invaluable knowledge that unquestionably contributed to my successes. I want to
also acknowledge Dr Jinglu Ai, whose creativity and hard work inspired the preliminary
data in the early days of this work back in 2006. Elaine Liu, our technician, is also worthy
of significant thanks. Elaine has been a beacon of unwavering support in my pursuit of
higher education, and made the downtime in the lab as well as lab get-togethers much
more enjoyable. Finally, I want to thank Dr Carlo Santaguida. Though he arrived when
my data collection was finished, his friendship and his exceedingly generous will in
providing medical advice to me during my thesis writing days expedited the process of
thesis completion significantly. He has been a constant alleviator of anxieties! I look
forward to further collaboration with Carlo and many more tasty shawarmas.
I want to thank Dr Beverley Orser, Dr Michael Fehlings, and Dr Peter
Pennefather for their participation in my Program Advisory Committee. The collective
advice that I have received has helped me appreciate the complexity of interpreting
results, designing experiments, and thoughtfully considering alternatives to my
hypotheses. It was significantly easier for me to mature as a scientist while working with
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such stunning examples of academic success. I feel truly lucky to have had the
opportunity to share my work with these scientists.
Lastly, I want to thank my family. There is no way that I could have completed
this work without Rachel, the love of my life, the maker of lunches on extremely busy
days, and the most unconditionally devoted person I have ever met. Rachel was a huge
part of this work, and as Dr Baker constantly says, this lab would fall apart without her. I
also want to thank my Dad and my brother. Though they didn’t understand a word of
what is written on these pages, they did flip through the thesis and humor me. Their
support has meant the world to me. Finally, this thesis is dedicated to my mom, Judi,
who never got to see or share in any of my successes. I hope I’ve made you proud.
Formal Acknowledgements:
Figures 7-8 are taken from the following manuscript:
Bell JD, Ai J, Chen Y, and Baker AJ. (2007) Mild in vitro trauma induces rapid Glur2
endocytosis, robustly augments calcium permeability and enhances susceptibility to
secondary excitotoxic insult in cultured Purkinje cells. Brain;130:2528-42.
Figures 11-24 (excluding 19, 21, and 23) are taken from the following manuscript:
Bell JD, Park E, Ai J, and Baker AJ (2009). PICK1-mediated GluR2 endocytosis contributes to cellular injury after neuronal trauma. Cell Death Differ 16:1665–1680.
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Table of Contents Abstract.......................................................................................................... ii Acknowledgements ...................................................................................... iv Table of Contents ......................................................................................... vi List of Abbreviations ................................................................................... xi Chapter 1: Introduction ............................................................................... 1
1.1 Clinical Overview and Epidemiology of Traumatic Brain Injury ............................ 1 1.1.1 TBI Epidemiology ............................................................................................. 1 1.1.2 Cost of TBI ........................................................................................................ 3 1.1.3 Classification of TBI Severity ........................................................................... 4
1.2 Pathophysiology of a traumatic brain injury............................................................. 6 1.2.1 Primary Injury.................................................................................................... 6 1.2.2 Mechanical forces affecting cerebral tissue after TBI ....................................... 9
1.3 Mechanisms of secondary injury after TBI ............................................................ 12 1.3.1 Intracranial pressure and secondary ischemia.................................................. 12 1.3.2 Sub-cellular mechanisms of secondary injury ................................................. 14
1.4 Glutamate Excitotoxicity ........................................................................................ 15 1.4.1 Glutamate......................................................................................................... 15 1.4.2 Glutamate Release ........................................................................................... 19 1.4.3 Glutamate Receptors........................................................................................ 24
1.4.3.1 NMDARs .................................................................................................. 25 1.4.3.2 AMPARs – Discovery and function ......................................................... 28 1.4.3.3 Kainate receptors ...................................................................................... 35 1.4.3.4 Metabotropic Glutamate Receptors (mGluRs) ......................................... 36
1.4.4 The concept of excitotoxicity........................................................................... 37 1.4.4.1 De-regulation of glutamate release ........................................................... 39 1.4.4.2 An alternative look at excitotoxicity:........................................................ 43 Post-synaptic glutamate receptor dysfunction ...................................................... 43 1.4.4.3 Consequences of excitotoxicity: Ca2+-dependent neurodegeneration ...... 45 1.4.4.4 Oxidative stress and Mitochondrial Injury ............................................... 46
1.5 AMPA Receptor Trafficking: GluR2-lacking AMPA Receptors as sources of calcium influx ............................................................................................................... 50
1.5.1 Modification of the AMPA Receptor GluR2 content. ..................................... 52 1.5.1.1 Epigenetic silencing of GluR2.................................................................. 52 1.5.1.2 Local trafficking of GluR2 protein ........................................................... 54
1.5.1.2.1 NSF/AP2 Site interactions in GluR2 trafficking ............................... 56 1.5.1.2.2 AMPA receptor c-terminal PDZ interactions .................................... 57 1.5.1.2.3 PDZ Interactions in GluR2 trafficking .............................................. 63
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1.5.1.2.4 GluR2 trafficking in synaptic plasticity............................................. 69 1.5.1.2.5 GluR2 trafficking in TBI ................................................................... 71
1.6 Rationale for proposed study .................................................................................. 73 1.7 Statement of Hypotheses......................................................................................... 76
1.7.1 General Hypotheses ......................................................................................... 76 1.7.2 Specific Hypotheses......................................................................................... 76
1.8 Statement of Objectives .......................................................................................... 78 Chapter 2 – Model Characterization and General.................................. 79 Methods........................................................................................................ 79 Chapter 2: General Methods ..................................................................... 80
2.1 Preface..................................................................................................................... 80 2.2 In vitro methods ...................................................................................................... 80
2.2.1 Isolation and dissociation of cortical cell cultures........................................... 80 2.2.2 In Vitro Model of TBI...................................................................................... 81
2.2.2.1 Use of stretch injury models in TBI literature .......................................... 82 2.2.2.2 The Stretch + NMDA model .................................................................... 83 2.2.2.3 Toxicity studies: Dose response characterization of stretch pressures ..... 86 2.2.2.4 Carboxyfluorescein assays of membrane permeability ............................ 90
2.2.3 Protein extraction and quantification............................................................... 94 2.2.4 Co-Immunoprecipitation of GluR2 endocytotic proteins ................................ 95 2.2.5 SDS-PAGE ...................................................................................................... 95 2.2.6 Immmunoblotting ............................................................................................ 96 2.2.7 Acid Strip Immunofluorescence ...................................................................... 97 2.2.8 [Ca2+] Measurement......................................................................................... 99 2.2.9 Secondary AMPA Toxicity............................................................................ 100 2.2.10 Whole cell electrophysiology ...................................................................... 101
2.3 TAT peptides ........................................................................................................ 102 2.3.1 The HIV-1 TAT protein transduction domain ............................................... 103 2.3.2 Design of PICK1 inhibitory TAT peptides.................................................... 104
2.4 In vivo Methods .................................................................................................... 111 2.4.1 Fluid percussion trauma................................................................................. 111 2.4.2 Slice Electrophysiology ................................................................................. 111 2.4.3 TUNEL staining............................................................................................. 112
2.5 Contributions......................................................................................................... 113 2.6 Statistics ................................................................................................................ 114
Chapter 3: GluR2 trafficking in modeled brain trauma ...................... 115 3.1 Preface................................................................................................................... 116 3.2 Phosphorylation of GluR2 serine 880 following in vitro trauma correlates with susceptibility to AMPA toxicity ................................................................................. 116
3.2.1 NMDA receptor dependence of GluR2 phosphorylation .............................. 118
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3.3 In vitro trauma increases PICK1-PKCa binding................................................... 119 3.4 PKCa is embedded in the NMDAR complex: ...................................................... 122
PKCa co-precipitates with PSD-95......................................................................... 122 3.5 Traumatic injury increases GluR2 endocytosis .................................................... 126 3.6 PICK1-mediated endocytosis of GluR2 following fluid percussion trauma ........ 130 3.7 Summary of results ............................................................................................... 133
Chapter 4: Phenotypic AMPAR changes in modeled brain trauma ... 137 4.1 Preface................................................................................................................... 138 4.2 AMPAR-mediated mEPSC activity following in vitro traumatic injury.............. 138 4.3 AMPA receptor-mediated calcium influx following in vitro trauma: .................. 140 4.4 Interfering with GluR2 endocytosis is cytoprotective in vitro.............................. 144 4.5 Hippocampal CA1 is hyperexcitable following fluid percussion trauma: Excitability is lowered with TAT-QSAV application ................................................ 147 4.6 Hippocampal CA1 Naspm sensitivity increases after FPI.................................... 150 4.7 Occluding GluR2 endocytosis reduces apoptotic cell death:................................ 156 4.8 Summary ............................................................................................................... 157
Chapter 5: Discussion, Limitations and Future Directions .................. 161 5.1 Preface................................................................................................................... 162 5.2 Corroborating studies............................................................................................ 162 5.3 Co-operation of Stretch + NMDA ........................................................................ 167 5.4 Limitations of the current study............................................................................ 168
5.4.1 Non-specific Tat peptide interactions ............................................................ 168 5.4.2 Non-specific effects of Tat peptide transduction ........................................... 169 5.4.3 Co-precipitation: What does it mean?............................................................ 170 5.4.4 TNFα-induced AMPA receptor trafficking: An alternative mechanism of calcium-permeable AMPA receptor expression ..................................................... 172
5.5 Future Directions .................................................................................................. 173 5.5.1 Total GluR2 levels are reduced by 24 hours following trauma ..................... 174 5.5.2 GluR1 trafficking may increase following trauma: ....................................... 175 5.5.3 Tat-QSAV treatment does not occlude induced synaptic plasticity: ............. 178 5.5.4 – Does inhibition of the PICK1 PDZ domain represent a future anti-excitotoxic therapy?................................................................................................................... 181
5.6 Significance of Findings ....................................................................................... 185 5.7 Conclusions........................................................................................................... 186
References Cited........................................................................................ 195
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List of Figures
Chapter 1: Introduction FIGURE 1. Mechanisms of cytotoxicity following traumatic brain injury. FIGURE 2. Schematic diagram of an AMPA receptor subunit. FIGURE 3. Processes leading to excitotoxicity after CNS injury. FIGURE 4. GluR2 subunit domain structure. FIGURE 5. Steps involved in the intracellular trafficking of the GluR2 subunit.
Chapter 2: General Methods FIGURE 6. The cell injury controller and schematic of experimental paradigm. FIGURE 7. Dose-Response Characterization of stretch injury model FIGURE 8. Mild injury does not increase non-specific neuronal cell membrane permeability. FIGURE 9. Design of PICK1-inhibitory Tat peptides and mechanisms of Tat-peptide uptake. FIGURE 10. Transduction of dansyl-Tat-QSAV into cultured cortical neurons and brain slices in vivo.
Chapter 3: GluR2 trafficking in modeled brain trauma
FIGURE 11. Stretch + NMDA increases S880 phosphorylation of GluR2 and vulnerability to secondary AMPA toxicity FIGURE 12. Stretch + NMDA confers association of PKCa with PICK1. FIGURE 13. PKCa co-precipitates with PSD-95: A potential link from the NMDAR to GluR2 endocytosis. FIGURE 14. Stretch + NMDA increases GluR2 endocytosis FIGURE 15. In vivo traumatic brain injury (TBI) promotes GluR2 phosphorylation and association with PICK1.
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Chapter 4: Phenotypic AMPAR changes in modeled brain trauma FIGURE 16. Traumatic injury in vitro increases AMPAR mEPSC amplitude and sensitivity to intracellular polyamines. FIGURE 17. Stretch + NMDA promotes calcium influx through calcium-permeable AMPARs. FIGURE 18. Inhibiting GluR2 endocytosis is neuroprotective FIGURE 19. Post-injury CA1 hyperexcitability is attenuated by Tat-QSAV treatment. FIGURE 20. CA1 hippocampal physiology is sensitive to antagonists of calcium-permeable AMPA receptors after TBI. FIGURE 21. Perturbing GluR2 endocytosis affords cytoprotection from apoptosis at 24 hours following fluid percussion trauma.
Chapter 5: Discussion and Limitations FIGURE 22. Summary of proposed signaling in TBI FIGURE 23. Total GluR2 protein levels are reduced at 24 hours following FPI. FIGURE 24. Stretch + NMDA increases GluR1 S845 phosphorylation FIGURE 25. Hippocampal LTP is preserved with PICK1 inhibition.
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List of Abbreviations ABP – AMPA receptor binding protein
AMPA - α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
AMPAR – AMPA receptor
ANOVA – Analysis of variance
AP2 – Adaptor protein 2
APAF-1 – Apoptosis peptidase activating factor 1
ATP – Adenosine Triphosphate
BAR domain - Bin–Amphiphysin–Rvs domain
CA1/3 – Cornus ammonis area 1/3
CNQX - 6-cyano-7-nitroquinoxaline-2,3-dione
CNS – Central Nervous System
CoIP – Co-immunoprecipitation
CP-AMPARs – Calcium-permeable AMPA receptors
CPP- Cerebral perfusion pressure
DAI – Diffuse Axonal Injury
D-MEM - Dulbecco’s modified eagle medium
EAA – Excitatory amino acid
FDU – (+)-5-fluor-2’-deoxyuridine
fEPSP – Field excitatory post-synaptic potential
GCS – Glasgow Coma Score
GluR2 – Glutamate receptor subunit, 2
GRIP – Glutamate receptor interacting protein
HBSS - Hank’s balanced salt solution
HIV – Human immunodeficiency virus
ICP – Intracranial pressure
IgG – Immunoglobulin G
L-NAME - γ-nitro-L-Arginine-Methyl Ester
LTP/LTD – Long term potentiation/depression
MAP – Mean arterial pressure
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mEPSC – Miniature excitatory post-synaptic current
mRNA – Messenger Ribonucleic Acid
Naspm – 1-naphthyl acetyl spermine
NCX – Sodium calcium exchanger
NMDA - N-methyl-D-aspartic acid
NMDAR – NMDA receptor
nNOS – Neuronal nitric oxide synthase
NSF - N-Ethylmaleimide-Sensitive Fusion Protein
OGD – Oxygen glucose deprivation
PDZ - Post synaptic density protein (PSD95), Drosophila disc large tumor suppressor
(DlgA), zonula occludens-1 protein (zo-1)
PBS - Phosphate buffered saline
PI – Propidium Iodide
PICK1- Protein interacting with C Kinase 1
PKCα – Protein kinase C, alpha
PMSF - Phenylmethylsulphonyl fluoride
PSD-95 - Post-synaptic density protein, 95 kDa
P.S.I – Pounds per square inch
PTD – Protein transduction domain
RIPA - Radio-Immunoprecipitation Assay
ROS – Reactive Oxygen Species
SDS-PAGE - Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SNARE - Soluble NSF Attachment Protein Receptors
TARP – Transmembrane AMPA receptor regulatory protein
TAT – Transacting activator of transcription
TBI – Traumatic Brain Injury
TUNEL - Terminal deoxynucleotidyl transferase dUTP nick end labeling
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Chapter 1: Introduction
1.1 Clinical Overview and Epidemiology of Traumatic Brain Injury
1.1.1 TBI Epidemiology
Traumatic brain injury (TBI) continues to be a leading cause of death and
disability in both developed and developing nations1-5. In North America, TBI is
recognized as the leading cause of mortality and morbidity in young adults (15 to 44
years of age)6, while incidences of brain trauma continue to rise in the developing world
as rates of vehicle use outpace the implementation of safety infrastructure and effective
neurosurgical critical care initiatives7-9. Thus, the acute management and chronic
treatment of head trauma is a global issue, with some estimates suggesting that by the
year 2020 TBI will rank as the third most prevalent cause of worldwide mortality and
disability10.
Epidemiological studies are highly varied with respect to statistical estimates of
TBI incidence, largely because of differing head injury inclusion criteria, and variability
in classification of hospital admission. The United States Center for Disease Control
(CDC) estimates that approximately 1.4 million Americans sustain a TBI each year11. Of
those, approximately 1.1 million people will be treated and subsequently released from an
emergency department, 235,000 will require long-term hospitalization, and 50,000 will
die11-13. These figures represent a startling statistic; the number of people hospitalized
each year for traumatic brain injuries exceed those diagnosed with multiple sclerosis,
breast cancer, and spinal cord injury combined14. In Europe, epidemiological data for TBI
is scarce, but most estimates indicate an annual aggregate incidence of hospitalized and
fatal TBI of approximately 235 per 100, 00015. Globally, patients that succumb to their
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injuries post TBI are thought to represent approximately one third of all injury-related
deaths11,12,16.
The major causes of head injury highlight the susceptibility of certain populations
to TBI. For example, falls are thought to represent approximately 30% of all brain
injuries, and occur most frequently in children aged 0-4 and in adults over the age of
7511,13. Events described as being “struck by or against” account for 20% of TBIs, and are
thought to represent most of the 475,000 injuries sustained in the United States by
children aged 0-14, largely from participation in youth sports and other recreational
activities11. Motor vehicle accidents account for 20% of TBIs, with the highest rate of
TBI-induced hospitalization from MVA occurring in adolescents aged 15-1911.
Collectively, these statistics highlight the susceptibility of young children and young
adults to TBI, and point to a need for new safety initiatives for young athletes and
inexperienced drivers. Interestingly however, while TBI related emergency department
(ED) visits are dominated by young children and adolescents (1696.1 per 100,000), the
highest rate of TBI-related hospitalization and death actually occurs in adults aged 75 and
older (322.7 per 100,000)11, indicating that health care costs are more significantly
affected by injuries to the elderly, despite the high number of emergency department
visits by children.
Additional causes of TBI include assault (11%), non-motor vehicle transport (e.g.,
cycling, rollerblading, skateboarding) (3%), idiopathic injuries (9%), and other causes of
brain trauma (e.g., blast injury and suicide) (7%). At all age groups and causes, TBI is
three times more likely to effect males than females17, primarily due to participation in
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violent athletic and recreational programs and the more aggressive risk-taking behaviours
of males.
1.1.2 Cost of TBI
Recent estimates suggest that approximately 5.3 million people in the United
States are currently living with a disability as a result of an acquired TBI5, which
highlights brain injury as a public heath issue with a significant economic burden. The
most recent estimate of the cost of TBI to the United States health care system is $37.8
billion USD18, which, when averaged across all injury severities (i.e., mild, moderate and
severe TBI), translates to a per person cost of $115,500 USD19,20. Approximately 65% of
the cost of traumatic brain injury is accrued by TBI survivors, and is related to direct
hospital costs, long-term care and rehabilitation, while 35% of the cost is associated with
head injury deaths20.
Overshadowed by the significant monetary drain on the health care system are the
intangible challenges facing the families of survivors, and the longer term psychosocial,
functional and neuro-cognitive disabilities suffered by survivors of TBI that impact
society on the whole. Some epidemiologists have relied on standardized scales such as
the functional capacity index (FCI) and life years lost to injury (LLI) to estimate these
more abstract costs21, which measure inabilities to perform basic tasks including eating,
hearing and speaking, and ambulation, and take into account both life expectancy and the
number of years of professional productivity lost to injury. One study estimated that TBI
results, on average, in 43 years of reduced functional capacity, a frightening statistic
when coupled with the data reflecting the number of individuals affected by a head injury
per year. These trends continue in Europe, where TBI accounts for the greatest number
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of total years lived with disability resulting from trauma22. Moreover, when the
psychosocial and emotional sequelae (including depression, anxiety, confusion and
loneliness23) in individuals sustaining TBI are taken into account, it is easy to grasp the
overall burden that brain trauma places on the individual and society as a collective, and
to gain an appreciation for the data suggesting that indirect TBI costs more than triple
those related to hospitalization and emergency treatment21.
1.1.3 Classification of TBI Severity
An individual is said to have sustained a traumatic brain injury if he/she has
cranio-cerebral trauma caused by an external force, and associated with neurological or
neuropsychological abnormalities, loss of consciousness, skull fracture, intracranial
lesions or death. However, beyond identifying a patient as having suffered acquired brain
trauma, the severity classification of TBI is of long-standing interest to both clinicians
and researchers interested in predicting outcome and providing post-acute medical care.
In most clinical settings, TBI is classified on the basis of single indicators including the
Glasgow Coma Scale (GCS), duration of post-traumatic amnesia (PTA) and duration of
loss of consciousness (LOC). Indeed all of these indices have demonstrated good
predictive value in classifying TBI, with higher GCS scores and brief losses of
consciousness associated with what is generally termed “mild” TBI24-31. However, recent
studies indicate a number of confounds in classifying TBI according to these individual
scales, citing issues in GCS predictability when patients are either intoxicated at the time
of injury or given roadside sedation. Further, systemic and psychological shock sustained
5
from poly-trauma has been shown to contribute significantly to durations of PTA,
skewing the validity of this measure as a predictor of TBI severity. Thus recent efforts
have aimed at classifying TBI according to more reliable measures, most of which focus
on a combination of GCS and neuro-anatomical pathologies.
For example, Malec et al., at the Mayo Clinic classify TBI as a) Moderate-Severe
(definite), b) Mild (probable) and c) Symptomatic (possible)32. Moderate-Severe TBI is
said to have occurred if one or more of the following is present: death, LOC greater than
30 minutes, PTA of greater than 24 hours, GCS below 13 and not invalidated by
confounding factors, and/or intra-parenchymal/subdural/epidural hematoma,
subarachnoid hemorrhage, cerebral or hemorrhagic contusion or brain stem injury. If
none of the above has occurred from injury, a mild TBI is diagnosed if there is LOC less
than 30 minutes, PTA less than 24 hours, or a depressed, basilar or linear skull fracture
with dura intact. If there is no skull fracture, LOC, or PTA at all, the patient is classified
as having a symptomatic TBI, if they experience one of dizziness, confusion, blurred
vision, nausea, or headache.
It is likely that mild and symptomatic TBI are considerably under-diagnosed, with
the reported values of 100-300 cases per 100,000 representing a highly conservative
estimate of the prevalence33. Accordingly, mild TBI is thought to be somewhat of a
“silent” epidemic, due to the under-diagnosis of the condition, coupled with the
frequency of residual deficits resulting from it. Indeed one study by Thornhill et al.,
(2000) identified that one year after TBI, 1260 of 1397 (90%) disabled patients included
in their analysis had sustained a mild injury34.
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The principal complaints from patients sustaining a mild TBI include mood
disturbances (including irritability and anxiety), loss of employment due to difficulty with
concentration, and increased fatigue34. These impairments, though mild when compared
with the physical disabilities sustained by those suffering from a more severe injury, can
have a profound impact on an individual’s socialization and quality of life, pointing to the
need for clinical hyper-vigilance when a case of mild TBI is suspected.
1.2 Pathophysiology of a traumatic brain injury
1.2.1 Primary Injury
A traumatic brain injury is not self-limiting, but rather is an evolving biological
injury that stems from an initial trauma. Accordingly, the pathophysiological mechanisms
that lead to neurological deterioration after a head injury can be classified into two
separate categories of insults: primary and secondary.
The primary injury that occurs following TBI consists of the physical perturbation
of the cerebral tissue and vasculature, and is the major determinant of functional
outcome35. The extent of primary injury depends almost exclusively on the type of
physical load (i.e., force) placed on the brain at the time of injury. For instance, TBI can
occur as a result of a blunt impact to the skull, rapid acceleration or deceleration, a
penetrating object (e.g., gunshot), or blast waves from an explosion, each of which will
produce a unique primary injury profile.
Generally speaking, the type of primary injury is classified as focal or diffuse by
radiological imaging of structural damage. Diffuse injuries, caused by inertial forces,
7
include macroscopic alterations such as white matter lesions and shearing (known as
diffuse axonal injury, DAI), brain swelling, and tearing of blood vessels causing micro-
hemmorhages. Focal injuries on the other hand primarily include contusions
(microvasculature injury) and hematomas (both intra-cerebral and extradural). In many
cases (e.g., MVAs), more than one of these pathologies is present, representing either a
multi-pronged primary injury from one impact (e.g., closed head impact causing both
contusion and DAI) or a manifestation of injury resulting from more than one external
force (e.g., rotational injury followed by direct impact).
The nature, intensity, direction, and duration of the external forces causing
primary injury will dictate the pattern and extent of damage, and accordingly has an
enormous impact on functional outcome after TBI. In static crush injuries and focal
trauma (e.g., a blow to the head), a large proportion of the energy is absorbed by the
skull, often limiting damage to superficial structures (e.g., a depressed skull fracture).
Extra-dural bleeds, although problematic if left untreated because they will increase
intracranial pressure, can often be removed through neurosurgical evacuation, and
outcome in these situations is favorable. The poorest outcome is usually associated with
diffuse axonal injury, resulting from rotational and inertial forces placed on the brain
(discussed in detail in the next section). DAI is characterized radiologically by multiple
lesions and disconnection of white matter tracts, appearing often throughout the deep and
subcortical white matter and in midline structures including the splenium of the corpus
callosum and brainstem. Usually, patients with DAI remain in a lengthy coma and, if they
regain consciousness, have significant neuropsychological sequelae and physical
disability. The acceleration-deceleration forces responsible for DAI can also have a
8
profound impact on cerebrovascular integrity, resulting in both vascular stenosis and
shearing of vessels, leading to multiple intra-parenchymal hemorrhages. In the most
severe cases, patients with DAI who survive rapidly lapse into coma, and remain
unconscious, vegetative, or severely disabled until life support is withdrawn.
Interestingly, the global demographics of the types of primary TBI are changing,
as contusions become more frequent than diffuse injuries. Some authors explain this
trend by citing an increase in the prevalence of falls in older patients, coupled with
decreases in the frequency of high-velocity traffic accidents in young adults due to
implementation of more effective safety measures and crackdown on “road-racing”.
Another type of primary injury that is increasing in frequency is that sustained from a
blast injury (i.e., shockwave-induced brain trauma following an explosion) as military
conflict in Afghanistan and Iraq continues to escalate. Although less understood than
penetrating injury sustained in combat, blast injuries result in early brain swelling, sub-
arachnoid hemorrhage, and vasospasm36,37, sparking increased research efforts aimed at
understanding the interplay between shockwave physics and the corresponding biological
injury.
Primary injury is irreversible, and is not amenable to therapeutic intervention.
Accordingly, the efforts that make the biggest difference in preventing primary TBI are
those of safety awareness, and changes to public policy. To this end, primary prevention
includes changes to speed limits, enforcement of seat-belt use, and improved road
engineering, primarily in underdeveloped countries. Further, socio-cultural attitudes play
an important role in prevention of TBI, and should include increased awareness of the
dangers of alcohol abuse when participating in certain activities, and increased helmet
9
use in both recreational activities and organized sports. Along this vein, a greater
understanding by coaching staff of return-to-play guidelines following concussion will
undoubtedly minimize incidents of TBI in both youth and professional athletics.
1.2.2 Mechanical forces affecting cerebral tissue after TBI An understanding of the biomechanics of traumatic brain injury is essential in the
development of effective treatment strategies, and will provide the theoretical knowledge
base necessary to understand the rationale and physics behind the in vitro and in vivo
injury devices described later in this thesis.
The compliant properties of cerebral tissue leave the brain susceptible to a variety
of mechanical deformations during an impact. Exactly how physical perturbation of grey
and white matter transfers to injury at the cellular level is unknown, but some of the basic
mechanisms of mechanical injury have been mapped out for decades. The first of these
mechanisms is pressure loading. The concussive effects of pressure on the brain were
identified in the early TBI literature38, and are now known to reflect the dissipation of
energy throughout the brain from pressure gradients generated in the intracranial space at
the time of injury39. The direction of propagation of this intracranial pressure front effects
the elastic deformation of cerebral tissue, and accordingly, impacts the type and level of
strain experienced by the structures within the brain40. To calculate tissue strain (i.e., a
deformation representing the relative displacement of tissue), some simple formulae can
be applied. Strain (e) is calculated as:
e = λ – 1
10
where λ represents the stretch ratio, a definition of tissue deformation expressed as:
λ = l/lo,
where l = the length of deformed tissue and lo = original tissue length
The calculation of tissue strain (as well as its regional distribution) induced by a uniform
pressure load is important in understanding how a blow to the head translates to tissue
injury. Clinically, it is impossible to measure the strain experienced by cerebral tissue
during impact, but experimental approaches have generated both simulated intracranial
pressure patterns produced during impact41 as well as calculated the corresponding
regional strain of brain tissue in response to said pressure loading40 (identified through
the use of finite element models, or FEMs). One such model identified that pressure
loading of 3.5 atmospheres (atm) produces brainstem strain (e) in excess of 10%, a level
of axonal strain higher than in any other regions, and similar to that produced during
herniation of the brain stem through the foramen magnum40. Indeed this level of loading
is reflective of a severe brain injury (calculated to occur when pressure loading exceeds
235 kPA, or 2.3 atm42) where brain stem herniation is a common response to rapidly
elevated ICP. Brainstem injury arising from shear stress plays a prominent role in
neurological dysfunction following pressure loading, and accounts for the vast majority
of TBI-induced death by neurological criteria43.
11
The identification of pressure as a major contributor to TBI pathophysiology led
to the development of animal models implementing a so-called “percussion-concussion”.
One such model, the fluid percussion injury device (FPI), will be discussed in detail in
the next chapter, and is used in this thesis as an in vivo TBI experimental paradigm.
The second type of load placed on the brain during TBI is an inertial load that
results from rapid head rotational motions, common in motor-vehicle accidents, and in
some cases, falls and assaults44. The human brain has a moment of inertia (I) – that is, a
resistance to change in its rotation rate. When the forces that resist the rotation of the
head are overcome by sufficient changes to rotational acceleration (i.e., angular velocity
over time), there is an instantaneous change to the angular momentum of the head, and
unrestricted movement causing dynamic shear, tensile, and compressive strains on
cerebral tissue44. Angular momentum is represented by the formula:
L = I ω
where L = angular momentum, I = moment of inertia, and ω = angular velocity. Thus
one can see the direct relationship between changes to angular velocity and
corresponding angular momentum. Rapid changes to angular velocity are responsible for
diffuse axonal injury (DAI), the shearing and stretching of neuronal white matter
discussed previously that result in a large number of swollen and disconnected axons.
The duration of this axonal stretching plays an important role in the resultant
injury. Under normal circumstances, human brain tissue is ductile to stretch, rapidly
regaining its original geometry when deformed (e.g., during a concussive force). This is
because the deformation in this scenario is generally quite slow. However, when axonal
strain is applied rapidly (e.g., during an MVA), the tissue acts stiffer, exhibiting a more
12
brittle character. This can be easily recapitulated when one stretches and ordinary piece
of plasticine (i.e., rapid stretching will break the material). This is a classic visco-elastic
response to rapid deformation, and occurs in the human brain as it does in other
materials, causing damage to the axonal cytoskeleton, and sometimes, physical
disconnection. The mass effects of the brain thus result in the white matter literally
pulling itself apart. The forces that result in this type of tensile elongation occur in 50 ms
or less45.
Because the mass effects of the human brain play such a large role in the impact
of rotational acceleration on axonal integrity, the reproduction of this phenomenon in an
animal model (where the brain is much smaller) has proven difficult. Indeed the
Holbourn scaling relationship (which summarizes the acceleration needed to produce
injury across varying brain sizes) predicts that the inertial forces necessary to produce
DAI in a rat (with a brain weighing just 2 g) would need to approach an unachievable
8000% of those that produce DAI in a human46. Thus, to produce a clinically relevant
level of axonal and neuronal stretching, investigators have relied on in vitro models of
tissue strain which are also utilized in this thesis, and are discussed in the next chapter.
1.3 Mechanisms of secondary injury after TBI
1.3.1 Intracranial pressure and secondary ischemia Over the last few decades, we have learned much about factors associated with
worse outcomes following traumatic brain injury. There is a substantial body of work that
has analyzed the systemic and intracranial physiologically targeted interventions that
might reduce secondary injury and make a difference in outcomes. The first such
13
intervention targets elevated intracranial pressure (ICP) after TBI. ICP is the pressure
measured within the skull, and therefore, exerted on the brain (ICP can also be measured
intra-parenchymally or intra-ventricularly, but all estimates suggest that these values
should be identical). Normal ICP in healthy adults is generally below 20 mmHg;
however, post-TBI edema of the brain or the development of an epidural hematoma or
subdural hemorrhage can dramatically raise ICP, causing internal or external herniation
of the brain, with distortion and pressure on cranial nerves and vital neurological centres.
To treat elevated ICP, both neurosurgical and physiologic approaches are employed.
Neurosurgically, a decompressive craniectomy can allow for the expansion of the brain
as it swells without increasing ICP, while an intraventricular catheter can relieve pressure
by removing cerebrospinal fluid. Evacuation of a hematoma will also relieve pressure.
Physiologically, osmotherapy (increasing the osmolarity of the blood) serves to draw
water out of tissues and reduce cerebral edema47,48 while simultaneously increasing blood
pressure to counteract the effects of ICP on cerebral perfusion (discussed next).
A second but related major physiological intervention targets hypotension and
ischemic injury. Ischemic brain damage (reduced blood flow) after TBI is frequently
superimposed on the primary injury, and can manifest as either widespread or peri-
lesional. Maintenance of cerebral blood flow depends on a balance between ICP and the
arterial pressure of the blood, mean arterial pressure (MAP). Indeed cerebral perfusion is
defined as the difference between mean arterial pressure and intracranial pressure:
CPP = MAP - ICP
14
From this relationship, it is easy to see that when ICP is increased, the perfusion of the
brain is decreased, resulting in inadequate tissue oxygenation and ischemic injury.
Normal CPP falls around 80 mmHg, but when reduced to 50 mmHg or lower, there is
metabolic evidence of impaired electrophysiology and tissue ischemia. Indeed clinical
studies have demonstrated a correlation between poor neurological outcome and a
reduction of CPP below 70 mmHg for a sustained period49,50. Notably, cerebral
oxygenation can also be impaired after TBI following more focal micro-vascular
destruction, coagulation and stenosis51-53, which results in smaller and more localized
infarction. Basic science investigations have corroborated this evidence, demonstrating
the sub-cellular expression of hypoxia-inducible factors after destruction of cerebral
microvasculature52 following TBI.
1.3.2 Sub-cellular mechanisms of secondary injury
In addition to complications of systemic and intracranial physiology, primary
injury after TBI is exacerbated by discrete secondary sub-cellular processes that are more
elusive to conventional imaging techniques and therapeutic intervention. An
understanding of these more complex mechanisms of cell death is integral in the
establishment of effective “neuroprotective” treatments for delayed cellular death and
dysfunction after TBI, as interventions in systemic or intracranial physiology provide
little protection against tissue injury at the cellular level. For example, even when ICP
and CPP are restored to normal levels, there remain ongoing sequelae of damage to
nervous tissue perpetuated by a number of cytotoxic processes. These include oxidative
and nitrosative injury54-65 (free radical injury, lipid peroxidation, DNA fragmentation)
glial proliferation and dysfunction66-68 (swelling of astrocytic foot processes, reversal of
15
neurotransmitter reuptake and reactive astrocytosis), inflammation69-80 (invasion of the
injury site by microglia and release of proinflammatory cytokines), white matter and
cytoskeletal deterioration81-94 (demyelination and proteolysis of the cytoskeleton),
apoptotic cell death89,95-106 (both intrinsic and extrinsic) and finally, excitotoxicity and
aberrant ionic homeostasis in neurons68,107-120.
Each of these interrelated processes contributes to known mechanisms of grey and
white matter injury after TBI and a number of comprehensive reviews exist for each
topic. Accordingly, emphasis in this section will be placed on excitotoxicity (cell death
mediated by hyper-activation of glutamate receptors) as it is a critical initiating factor in
the progression of a number of these cascades and is the focus of the cell signaling
studied in this particular thesis.
1.4 Glutamate Excitotoxicity
1.4.1 Glutamate Glutamate is the major excitatory neurotransmitter in the mammalian central
nervous system, an observation that dates back to the 1950’s121-123. It is a ubiquitous
amino acid (estimated to participate in signaling at over half of all brain synapses124) with
two stereoisomeric configurations, L and D. In mammals, L-glutamate is the only
physiologically relevant conformation of the molecule, and thus any further reference to
glutamate refers to the L-glutamate stereoisomer. To gain a full appreciation for the
process of excitotoxicity, it is necessary to review glutamatergic pharmacology and
physiology, beginning with the synthesis of glutamate and ultimately concluding with
16
Figure 1. Mechanisms of subcellular injury following brain trauma. Microcirculatory
derangements involve stenosis (1) and loss of microvasculature, and the blood–brain
barrier may break down as a result of astrocyte foot processes swelling (2). Proliferation
of astrocytes ("astrogliosis") (3) is a characteristic of injuries to the central nervous
system, and their dysfunction results in a reversal of glutamate uptake (4) and neuronal
depolarization through excitotoxic mechanisms. In injuries to white and grey matter,
calcium influx (5) is a key initiating event in a molecular cascades resulting in delayed
cell death or dysfunction as well as delayed axonal disconnection. In neurons, calcium
and zinc influx though channels in the AMPA and NMDA receptors results in
excitotoxicity (6), generation of free radicals, mitochondrial dysfunction and postsynaptic
receptor modifications. These mechanisms are not ubiquitous in the traumatized brain but
are dependent on the subcellular routes of calcium influx and the degree of injury.
Calcium influx into axons (7) initiates a series of protein degradation cascades that result
in axonal disconnection (8). Inflammatory cells also mediate secondary injury, through
the release of proinflammatory cytokines (9) that contribute to the activation of cell-death
cascades or postsynaptic receptor modifications.
17
Figure 1. Mechanisms of subcellular injury following brain trauma. Adapted with permission from Park, Bell and Baker, 2008, CMAJ, “Traumatic Brain Injury: Can the consequences be stopped”. 178 (9), 1163-1170. Copied under license from Access Copyright. Further reproduction prohibited.
18
mechanisms of glutamate-induced neuronal death. This will highlight both the
physiological action of glutamate and its importance in regulating neuronal transmission,
as well as the pathological nature of aberrant glutamatergic signaling.
The structure of glutamate is that of any other amino acid found in the human
body; that is, a central carbon atom bonded to 3 moieties: 1) a carboxyl group (COOH),
2) an amino group (NH3), and 3) a distinctive side-chain, termed an R group. In
glutamate, this R group is CH2CH2COO-, an ionized form of CH2CH2COOH (pKa 4.1)
that exists at physiological pH levels125. Notably, the ionized R group is what
distinguishes the nomenclature of L-glutamic acid (unionized) and the more common
term, glutamate (ionized).
To use glutamate as an intercellular signal, neurons and glia have collectively
developed a system which comprises an input, output, and termination of glutamate
signaling. Glutamate does not cross the blood-brain barrier, and thus must be synthesized
in neurons from local precursors124. Of these, the precursor with the highest prevalence is
glutamine, the most abundant free amino acid in the body (500-900 µmol/l) and released
primarily by astrocytes in the brain126. Peri-synaptic glutamine is taken up by neurons
through pre-synaptic excitatory amino acid transporters (EAAT1-5, discussed later), and
metabolized to glutamate by the mitochondrial enzyme glutaminase. An alternative form
of glutamate synthesis involves phosphate-activated transamination (transfer of an amino
group from an amino acid to an α-keto acid) of 2-oxoglutarate (also termed α-ketoglutaric
acid), an intermediate of the tricarboxylic acid (Krebs) cycle127. Indirectly then, neuronal
glucose metabolism also plays a key role in glutamate synthesis.
19
Glutamate, similar to all neurotransmitters, is stored pre-synaptically in cytosolic
vesicles, a process which is dependent on the activity of another transmembrane
glutamate transporter, VGLUT (vesicular glutamate transporter). VGLUTs (3 genes have
been identified, VGLUT1-3) regulate the packaging of glutamate into vesicles using an
electrochemical proton gradient, established by vacuolar-type proton ATPase127. Because
of the remarkably strict substrate recognition ability of VGLUT (i.e., the protein only
recognizes L-glutamate and a few cyclic glutamate analogues), it is frequently used as an
immunocytochemical marker of glutamatergic nerve terminals.
Glutamatergic vesicles (with a glutamate concentration of ~ 100 mmol/l) are
transported along axonal microtubules to the presynaptic plasma membrane, where they
fuse with exocytotic machinery and form the SNARE complex, a protein-protein
interaction involving vesicular synaptobrevin and synaptotagmin, and membrane bound
syntaxin and SNAP-25. This anchors the glutamatergic vesicle to the plasma membrane,
allowing for subsequent exocytosis of the vesicle’s constituents.
1.4.2 Glutamate Release
An understanding of glutamatergic vesicle release is critical in the discussion of
excitotoxic processes, as aberrant vesicle fusion is thought to be an important initiating
factor in excitotoxic neuron death. Vesicle release is a calcium-dependent process, with
vesicle-bound synaptotagmin serving as an intracellular calcium sensor. The calcium
responsible for glutamatergic vesicle release is thought to originate from pre-synaptic N
and P/Q-type (n, representing neural, p/q meaning purkinje) calcium channels126,128,129,
voltage-gated ion channels found in excitable cells. These channels -- which are activated
at depolarized membrane potentials and are responsible for the fidelity of synaptic
20
transmission from neuron to neuron -- were identified to be in close proximity to
glutamatergic vesicle docking sites, creating a calcium micro-domain that serves as an
immediate trigger for vesicle fusion and exocytosis. As such, the probability of release of
a glutamatergic vesicle is dependent on the type and density of these pre-synaptic Ca2+
channels expressed and their individual proximity to and interaction with neighbouring
transmitter release machinery. Of the calcium channel subtypes, it has been demonstrated
that P/Q-type calcium channels contribute to approximately 50% of the presynaptic
calcium influx responsible for glutamatergic vesicle fusion, evidenced by a marked
inhibition of glutamate release by presynaptic blockade of these channels with -
Agatoxin IVA130. N-type calcium channels by contrast contribute to only 30% of the total
pre-synaptic calcium entry, leading some authors to conclude that the P/Q-type channel
interacts more tightly with the release machinery than does the N-type channel at
glutamatergic synapses130-132.
Mutations in these pre-synaptic calcium channels have a profound impact on
neuronal functioning due to their influence on glutamatergic vesicle release. Mutations in
the 1A subunit of pre-synaptic voltage-gated P/Q-type channels have been identified in
two strains of mice, known as the tottering and leaner mice133,134. The mutations which
occur at the S4-S5 linker region of the third transmembrane domain near the pore-
forming region of the channel, markedly reduce voltage-dependent inactivation of the
calcium channels during prolonged depolarization, increase glutamate release, and
produce a behavioural phenotype of motor seizures135. Accordingly, de-regulation of
pre-synaptic calcium channel activation is a critical contributor to aberrations in
glutamatergic vesicle fusion, plays a key role in de-regulation of cortical and
21
hippocampal circuitry, and –as will be discussed – contributes significantly to excitotoxic
neuronal injury.
These pre-synaptic, voltage-gated calcium channels, (and indirectly,
glutamatergic vesicle release) are also profoundly regulated by another pre-synaptic
protein, the G-protein coupled metabotropic glutamate auto-receptors. Metabotropic
glutamate receptors (mGluRs, discussed briefly in the next section Glutamate Receptors)
are seven transmembrane domain-containing proteins that bind synaptic glutamate both
pre and post-synaptically (the latter of which is discussed later, along with description of
the receptor itself). Pre-synaptic mGluRs serve the unique function of acting as a
glutamatergic autoreceptor; that is, a glutamate receptor that, upon binding glutamate,
provides negative feedback onto transmitter release machinery, thereby reducing
glutamatergic vesicle fusion and synaptic transmission. Indeed throughout the CNS,
mGluR agonists consistently reduce transmission at glutamatergic synapses (reviewed
extensively by 136,137).
Some of the precise mechanisms by which pre-synaptic mGluRs inhibit glutamate
release are known, and a large body of evidence describes the effects of mGluR
activation on pre-synaptic voltage-gated calcium channel activation. Agonists acting on
mGluRs reduce current density and calcium influx originating from N, L, and P/Q-type
calcium channels found in isolated neocortical, striatal, cerebellar, hippocampal, and
retinal ganglion neurons, thereby preventing glutamatergic vesicle fusion in all of these
cell types138-142. The mechanism of this inhibition involves translocation of the G-protein
βγ moiety, an observation that was made through an elegant experiment that injected Gβγ
cDNA into adult rat sympathetic neurons and observed tonic inhibition of N-type calcium
22
channel current143 (represented by a positive shift in the voltage dependence and a
slowing of channel activation). At around the same time, a separate investigation found
that transfection of neurons with Gβγ, but not Gα, induced a marked inhibition of P/Q-
type voltage-gated calcium channels, corroborating the evidence for this moiety in
channel inhibition144.
However, other studies suggest that the mechanism by which mGluRs inhibit
glutamate vesicle release is independent of voltage-gated calcium channel modulation.
For instance, L-AP4, a phosphonic derivative of glutamate, potent mGluR agonist and
synaptic depressant, induces a marked reduction in miniature excitatory postsynaptic
current (mEPSC) frequency in hippocampal CA1 pyramidal cells, while the broad
spectrum voltage-gated calcium channel antagonist cadmium completely abolishes
mEPSC activity in this cell type145-147. These results have lead authors to suggest that the
mechanism of mGluR-mediated inhibition of glutamate release is in fact quite different
from voltage-gated channel blockade. To address this discrepancy, other studies have
examined the influence of mGluR activation on pre-synaptic potassium channel
activation, a modulatory effect that would also decrease glutamate release. Indeed it has
been observed that mGluR activation activates pre-synaptic outward potassium
conductances in visual cortex, raising the possibility that mGluR activation reduces
glutamatergic signaling by a mechanism involving pre-synaptic potassium channels148.
A third mechanism through which pre-synaptic calcium is kept in homeostatic
balance is through the activity of the sodium/calcium (Na+/Ca2+) exchanger, another
protein that dynamically modulates the release of glutamate. Na+/Ca2+ exchangers are 11
transmembrane domain ion transporters found in almost all tissues of the body including
23
the brain, where their mRNA is abundant in the cortex, hippocampus, dentate gyrus,
thalamus, and cerebellum149. These exchangers play a critical role in the maintenance of
cytosolic calcium by pumping calcium ions out of the cell, using an electrogenic sodium
gradient as energy and thereby making this protein an anti-porter. The majority of
Na+/Ca2+ exchangers have a transport stoichiometry of 3Na+:1Ca2+, pumping 3 sodium
ions into the cell for every one calcium ion pumped out150.
At the cellular level, Na+/Ca2+ exchangers play a role both pre and post-
synaptically at glutamatergic synapses. Presynaptically – where the exchanger is most
abundant relative to other sites151-153 – the protein plays a role in calcium-dependent
neurotransmitter release by regulating [Ca2+] at nerve terminals152. When calcium enters
the pre-synaptic terminal, it is only required for a brief period of time (pre-synaptic
calcium transients last less than a millisecond154-156), and must be rapidly extruded157 to
prevent the aberrant and pathological event of uncontrolled vesicle fusion. After
depolarization-induced Ca2+ entry, Ca2+ efflux from isolated nerve terminals
(synaptosomes) is markedly slowed by the removal of extracellular sodium158,159,
suggesting that if the electrochemical gradient required for function of the Na+/Ca2+
exchanger is altered, so too are pre-synaptic calcium dynamics, and by association,
transmitter release. Pre-synaptic calcium extrusion by the Na+/Ca2+ exchangers is
therefore among the most critical regulators of neurotransmission, and a dysfunction of
this protein has dire consequences on neuronal cell viability and function.
As will be discussed in the section on excitotoxicity, dysfunction of the sodium-
calcium exchanger can lead to significantly augmented glutamate release at nerve
terminals, manifest through both spontaneous vesicular exocytosis and synaptic
24
facilitation160. Usually this is observed by loading the presynaptic terminal with sodium,
through ionophores or other compounds. The augmentation of pre-synaptic vesicle
release by high sodium is a phenomenon that has been observed in synapses of
invertebrates161-164, the frog neuromuscular junction165-168, and at both peripheral169,170
and central mammalian synapses153,171-173. In TBI, NCX dysfunction occurs through two
processes that will be discussed: proteolysis of the exchanger by activated proteases, and
reversal of the exchanger due to uncontrolled loading of presynaptic sodium, thereby
reversing the electrogenic gradient required for exchanger function. When uncontrolled,
this is turn leads to a number of rapidly fatal cellular processes and progression of
secondary insult caused by hyperactivation of glutamate receptors.
1.4.3 Glutamate Receptors Fast synaptic communication between nerve cells involves the control of
transmembrane electrostatic potential by a host of ion channels, including glutamate
receptors. Glutamate receptors are located in the postsynaptic membrane, and activated
by neurotransmitters (specifically, glutamate) that are released from the presynaptic cell.
Generally, glutamate receptors are closed in the resting state, but open in response to the
binding of agonist (i.e., they are ligand-gated), allowing selected ions to flow down their
electrochemical gradients through an internal pore (i.e., they are also ionotropic). This ion
flux mediates a local depolarization (positive change in membrane potential),
representing an excitatory signal that can be further processed by the post-synaptic cell.
The magnitude, duration, and type of signal depends on the subtype of glutamate receptor
passing current, as each channel has distinct kinetics and permeability (i.e., ionic
selectivity) that will characterize the depolarization.
25
Glutamate receptors are responsible for most excitatory signaling in the brain, and
are thought to play an instrumental role in the synaptic plasticity that mediates learning
and memory formation. Similarly, the physiological significance of glutamate receptor
function is highlighted by the involvement of these receptors in a number of CNS disease
states, including motor neuron disease, pain, epilepsy, stroke, and as discussed in this
thesis, brain trauma. On the basis of their response to synthetic chemical agonists and
sequence-homology criteria, three ionotropic glutamate receptor subtypes have been
identified: the N-methyl-D-aspartate (NMDA) receptor, the kainate receptor, and the α-
amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor. The general
characteristics of each channel will be discussed in this section, as pathophysiological
activity at all of these receptors has been implicated in excitotoxic injury following TBI.
However, as this thesis examines the specific role of the AMPA receptor in mediating
excitotoxic injury, a more thorough introduction to AMPA receptor function is necessary
to accurately clarify and justify the specific aims and hypothesis of this thesis. This will
follow this section.
1.4.3.1 NMDARs The NMDA receptor is a hetero-oligomeric assembly of integral membrane
protein subunits. This modular construction has aided in the identification of receptor
makeup and influence of subunit composition on the electrophysiological and
pharmacological properties of the channel.
The NMDA receptor is generally accepted as a hetero-tetrameric assembly of four
subunits, two of which are known as obligatory NR1-type subunits, and the other two of
the regionally localized NR2-type. In certain developmental periods and in restricted
26
brain regions, NR2 can be replaced with subunits of the NR3 subtype. Receptor isoforms
result from incorporating more than eight alternatively spliced variants of NR1 (a-h), and
peptides encoded by four separate NR2 genes (A-D)174-176. As a result, the receptor is
termed a dimer of dimers, with one dimer homomeric for NR1, and the other for NR2.
Each NMDA receptor subunit has two extracellular, globular domains: a ligand
binding domain (LBD) for binding of agonist (e.g., glutamate on NR2, glycine on NR1
and NR3) and an n-terminal domain (NTD)177,178. All subunits also contain three
transmembrane domains, and an intracellular c-terminal (CT) domain, which contains a
number of serine and tyrosine kinase phosphorylation sites that regulate channel gating
and receptor trafficking. Many of these phosphorylation sites are neighboured by PDZ-
domains (discussed later), which serve as protein:protein interaction motif’s necessary to
keep intracellular scaffolds close to the receptor complex. One such PDZ interaction
occurs through binding of the NR2B c-terminus to post-synaptic density protein, 95 kDa
(PSD-95), an interaction that regulates the post-synaptic production of nitric oxide, and
activation of Ras GTPases among other notable downstream effectors.
The NMDA receptor plays in integral role in physiological excitatory CNS
neurotransmission as well as pathological disease states. Two main signals generated
simultaneously by the receptor complex are responsible for the information conveyed at
these channels; the first is a depolarizing current, and the second is a biochemical signal
of calcium influx. Upon glutamate binding to the ligand binding domain in the presence
of glycine, the channel opens a cation-permeable pore causing a transient membrane
depolarization. However, in addition to this dependence of channel opening on agonist
binding, the NMDA receptor has a second dependence; membrane potential. At
27
hyperpolarized membrane potentials (including resting membrane potential), NMDA
receptors are blocked by sub-millimolar extracellular concentrations of magnesium
(Mg2+). Magnesium tightly binds the channel pore, and consequently reduces the NMDA
receptor component of synaptic currents considerably. However, when neurons are
depolarized (e.g., by activation of neighbouring glutamate receptors of the non-NMDA
subtype – see below), the magnesium block is partially expelled, allowing both sodium
and calcium influx through the receptor complex. This unique property renders the Ca2+
influx through NMDA receptors a type of neuronal coincidence detector for the
simultaneous occurrence of both depolarization and synaptic release of glutamate.
Calcium influx from the NMDA receptor triggers events crucial to neuronal
survival and plasticity. For example, calcium micro-domains located near the NMDA
receptor play an important role in synapse to nucleus signaling, triggering the
transcription of many pro-survival neuronal proteins. This occurs through a cascade
involving extracellular signal related kinase (ERK1/2), which undergoes nuclear
translocation in response to NMDA receptor activation and phosphorylates the cyclic-
adenosine-monophosphate (cAMP)-response element binding protein (CREB)179,180.
CREB is ubiquitously expressed transcription factor that initiates the transcription of a
number of anti-apoptotic factors, including brain derived neurotrophic factor (BDNF)181,
as well as anti-apoptotic bcl-2 proteins that inhibit the initiation of programmed cell death
(apoptosis)182. NMDAR-derived calcium binding to cytosolic calmodulin also initiates
the transcription of CREB-dependent proteins, since CREB phosphorylation also occurs
via activation of calmodulin-dependent activation of calmodulin kinase IV (CaMKIV)183.
28
NMDA receptor activation is also responsible for remodeling the synapse during
period of synaptic plasticity. The most thoroughly characterized examples of such
synaptic plasticity in the mammalian nervous system are long-term potentiation (LTP)
and long-term depression (LTD), which involve changes to the post-synaptic response of
neurons following various patterns of electrophysiological or chemical stimulation.
These events will be discussed in detail in the section on AMPA receptors, as although
they are initiated by activation of NMDARs, they primarily involve the trafficking of
AMPA receptors from cytosolic and extrasynaptic sites to the plasma membrane, and
vice versa.
Pathological activation of the NMDA receptor is implicated in numerous diseases
of the central nervous system, though the clinical failure of NMDA receptor antagonists
has brought this hypothesis under much scientific scrutiny in the last few years. The
section that follows this will discuss the involvement of the NMDAR in traumatic brain
injury-induced neuronal death and dysfunction.
1.4.3.2 AMPARs – Discovery and function Similar to the NMDA receptor, AMPA receptors are hetero-oligomeric proteins
made up of globular subunits, in this case termed GluR1-4 (also termed GluRA-D). The
polypeptides encoding AMPAR subunit makeup were first identified through expression
cloning in oocytes in 1994184, and the sequence predicted the functional domains that
each subunit is now known to contain. The cloned GluR1 polypeptides contained a
hydrophobic signal sequence, and four hydrophobic regions, which correspond to the
four transmembrane domains that span the plasma membrane as α helices.
29
Epitope tagging185 and glycosylation analysis184 subsequently identified the rest of
the AMPA receptor subunit topology. A large extracellular N-terminal region was
identified, which is followed by the first transmembrane domain. Following this, the
second transmembrane domain was identified as the channel pore region, which does not
actually traverse the membrane, but rather dips into it from the cytosolic side. The pore-
forming domain is followed by a true transmembrane domain, an extracellular loop, the
third transmembrane domain, and finally, the cytoplasmic tail, otherwise known as the c-
terminus region. Each subunit also contains an extracellular domain known as the S1-S2
site, which is the primary binding site for the endogenous agonist glutamate (Figure 2).
GluR1 mRNA is expressed in most brain regions, but is absent from the thalamus
and mesencephalon, anatomical locations which are known to express AMPA sensitive
channels184. This led to the subsequent homology cloning of three additional AMPA
receptor subunits, GluR2, GluR3 and GluR4, which were found to be highly related to
the originally cloned GluR1184,186.
AMPA receptor subunit mRNA is initially translated on the rough endoplasmic
reticulum (ER), where subunit dimerization occurs, and a high mannose glycosylation
attaches to specific asparagine residues in the first extracellular domain. Following ER
synthesis, the receptors transit through the golgi network, where the high mannose sugars
are modified to the complex carbohydrates seen in mature receptors. Receptors are
further trafficked to dendrites or axons, where they are inserted either extrasynaptically
(for GluR1187,188) or directly into the synapse (as is the case for GluR2188). Unlike the
NMDA receptor, AMPA receptor subunits are also sorted and stored in cytoplasmic
vesicles, which allows for the dynamic trafficking of receptors both to (exocytosis) and
30
from (endocytosis) the membrane during synaptic plasticity (discussed in the next
section, AMPA receptor trafficking).
Like the NMDA receptor, AMPA receptors are also a dimer of dimers. In the
forebrain, including the hippocampus and cerebral neocortex, the predominantly
expressed subunits are GluR1 and GluR2, with low levels of GluR3 and GluR4. Thus, the
major neuronal population -- pyramidal cells -- expresses AMPARs primarily comprised
of hetero-tetramers of GluR1 and GluR2. At one point it was hypothesized that GluR2/3
was the other major heteromer in cortical neurons, but the expression of GluR3 is low in
this cell type (i.e., ~ 10% of GluR1 or GluR2 levels), suggesting that GluR2/3 is not a
predominant subunit combination189.
All AMPA receptors are glutamate-gated channels whose post-synaptic activation
provides the primary sodium-dependent depolarization during excitatory
neurotransmission in the brain. Indeed synaptic strength is almost entirely mediated by
the ultimate density of AMPA receptors that accumulate at dendritic synapses190.
However, of all of the AMPA receptor subunits, GluR2 is responsible for dictating the
channel biophysics as well as ionic permeability. AMPA receptors that contain GluR2 (in
contrast to those lacking GluR2, for example GluR1 homomeric channels) have a number
of identifiable properties: 1) they are impermeable to divalent cations (including calcium
and zinc); 2) they have a lower single channel conductance than receptors lacking GluR2;
3) they exhibit linear current-voltage relationships and 4) they are not subject to blockade
by intracellular polyamines.
GluR2 dictates these processes as a result of its amino acid makeup. Most mature
GluR2 protein contains a positively charged arginine residue (R+) within the re-entrant
31
membrane loop (i.e., the channel pore region) at position 607 in place of the genomically
encoded neutral glutamine (Q) residue. This change arises from hydrolytic RNA editing
of a single adenosine base to inosine by the adenosine deaminase enzyme ADAR2.
Notably, this Q/R editing is exclusive to GluR2, and therefore does not occur in any of
the other AMPAR subunits. The addition of this positive charge into the pore of AMPA
receptor channels containing GluR2 lowers the single-channel conductance, prevents the
passage of divalent cations through the receptor, and also repulses the intracellular
blockade of the channel by similarly charged polyamines (e.g., spermine) at positive
voltages, thereby sustaining a linear relationship between membrane voltage and current
amplitude (as opposed to the inwardly rectifying relationship observed when patching
AMPA receptors lacking GluR2).
In addition to RNA editing, AMPA receptor molecular diversity is further
complicated by alternative RNA splicing of GluR1-4. Each AMPA receptor subunit
exists as either of two distinct isoforms, termed “flip” and “flop”, both of which are
generated by alternative splicing of a 114 base pair region immediately adjacent to
another RNA editing site, the R/G site. This splicing process introduces a functionally
critical cassette of 38 amino acids (either flip or flop) into the extracellular loop, which
controls AMPAR desensitization and recovery following agonist binding. Differentially
spliced subunits also exhibit varying sensitivity to allosteric modulators; for example,
cyclothiazide, which reduces AMPA receptor desensitization, is only active in flip, but
not flop variants of recombinant receptors. RNA splicing is also developmentally
regulated, with only flip splice forms expressed in early postnatal mammalian life,
followed by expression of GluR flop isoforms later in development.
32
AMPA receptors are also complemented on the plasma membrane by
transmembrane AMPA receptor regulatory proteins, or TARPs. These proteins, including
the most well characterized stargazin, co-assemble stoichiometrically with native
receptors, acting as auxiliary subunits that are required for receptor maturation,
trafficking, and other channel functions191,192. Further detail of TARP-mediated AMPA
receptor modulation are reviewed elsewhere192.
The ionic permeability of > 95% of native AMPA receptors is exclusively
monovalent due to the presence of edited GluR2 in the receptor complex; however, there
are a number of neuronal inputs that are capable of modifying AMPA receptor ionic
permeability to include passage of divalent cations, via the removal of GluR2. This
modification of AMPA receptor ionic permeability to include calcium influx is a critical
mediator of both synaptic plasticity and excitotoxic neuron death in a number of CNS
diseases, including ischemia, brain trauma, epilepsy, and motor neuron disease. The
mechanisms through which GluR2 expression is altered under both physiological and
pathological conditions are highly complex -- involving epigenetic changes to mRNA
editing as well as intracellular protein:protein interactions between PDZ domains -- and
are discussed at length in the sections that follow.
33
Figure 2. Schematic diagram of an AMPA receptor subunit. All receptor subunits have a
similar structure and topology. The N-terminal domain (NTD) is followed by S1, which
together with S2 forms the glutamate binding site (Glu). Of the four hydrophobic
segments, three span the membrane, while one (domain 2) dips into the membrane from
the cytoplasmic face and contributes to the channel pore. The alternatively spliced flip/
flop region and the C-terminal PDZ ligand, which interacts with intracellular PDZ
domains, are shown.
34
Figure 2. Schematic diagram of an AMPA receptor subunit. Modified with permission from Bredt & Nicoll, 2003. “AMPA receptor trafficking at excitatory synapses”. Neuron. 40, 361-379.
35
1.4.3.3 Kainate receptors Kainate receptors are another class of ionotropic glutamate receptor, made up of
subunits KA1-2, and GluR5-7. KA1 and KA2 on their own do not form functional ion
channels, but when expressed in conjunction with GluR5-7 will form a channel that
allows ion flux in response to glutamate stimulation184. Kainate receptor mRNA can be
detected in a number of brain regions including the hippocampus, cerebellum, amygdala,
and spinal cord. Generally speaking, kainate receptors are sodium channels, triggering a
local depolarization of membrane potential upon agonist binding. However, these
receptors have demonstrated calcium permeability in recombinant systems when certain
subunit combinations are applied, alluding to the possibility that endogenous kainate
receptor subtypes might also play an important role in post-synaptic calcium
signaling193,194. Similar to other glutamate receptors, kainate receptor electrophysiology
can also be modulated by intracellular effectors. For example, protein kinase A-
dependent phosphorylation of GluR6 increases kainate receptor single channel
conductance, by increasing the coupling efficiency of glutamate binding and channel
opening195.
Kainate receptors also mediate glutamate release and contribute to excitotoxic
neuronal damage, particularly the death of oligodendroglial cells196. Pre-synaptically,
investigators report that kainate receptors reduce glutamate exocytosis197, while post-
synaptically, kainate receptors couple to c-Jun N-terminal kinase (JNK) activation,
initiating an apoptotic cascade that contributes to neuronal and glial cell death in both
epilepsy and cerebral ischemia198,199. This has prompted emerging therapies aimed at
36
uncoupling the kainate receptor from its downstream apoptotic machinery with the use of
inhibitory peptides198,199.
1.4.3.4 Metabotropic Glutamate Receptors (mGluRs)
Metabotropic glutamate receptors have been discussed previously in the context
of regulating synaptic transmission, through their modulation of pre-synaptic voltage
gated calcium channels. Post-synaptically however, these 7-transmembrane domain
single peptide proteins couple to G-protein activation, resulting in slow, modulatory
effects on neurotransmission. Therefore, unlike the ionotropic NMDA and AMPA
receptors, mGluRs are not ion channels. However, their modulation of neuronal
physiology is responsible for many types of synaptic plasticity, including long-term
depression of post-synaptic glutamatergic EPSCs. Their effect on glutamate dependent
ion flux is therefore indirect, but nonetheless critical to CNS excitatory signaling.
There are 8 different mGluR subtypes that have been identified (mGluR1-8),
which are subdivided into three groups based on their sequence homology and their
associated signal transduction pathways. Group I mGluRs consist of mGluR1 and
mGluR5, which couple intracellularly to phospholipase C and generation of inositol
triphosphate (IP3)200,201. This cascade is responsible for liberation of intracellular calcium
stores from the endoplasmic reticulum. Group I mGluRs have also demonstrated
inhibitory activity on excitatory EPSCs in the hippocampus, through G-protein
independent activation of tyrosine kinases202. Group II mGluRs consist of mGluR2 and
mGluR3, and are largely responsible for the pre-syaptic effects on N and P/Q-type
voltage gated calcium channels discussed previously203,204. These receptors also have an
37
inhibitory effect on adenylyl cyclase signaling, reducing intracellular levels of cyclic
AMP (cAMP) and activation of its downstream effectors including voltage-gated calcium
channels, protein kinase A (PKA) and cyclic nucleotide gated ion channels137. Group III
mGluRs include mGluR4,6,7, and 8, and have modulatory properties similar to the group
II mGluRs. They also act as glutamatergic autoreceptors, reducing pre-synaptic glutamate
release through modulation of voltage-gated calcium channels137.
Because of the inhibitory effect of mGluRs on pre-synaptic glutamate release, the
activation of these receptors has gained much attention for the treatment of a number of
CNS disorders involving excitotoxicity, including TBI205-208. In the following section,
evidence will be presented that suggests that reduced activity of pre-synaptic mGluRs
might contribute to excitotoxic glutamatergic signaling following CNS trauma.
1.4.4 The concept of excitotoxicity The neurotoxic potential of glutamate was first proposed by Lucas and Newhouse
in 1957, when they discovered that injections of L-glutamate could destroy the inner
layers of the mouse retina209. Twenty years later, Olney described the cerebral lesions
associated with injection of kainate (structurally related to glutamate) to young animals
lacking an intact blood-brain barrier. Olney’s initial findings were also critical to our
modern understanding of how glutamate kills neurons, as his data described rapid cellular
swelling near dendrosomal components, now known to be particularly enriched in the
excitatory amino acid (EAA) receptors which were just discussed. It was in 1969 that he
coined the term “excitotoxicity”, to refer to neuronal death induced by excitatory amino
acids.
38
Our understanding of how glutamate receptor over-activation induces neuronal
death is rooted in important ion substitution experiments performed in the late 80’s. It has
long been understood that stimulation of glutamate receptors increases the post-synaptic
concentrations of both intracellular sodium and calcium, and a separate role for these ions
has been established in excitotoxic neuron death. First, investigators have demonstrated
that neuronal cultures exposed to glutamate exhibit immediate and irreversible sodium-
mediated cell swelling, even in the absence of extracellular calcium210. However, a role
for calcium was identified later when other groups described delayed (i..e., long term)
glutamate-induced neuronal death when extracellular sodium was removed211. Indeed
cell death in this model was attenuated only in the absence of both extracellular sodium
and calcium. These observations provided for a simple model of excitotoxicity consisting
of two components: an early sodium-mediated cell swelling, and a more delayed,
calcium-dependent neuronal degeneration, which can be reproduced through the use of
calcium ionophores115.
Little debate exists that there is a strong correlation between intracellular calcium
concentrations and neuronal injury induced by glutamate. It is well understood that
elevated intracellular calcium is the initiating factor in many neurotoxic cascades,
including the uncoupling of mitochondrial electron transport from ATP synthesis, the
activation of proteolytic enzymes (e.g., calpains) that cleave the neuronal cytoskeleton,
endonucleases that fragment nuclear DNA, production of reactive oxygen and reactive
nitrogen species, and the initiation of programmed cell death (apoptosis). However, there
are two schools of thought directed at understanding how glutamate shifts from an
important mediator of neuronal excitatory physiology to an endogenous neurotoxin
39
following injury to the central nervous system. The first hypothesis is that following CNS
trauma (including ischemia and TBI), there exists prolonged activation of glutamate
receptors due to elevated levels of extracellular glutamate, resulting in increased calcium
influx. The second hypothesis suggests that injury to the CNS induces changes to
glutamate receptor function, allowing excessive entry of extracellular calcium. In TBI,
there exists evidence for the involvement of both of these phenomena, which may in fact
also occur at the same time.
1.4.4.1 De-regulation of glutamate release
Microdialysis studies have reported that following traumatic brain injury,
extracellular glutamate is markedly increased113,212-217, in some cases up to 9 days
following injury218. Indeed these clinical observations have been recapitulated by animal
models of TBI219-221. Accordingly, there have been a number of hypotheses put forward
to explain how glutamate release and/or reuptake are altered following TBI, resulting in
excessive extracellular glutamate accumulation. The first relates to dysfunction of pre-
synaptic calcium extrusion and subsequent glutamate vesicle fusion caused by reversal
and failure of the sodium-calcium exchanger. TBI, as discussed, causes a reduction of
cerebral perfusion pressure when intracranial pressure increases. This hypoperfusion
deprives the cell of both oxygen and glucose. As is well understood from the basics of
cellular respiration, glucose is the primary method of ATP production. When ATP levels
are depleted, there is a dysfunction of the sodium-potassium exchanger (the membrane-
bound ion pump responsible for maintaining neuronal resting membrane potential). This
results in neuronal depolarization and accumulation of intracellular sodium. As was
40
discussed previously, the sodium-calcium exchanger operates on an electrogenic sodium
gradient, such that when intracellular sodium is markedly increased, operation of the
pump ceases (failing to extrude calcium) and in some cases will reverse, pumping
calcium into the cell. Calcium will also enter the cell through the activation of voltage-
gated calcium channels. This elevation of pre-synaptic calcium in turn triggers the fusion
of glutamatergic vesicles in an unregulated fashion, resulting in excessive glutamate
exocytosis and toxic concentrations of the transmitter in the synaptic cleft.
There is also some evidence that sodium-calcium exchanger (NCX) function
ceases due to proteolytic cleavage. Proteolytic inactivation of NCX has been
demonstrated in cellular models of excitotoxicity, as well as in whole animal CNS injury,
where it was noted that calpain inhibition (preventing NCX cleavage) or expression of
NCX lacking the calpain cleavage moiety protects against excitotoxicity222. Further,
inhibition of the sodium-calcium exchanger activity has shown neuroprotective properties
in both an animal model of TBI, as well as following cellular strain injury, suggesting
perhaps that the reverse operation of the protein contributes to neuronal death223,224.
A second mechanism by which extracellular glutamate is thought to increase is
through dysfunction of astrocytic glutamate transporters, known as excitatory amino acid
transporters (EAAT). Five EAAT subtypes have been cloned to date (EAAT1-5), two of
which (EAAT1-2) exist primarily in astrocytes225. Astrocytic EAAT2 accounts for > 90%
of total glutamate transport in the brain226-228, the majority of which is involved in
clearance of synaptic glutamate following regulated excitatory neurotransmission68,226.
A number of studies have identified both dysfunction and reversal of EAATs
following CNS injury, leading to the hypothesis that impaired clearance of synaptic
41
glutamate, or reversal of astrocytic transporters leads to increases in extracellular
glutamate levels following TBI. Firstly, transient down-regulation of EAAT1 and
EAAT2 has been reported in the ipsilateral cerebral cortex following controlled cortical
impact (CCI, an in vivo experimental model of TBI), concomitant with a reduction in
[3H]-D-aspartate binding229. This study is corroborated by evidence that down-regulation
of EAAT1&2 levels in the ipsilateral and contralateral cortex after CCI are associated
with a rise in CSF glutamate levels, reaching a maximum at 48 h following the injury230.
Other mechanisms aside from protein down-regulation are also thought to be involved in
EAAT dysfunction. Notably, inhibition of astrocytic glycolysis, a key component of
glucose metabolism, causes reversal of glutamate transporter activity. Indeed as discussed
glucose delivery is impaired following TBI, and this mechanism may play a role in
aberrant extracellular glutamate accumulation. EAAT transporter activity has also been
shown to reverse under ischemic conditions231,232, which is frequently an insult
superimposed on cerebral tissue following TBI. Collectively, the data suggesting that
EAAT protein is lost following traumatic injury coupled with reversal of transporter
activity during ischemia suggest that the activity of these proteins may play a critical role
in excitotoxic neuron death following TBI.
Accumulation of extracellular glutamate might also occur via cytoplasmic leakage
through damaged cellular membranes. The intra to extracellular ration of glutamate is
approximately 1000:1, suggesting that membrane shearing or cellular lysis from
cytotoxic edema might contribute to the early rise of glutamate into the extracellular
space. Indeed very high levels of dialysate glutamate are reported as a microdialysis
probe is lowered into the brain’s parenchyma, producing a laceration injury. A similar
42
cellular leakage is thought to occur in the shear stress zone produced after tissue
compression or contusion.
Leakage of plasmatic glutamate through disrupted blood-brain barrier (BBB)
dysfunction has also been proposed as a mechanism, albeit minor, of augmented
extracellular glutamate. It is well established that TBI induces a disruption in blood-brain
barrier integrity, and as the plasma concentration of glutamate is ~ 50 μM (i.e., 50x that
of the extracellular space), plasmatic glutamate can leak into the interstitial space after
injury233,234.
Finally, as discussed, glutamate release is profoundly affected by pre-synaptic
activation of group II metabotropic glutamate autoreceptors, which slow vesicular
exocytosis through inhibition of voltage-gated calcium channel activity. A number of
studies have identified a loss of this inhibitory activity following TBI, leading to the
hypothesis that pre-synaptic mGluR dysfunction contributes to excitotoxic glutamate
release. Indeed loss of group II mGluR mRNA and protein were reported following
experimental diffuse brain injury and lateral fluid percussion, a phenomena reported up to
7 days following trauma206,235. Accordingly, authors have tested the efficacy of group II
mGluR activation following TBI in attenuating neuronal injury. Indeed administration of
both a group II and III mGluR agonist 30 min after lateral FPI has attenuated both
neurotoxic extracellular glutamate accumulation and improved functional outcome
following the injury236,237. This approach has also improved neuronal survival in cellular
models of TBI, suggesting that augmentation of glutamatergic autoreceptor activity can
attenuate excitotoxic neuronal death.
43
1.4.4.2 An alternative look at excitotoxicity:
Post-synaptic glutamate receptor dysfunction The second hypothesis of how excitotoxicity occurs relates to the post-traumatic
dysfunction of glutamate receptor activity, and has emerged out of some contradictions of
the initial hypotheses related to augmented glutamate release as the cause of
excitotoxicity per se. According to some authors, the concept that high extracellular
glutamate is the key to excitotoxicity in TBI conflicts with important and convincing
experimental data. A number of studies employing intracerebral microdialysis have
indeed shown that cortical injury markedly increases the concentration of extracellular
glutamate (discussed in the previous section). However, it has also been demonstrated
through rapid sample collection at 2 minute intervals that this increase is often transient,
peaking within five minutes of impact and rapidly declining to control levels238-240.
Notably, much of the data reporting augmented extracellular glutamate levels following
trauma are not specific to excitatory amino acids, with similar abnormalities reported for
gamma-aminobutyric acid (GABA), taurine, ascorbate, and adenosine233,241,242.
Importantly, these increases occurred on the same time scale and at the same magnitude
as glutamate release. Thus, research efforts have also focused on identifying injury-
induced changes to glutamate receptor function, in an effort to understand how glutamate
might prove neurotoxic in the absence of extracellular accumulation, or in the presence of
only moderately elevated glutamate levels.
Augmented glutamate receptor function has deleterious effects on neuronal
survival and function in two ways; it imparts a vulnerability to secondary excitotoxicity,
and it may interfere with constitutive glutamatergic physiology, such that ordinarily
44
innocuous stimulation of glutamate receptors is rendered deleterious to cellular survival.
The concept that a traumatic injury to the CNS can change the ionic permeability,
kinetics, or subunit composition of glutamate receptors is supported by a number of
investigations. Nearly 15 years ago, it was demonstrated that mechanically injured
neurons exhibit a reduced voltage-dependent magnesium blockade of the NMDA
receptor243. Indeed this loss of magnesium blockade resulted in substantially larger
NMDA-induced calcium influx, equivalent to stimulating control neurons in magnesium-
free extracellular solution. This result has been corroborated by other studies that have
shown that mechanical stretch injury initiates large calcium transients that originate from
the NMDA receptor, and are accordingly antagonized by AP-5244-246, and that neuronal
stretch enhances NMDAR activity by increasing maximal NMDAR current, and steady-
state current density247. Unsurprisingly, this enhanced NMDAR current translates to a
marked vulnerability to otherwise innocuous levels of both glutamate and NMDA.
Investigators have repeatedly shown that treatment of sub-lethally stretched neuronal
cultures with L-glutamate augments cell death via the influx of NMDAR-derived
calcium117,119,120,248. Thus, in the absence of abnormally high levels of extracellular
glutamate, traumatic injury can impart a change to post-synaptic receptor function that
translates to increased susceptibility to glutamate receptor stimulation.
These findings are not limited to activity at the NMDA receptor. Much attention
has been paid to trauma-induced changes to AMPA receptor function as well, as these
receptors mediate the majority of ionotropic neurotransmission. Changes to AMPA
receptor function and ionic permeability following traumatic injury have been reported.
Agonist (i.e., AMPA)-activated currents recorded from traumatically injured neurons
45
exhibit marked potentiation, with increases in both AMPA and kainate mean steady-state
current density. AMPA receptor kinetics are also affected by stretch injury, with trauma
resulting in a significant increase in both the 20-80% activation rate and desensitization
time constant (τ)249,250. Further, traumatic injury to cortical neurons has twice been shown
to augment AMPAR-mediated calcium influx251,252, suggesting that traumatic injury is
capable of increasing the divalent ion permeability of an ordinarily calcium-impermeable
receptor. Notably, these changes to AMPA receptor function appeared to be mediated by
regulated signaling pathways, as the effects of trauma on AMPAR current density were
abolished by application of NMDA receptor antagonists and inhibitors of protein kinase
C, suggesting a potential calcium-dependent modification of AMPA receptor function.
However, though the phenomenon of trauma-induced increases in AMPA receptor
function has been consistently reported, the mechanism through which trauma modifies
AMPA receptor function has not been mapped out.
1.4.4.3 Consequences of excitotoxicity: Ca2+-dependent neurodegeneration The excessive stimulation of glutamate receptors following TBI from either
augmented glutamate release or altered post-synaptic glutamate receptor function can
only be tolerated for a short period of time due to the cytotoxic effects of elevated
intracellular calcium. As discussed, neurons possess specialized homeostatic mechanisms
to ensure the strict regulation of cytosolic free calcium, which include the activity of
sodium-calcium exchangers and calcium buffering proteins, as well as calcium
sequestration into organelles. However, excessive calcium influx can override these
regulatory processes, leading to the inappropriate activation of Ca2+-dependent processes
46
that are normally dormant or operate at low levels, causing metabolic derangements and
eventual cell death.
1.4.4.4 Oxidative stress and Mitochondrial Injury One mechanism through which stimulation of glutamate receptors causes calcum-
dependent cell death is through the production of reactive oxygen and nitrogen species
(ROS/RNS) downstream of the NMDA receptor. The NMDAR is structurally connected
to the intracellular, calcium-dependent synthase responsible for the generation of
neuronal nitric oxide (nNOS), via a membrane associated guanylate kinase (MAGUK)
protein scaffold. This protein, known as post-synaptic density, 95 kDa (PSD-95) binds
to both nNOS as well as the c-terminus of the NR2B NMDA receptor subunit. As such,
calcium influx from the NMDA receptor is placed in close proximity to nNOS,
effectively coupling activity of the NMDAR to generation of post-synaptic nitric oxide
(NO). While NO (by definition a free radical) is an important second messenger involved
in a number of constitutive neuronal regulatory pathways and reacts slowly with most
biological molecules, when combined with other free radicals it is remarkably reactive
and has acutely cytotoxic effects.
When excessive free calcium is sequestered by the mitochondria in an effort to
restore intracellular calcium homeostasis, the elevated calcium level in the mitochondria
increases the production of the superoxide anion. The reaction of this mitochondrial-
derived superoxide with NMDA-derived nitric oxide produces the highly reactive
nitrating species peroxynitrite (an oxidant with activities similar to that of the hydroxyl
radical and nitrogen dioxide radical). Peroxynitrite, which investigators have shown is
produced in excess following experimental traumatic brain injury119,248,253 as well as
47
following the exposure of neuronal cultures to excessive L-glutamate, produces nitration
of amino acid aromatic rings254,255, lipid peroxidation254,255 and DNA fragmentation248,254-
256 — all of which are rapidly fatal cellular processes responsible for excitotoxic cell
death.
Calcium overload from glutamate receptor over-activation also plays a critical
role in early mitochondrial swelling257-260. The excessive sequestration of calcium by
mitochondria causes not only superoxide generation, but also mitochondrial membrane
depolarization, the opening of membrane permeability transition pores and the release of
initiating factors of programmed cell death (apoptosis), including for example
cytochrome C. Once released into the cytosol, cytochrome C, through binding
apoptosome activating factor 1 (APAF-1), activates and recruits caspase-9261-263, a
cysteine protease responsible for the progression of apoptotic cascades to the point of cell
death.
The loss of mitochondrial function during excitotoxicity is cyclical in nature, as it
not only eliminates calcium buffering capacity and initiates apoptosis, but it also
contributes indirectly (via loss of ATP synthesis) to the influx of calcium resulting from
bioenergetic failure of the previously discussed ATP-dependent ion pumps.
Cytoprotective approaches to excitotoxic degeneration have therefore targeted
mitochondrial function to attenuate the multi-pronged effects of mitochondrial damage.
For example, cyclosporin A, an immunosuppressant and inhibitor of the mitochondrial
membrane-permeability-transition pore, has been shown to significantly reduce neuronal
cell loss following TBI, thus illustrating the importance of these processes.
48
Figure 3. Processes leading to excitotoxicity after CNS injury. A) Excitotoxicity can
occur following pre-synaptic depolarization caused by failure of ATP-dependent sodium
and calcium extrusion. Excessive calcium accumulating via voltage-gated calcium
channels and reversal of sodium/calcium exchangers leads to a deregulation of
glutamatergic vesicle fusion and massive glutamate exocytosis. Other sources of
synaptic glutamate accumulation include plasmatic leakage through disrupted blood-brain
barrier dysfunction, impaired glial-dependent glutamate uptake, as well as leakage
through damaged cellular membranes following cytotoxic edema. B) An alternative
hypothesis proposes that excitotoxicity can occur via a dysfunction of post-synaptic
receptors. These aberrations can include intracellular modifications leading to increased
receptor calcium permeability, decreased receptor desensitization, and increases in mean
steady-state current densities. Together these processes also lead to cell swelling and
calcium accumulation. Both theories of excitotoxicity involve a cytotoxic role for
calcium, which leads to potent oxidative injury, DNA fragmentation, cytoskeletal
proteolysis, and the initiation of apoptosis.
49
Figure 3. Processes leading to excitotoxicity after CNS injury
50
There is unquestionably a role for the NMDA receptor in the pathophysiology of
excitotoxic injury after TBI. However, treatments aimed at reducing NMDA receptor
functioning have proved to be impractical, due to interference with physiologic receptor
function and suppression of the pro-survival NMDA receptor signaling discussed
previously. Indeed all clinical trials for TBI employing NMDA receptor antagonists were
stopped prematurely due to adverse side effects and a lack of efficacy in improving
functional outcome. Rather, there has emerged a general consensus that antagonists of
AMPA receptors, (e.g., the quinoxalinedione NBQX) are much more effective than
NMDA receptor antagonists in attenuating neuronal cell death during periods of
excitotoxicity, even when given as late as 24 hours following injury to the CNS264-266.
However, it is not completely understood how native AMPA receptors mediate
excitotoxic neuronal cell death, due to their generally poor permeability to calcium ions.
1.5 AMPA Receptor Trafficking: GluR2-lacking AMPA Receptors as sources of calcium influx
Indeed the vast majority of AMPA receptors in the CNS exhibit a low
permeability to divalent cations, due to the presence of the GluR2 subunit in the receptor
heteromer. Accordingly, a reduction of the AMPA receptor GluR2 content would be
expected to have a dramatic impact on neuronal physiology and resistance to excitotoxic
injury. AMPA receptors lacking GluR2, as discussed earlier, exhibit higher single
channel conductances as well as permeability to both calcium and zinc. These attributes
make the GluR2-lacking AMPA receptor a powerful mediator of neuronal signaling.
Many physiological and pathophysiological processes involve the dynamic regulation of
51
the AMPA receptor GluR2 content, which itself is not static but subject to remodeling
from a variety of neuronal inputs. Changes to AMPA receptor GluR2 levels have been
observed during synaptic plasticity (including the induction of LTP267-269 where GluR2-
lacking AMPARs play a role in increasing basal synaptic strength), but also in disease
states, including drug abuse270-275, epilepsy276-278 and ischemia279-286.
In the latter scenarios, it is clear that the expression of calcium-permeable AMPA
receptors during periods of excitotoxicity imparts neuronal vulnerability to cellular injury
due to augmented cytosolic Ca2+ loads. While some investigators have suggested that the
NMDA receptor mediates the majority of glutamate-dependent excitotoxicity, it has been
shown that in fact over-activation of calcium-permeable AMPA receptors – when they
are expressed – results in levels of neuronal cell death similar to loading cells with Ca2+
via NMDA receptor activation287. Investigators have also repeatedly demonstrated that
calcium entry through calcium-permeable AMPA receptors triggers marked intracellular
production of reactive oxygen species as well as severe mitochondrial depolarization and
injury comparable to that produced by excessive stimulation of the NMDA receptor288-290.
Accordingly, oxygen radical scavengers and inhibitors of oxygen radical production have
demonstrated a marked cytoprotective efficacy against cell death mediated by AMPA
receptors291,292.
Thus, while GluR2-containing AMPARs require excessive amounts of
stimulation to induce neuronal death (likely due to neuronal depolarization and secondary
Ca2+ influx through voltage-sensitive Ca2+ channels293-296), the subset of AMPA receptors
lacking GluR2 appear to be particularly lethal sources of calcium. Indeed this can also be
observed with the widespread neuronal damage that follows the glutamatergic stimulation
52
of certain types of neurons that express endogenous calcium-permeable AMPARs287-290,
and the marked neuroprotection afforded by AMPA receptor antagonists in experimental
disease states involving excitotoxicity (reviewed by 297). Accordingly, this has promoted
a tremendous amount of interest in identifying the mechanisms responsible for the
aberrant expression of calcium-permeable AMPA receptors in neuronal populations
ordinarily expressing GluR2-containing channels, as this may shed important insight into
how AMPA receptors mediate excitotoxic neuronal death.
1.5.1 Modification of the AMPA Receptor GluR2 content. It is clear that the cytotoxic potential of AMPA receptor stimulation is almost
entirely dependent on the presence or absence of GluR2 in the receptor complex. Recent
studies have demonstrated that the remodeling of the AMPA receptor GluR2 content is a
consequence of the redistribution and trafficking of AMPA receptor subunits, as well as
epigenetic reprogramming of RNA editing (reviewed by 298). The molecular mechanisms
underlying activity-dependent remodeling of the subunit composition and permeability of
synaptic AMPA receptors are being further examined as potential targets of anti-
excitotoxic therapy, as it appears many of them contribute in critical ways to glutamate-
dependent neuronal death following CNS injury.
1.5.1.1 Epigenetic silencing of GluR2 Insights from studies of global or transient forebrain ischemia have shed
important light on one mechanism of GluR2 mRNA and protein loss in certain neuronal
populations during excitotoxicity. Ischemia -- which shares with brain trauma the
53
involvement of excitotoxicity in expanding the primary lesion into widespread neuronal
damage – involves an early rise in intracellular calcium and a delayed rise in free zinc,
similar to observations made in dying neurons following TBI. The neurons exhibiting
these rises in free Ca2+ and Zn2+ (primarily CA1 hippocampal cells) also demonstrate a
concomitant reduction in GluR2 mRNA and protein abundance281,285,299,300, inducing a
long-lasting switch in AMPA receptor phenotype, from GluR2-containing, to GluR2-
lacking280,283. Indeed following the excitotoxic input delivered during ischemia, AMPA
receptors physiology exhibits marked inward rectification of EPSCs, calcium
permeability, as well as sensitivity to polyamine antagonism280,283, three physiological
hallmarks of GluR2-lacking receptors. Following ischemia, antagonism of GluR2-lacking
AMPA receptors (but not GluR2-containing or NMDA receptors) affords significant
neuroprotection, suggesting that excessive activity at these receptors can initiate
substantial neuronal loss. This, along with the evidence that acute knockdown of GluR2
protein by anti-sense oligonucleotides causes death of hippocampal neurons even in the
absence of CNS injury301, has perpetuated the hypothesis that a reduction of GluR2
protein is a causal mechanism of cell death during excitotoxic injury.
The loss of GluR2 in ischemic injury is thought to involve transcriptional
silencing of its mRNA production. GluR2 mRNA transcription is under strict control by
repressor element-1 silencing transcription factor (REST), a transcriptional repressor that
actively represses neural specific genes important to synaptic plasticity and
development302-304. For example, REST functions, using epigenetic modifications, to
silence target genes in neural progenitor cells during development to maintain particular
receptor phenotypes305,306. However, under pathological scenarios where a loss of a
54
particular gene is undesirable, REST can mediate neuronal death. In CNS injury, REST
binds the GluR2 promoter, and functions through chromatin remodeling to suppress
GluR2 protein in neurons destined to die from excitotoxic insult281,285. Indeed it has been
shown that acute knockdown of REST affords significant cytoprotection in ischemia, and
preserves GluR2 protein levels281,285.
GluR2 editing is also affected by neuronal injury. As discussed, the RNA-editing
enzyme adenosine deaminase acting on RNA 2 (ADAR2) is responsible for editing
GluR2 RNA to contain the positively charged arginine residue in place of the genomic
glutamine, thereby governing the ionic permeability of the AMPA receptor channel pore.
Accordingly, investigators have shown that ischemic injury inhibits the activity of
ADAR2, rendering a substantial portion of GluR2 RNA in its unedited form, and thereby
increasing the proportion of calcium-permeable AMPA receptors307. Indeed this
increased population of GluR2-lacking AMPARs imparts neuronal vulnerability to
delayed cell death. Direct delivery of ADAR2 or constitutively active cAMP response
element binding protein (CREB), which induces ADAR2 expression, restores Q/R editing
and protects vulnerable neurons from cell death307. Thus, reduced GluR2 Q/R editing
further contributes to neuronal vulnerability in excitotoxic injury.
1.5.1.2 Local trafficking of GluR2 protein
The mechanisms discussed above are intriguing examples of how total GluR2
protein expression can be altered by injury to the brain, and how this can manifest as a
susceptibility to delayed excitotoxic injury. However, there are other important
mechanisms of GluR2 regulation that do not involve suppression of the protein’s
55
expression, but rather incorporate endo and exocytotic trafficking of the protein both to
and from the plasma membrane.
Much of our understanding of GluR2 trafficking comes from studies of synaptic
plasticity and the induction of long-term potentiation (LTP). LTP involves a long-lasting
increase in the efficacy of synaptic transmission, and is generally induced by high-
frequency stimulation of afferent fibers. One mechanism through which LTP has been
proposed to involve is an activity-dependent switch in AMPA receptor subtype. For
example, high frequency stimulation of the Schaffer collateral-CA1 synapse, which
ordinarily expresses GluR2-containing, calcium impermeable receptors, causes a
transient incorporation of GluR2-lacking receptors, thereby increasing both local calcium
influx as well as single channel conductance. Together, these two properties of calcium-
permeable AMPA receptors increase basal synaptic strength, initiating a larger post-
synaptic depolarization per quanta of pre-synaptic vesicle released.
The time scales on which these changes occur (i.e., within minutes) do not favor
the hypothesis that these phenomena are observed due to transcriptional silencing of
GluR2 mRNA. It is also unlikely that de novo GluR1 protein translation can occur on this
time scale. Indeed even with exogenous expression of AMPA receptor mRNA on isolated
dendrites, it takes hours for protein to be translated, folded, assembled, exported, and
trafficked to the post-synaptic membrane via secretory machinery308. Thus, recent
efforts have focused on identifying a more rapid mechanism of modifying the AMPA
receptor GluR2 content.
It is now known that mechanisms exist in neurons for the subunit specific
trafficking of AMPA receptors to and from synapses309. This was first illustrated by
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recombinant expression of GluR2 homomeric AMPA receptors in hippocampal CA1
neurons, where the channels were found to be constitutively incorporated into synaptic
sites310,311. Further work identified that the proteins responsible for GluR2 trafficking
regulated the movement of the subunit via protein-protein interactions with the c-
terminus of the subunit. The best characterized of these interactions are at the proximal
N-ethylamide-sensitive fusion protein (NSF)/Adaptor protein 2 (AP2) site, and at the
distal post synaptic density protein (PSD95), Drosophila disc large tumor suppressor
(DlgA), and zonula occludens-1 protein (zo-1) (PDZ) site (Figure 2).
1.5.1.2.1 NSF/AP2 Site interactions in GluR2 trafficking
NSF -- an ATPase that is involved in a number of membrane fusion events312 –
interacts directly with the GluR2 carboxy (c)-terminus313-315, and is thought to play a
critical role in the stabilization of the subunit’s surface expression. The interaction with
NSF on the GluR2 c-terminus is located at a membrane proximal site313. Though the
binding site involves a motif that is completely novel to this protein interaction, other
proteins may coassemble with the GluR2-NSF complex, including α and β SNAPs314,316
(soluble NSF attachment proteins). At the same site, AP2, an adaptor protein critical for
clathrin-mediated endocytosis317, also associates with GluR2318,319. The AP2 binding
motif overlaps, but is not identical to the NSF site318,319.
The role of these protein interactions was identified using targeted disruption with
dominant negative peptide decoys mimicking the binding sites. These experiments
revealed that these binding partners are involved in the constitutive and activity-
57
dependent regulation of AMPA receptor surface expression313,315,318,319. Indeed the
GluR2-NSF interaction is required to maintain receptor surface expression, with virally-
expressed or intracellularly delivered inhibitory peptides (the most well recognized being
pep2m) resulting in either a complete loss of AMPA receptor surface expression320, or a
~40% reduction of AMPA receptor EPSC amplitude313, depending on the experimental
preparation. It has also been shown that the loss of this protein interaction is involved in
certain types of endogenous synaptic plasticity, including NMDA-receptor dependent
long-term depression (LTD). This was noted by a marked occlusion of NMDAR-
mediated LTD by prior treatment of neurons with pep2m321,322. Similar effects have been
reported for the AP2 site, which is thought to mediate the recruitment and subsequent
formation of clathrin-coated pits during AMPA receptor endocytosis323,324, which also
occurs during NMDA receptor-dependent LTD. Some work has hypothesized that during
this process cytosolic hippocalcin acts as a calcium sensor, linking NMDAR-derived
calcium to AP2-dependent internalization of AMPA receptors325.
1.5.1.2.2 AMPA receptor c-terminal PDZ interactions
Three proteins, glutamate receptor interacting protein (GRIP)326, AMPA receptor
binding protein (ABP, also known as GRIP2)327, and protein interacting with C kinase 1
(PICK1)328,329 interact with the AMPA receptor at the extreme c-terminal PDZ binding
site. Prior to describing the nature of these interactions and their role in AMPA receptor
trafficking, a brief discussion of PDZ domains is necessary.
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Figure 4. GluR2 subunit domain structure. The C-terminal amino acids are shown, to
help identify the binding sites for NSF/AP-2 at the membrane proximal site (blue) and the
distal PICK1 binding site (at the PDZ ligand, green). Transmembrane domains are shown
in yellow and the flip/flop alternative splicing region is shown in grey shade.
Palmitoylation sites are noted by arrowheads, as is the Q/R editing site in transmembrane
domain 2, where the critical RNA editing occurs in the GluR2 pore, controlling ionic
permeability. Other alternative splicing regions are underlined, and in vivo
phosphorylation sites are denoted by bolded residues.
59
Figure 4. GluR2 subunit domain structure Modified with permission from Isaac et al., 2007. “The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity”. Neuron, 54, 859-871.
60
PDZ domains are modular protein-interaction domains of approximately 90
amino acids that function in specialized binding to the extreme c-terminal sequences of
other proteins. PDZ domains (of which ~ 440 have been identified in 259 different
proteins in humans330,331), are named after the first three proteins identified as carrying
them; the postsynaptic density protein PSD-95/SAP90, the Drosophila septate junction
protein Discs-large, and the tight junction protein zonula occludens-1 (ZO-1). Since their
initial identification, PDZ and PDZ-like domains have been recognized in numerous
proteins from organisms as diverse as bacteria, plants, yeast, metazoans, and Drosophila
and are among the most common protein domains represented in sequenced genomes331.
PDZ domains generally function as scaffolds as part of an assembly of large
multimeric protein complexes, involved in signal transduction and protein trafficking.
Any one protein may contain more than one PDZ domain, and may also contain PDZ
domains of differing specificity.
Based on their general ligand specificity, PDZ domains can be broadly divided
into several categories. Type I PDZ domains, including those found on PSD-95 and its
homologous family members, bind carboxy termini with the following consensus amino
acid sequence332,333:
Type I PDZ domain: (S/T)-2X-1(V/I/L)0
where S represents serine, T represents threonine, V represents valine, I represents
isoleucine, and L represents leucine. X in this scenario can represent any amino acid.
The superscript numbers above the amino acid symbols represent the relative position of
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the amino acid relative to the c-terminal end (i.e., 0 represents the most c-terminal amino
acid in the entire protein sequence). An example of a –COOH terminus containing this
sequence would be that of the NMDA NR2B subunit, which binds the PDZ domain of
PSD-95 via its KLSSIESDV sequence334, thus classifying this interaction as a type I PDZ
interaction.
Conversely, type II PDZ domains are found in proteins such as the fourth and
fifth PDZ domain of GRIP and the PDZ domain of calmodulin-sensitive kinase (CASK),
and bind a consensus sequence of333:
Type II PDZ domain: X-3-Φ-2- X-1-Φ0
where X represents any amino acid, and Φ represents a hydrophobic residue, (preferably
tyrosine or phenylalanine at P−2)335,336.
A third type of PDZ domain present in neuronal nitric-oxide synthase shows a
preference for aspartate at P−2 (i.e., a DXV c-terminal motif)337,338, although it also
accepts other residues (e.g. isoleucine)339. Additionally, another kind of binding has been
described and is exemplified by an internal (non c-terminal) sequence in neuronal nitric-
oxide synthase that binds to syntrophin's PDZ domain and the second PDZ domain of
PSD-95340,341.
It should be noted that possession of these consensus sequences does not
guarantee that the protein is involved in a PDZ interaction. Similarly, it is clear that other
residues must contribute to specificity for a given PDZ domain. Notably, several proteins
and ion channels that have an (S/T)XV motif do not bind PDZ domains under conditions
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in which certain other ligands do. For example, the neuronal inwardly rectifying K+
channels Kir3.2 and Kir3.3 – which possess COOH-terminal sequences in both cases of
ESKV – the Na+ channel Nav1.5 (c-terminus of ESIV), and diacylglycerol kinase ζ (c-
terminus of ETAV) do not bind to PSD-95342-344, which is well recognized for accepting
type I PDZ ligands as binding partners. Additionally, the β1 adrenergic receptor does not
interact with either of the first two PDZ domains of PSD-95, despite conforming to the
(S/T)XV motif345. Thus, beyond these consensus sequences, there are other auxiliary
factors involved in stabilization of a PDZ interaction. These factors are reviewed
elsewhere330.
The common structure of PDZ domains contains six β strands, (βA–βF), and two
α helices (αA and αB), which fold in an overall six-stranded β sandwich. The c-terminal
peptides discussed above bind the PDZ domain as an anti-parallel β strand, in a groove
between the βB strand and the αB helix. Within the βA–βB connecting loop, there is a
conserved sequence of Gly-Leu-Gly-Phe (GLGF), which participates in hydrogen bond
co-ordination of the c-terminal carboxylate group. For example, in a type I interaction,
the serine or threonine residue on the ligand c-terminus occupies the −2 position, where
the side chain hydroxyl group forms a hydrogen bond with the N-3 nitrogen of a histidine
residue at position αB1 in the PDZ domain itself. In type II interactions, the hydrophobic
residue at the −2 position of the peptide ligand interacts with a similar hydrophobic
amino acid in the αB1 position of the PDZ domain.
Finally, PDZ protein-protein interactions can be modulated through
phosphorylation of certain residues on the c-terminal ligand. For example, serine
phosphorylation at position −2 in the inward rectifier K+ channel Kir2.3 by protein kinase
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A (PKA) disrupts binding to the PDZ domains of PSD-95. The association of β2-
adrenergic receptor with Na+/H+ exchanger regulatory factor (NHERF) is abolished in a
similar fashion by phosphorylation at position −2 by G-protein-coupled receptor kinase 5
(GRK5). Not only can phosphorylation disrupt PDZ binding, but it can also regulate
specificity of PDZ protein interactions, with certain PDZ substrates preferring
phosphorylated moieties. A well characterized example of this is in the phosphorylation
of the GluR2 c-terminal serine residue at P-3, which enhances the binding of its c-terminal
PDZ ligand (SVKI) to PICK1, while disrupting the interaction with GRIP (discussed in
detail below).
1.5.1.2.3 PDZ Interactions in GluR2 trafficking
Because AMPA receptors themselves lack motor domains, the receptors must
associate with protein partners that assist in their trafficking. The extreme c-terminus of
GluR2 contains a type II PDZ binding motif (SVKI) that is involved in the trafficking of
the subunit both to and from the plasma membrane. The interaction of this PDZ ligand
with its various PDZ binding partners is regulated by the phosphorylation state of the P-3
serine residue. Constitutively, this serine is not phosphorylated, stabilizing the interaction
of the SVKI motif with the 5th PDZ domain of membrane-bound GRIP, a 7 PDZ domain-
containing AMPA receptor anchoring protein lacking a catalytic domain. The 4th PDZ
domain of GRIP plays a role, through intramolecular interactions, in stabilization of
SVKI-GRIP binding. It was through mutagenesis analysis that the role for the GRIP-
GluR2 interaction was first revealed. In transfected hippocampal neurons, GluR2 mutants
lacking the PDZ binding motif did not accumulate at synapses in the manner seen with
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wild-type subunits. Moreover, mutating a single residue, preventing GluR2-GRIP
binding, reduced synaptic accumulation of GluR2, suggesting that the role of the GluR2-
GRIP PDZ interaction is in preventing endocytosis of the subunit. Therefore, following
NSF facilitated fusion of GluR2-containing vesicles with the post-synaptic membrane
(discussed previously), GRIP acts as an anchor, to stabilize surface subunit expression346-
349.
To complicate matters, the synaptic expression of GRIP itself is regulated through
N-terminal cysteine palmitoylation350,351 (i.e., the covalent attachment of a 16 carbon
saturated fatty acid352). This modification of the GRIP N-terminus is required for
trafficking of the protein to synapses, with unpalmitoylated isoforms of GRIP residing
exclusively in the cytosol353.
The GluR2 c-terminal PDZ ligand also interacts with another PDZ-domain
containing protein known as protein interacting with C kinase alpha 1 (PICK1). PICK1 is
a peripheral membrane protein initially cloned as one of the proteins interacting with
protein kinase C α (PKCα) from a yeast two-hybrid screen354. It contains two structurally
known domains, the PDZ domain, as well as a crescent shaped dimeric Bin–
Amphiphysin–Rvs (BAR) domain355, which interacts with negatively charged curved
membranes during membrane fusion events356,357, and intramolecularly binds the PDZ
domain in an auto-inhibitory fashion358. In addition, there are three regions that border
these two domains: a short N-terminal region of 18 residues before the PDZ domain
enriched with acidic residues, a linker region of 40 residues between the PDZ and BAR
domains, and a c-terminal region characterized with a stretch of acidic residues355.
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The structure and function of PICK1 are quite unique among the human genome;
PICK1 is the only known protein to contain both a BAR domain and a PDZ domain, and
it is also the only known protein to contain a PDZ domain that accepts both Type I and
Type II PDZ ligands as binding partners (due to the presence of a lysine, K83, in the
critical α B1 position)355. To date, PICK1 has been shown to interact with over 40
proteins, most of which are membrane proteins, including receptors, transporters, and ion
channels. Of these interactions, the interaction of PICK1 with the c-terminus of GluR2 is
the best characterized, both in terms of its biochemical regulation and impact on neuronal
physiology.
The interaction of PICK1 with GluR2 was first identified through yeast two
hybrid screening328,329, and has subsequently been verified through co-
immunoprecipitation (CoIP) from heterologous cells359 and later through in vivo CoIP in
rat brain homogenates358,360. Indeed when expressed in heterologous cells, PICK1 and
GluR2 form many co-clusters that are abolished upon mutation of the PICK1 PDZ
domain (K27D28 to AA) or deletion of the GluR2 c-terminal PDZ ligand329,357.
It is now known that the function of the PICK1-GluR2 interaction is in
modification of GluR2 surface expression, with the vast majority of studies in this area
pointing to a role for this interaction in GluR2 endocytosis. That is, binding of PICK1 to
the GluR2 c-terminus has been identified as a critical event in the internalization of this
subunit from the post-synaptic plasma membrane. The evidence supporting this
hypothesis is extensive. Through immunohistochemistry, surface biotinylation, and
subcellular fractionation of membrane components, many investigators have shown that
PICK1 transfection into hippocampal neurons and heterologous cell lines reduces surface
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GluR2 protein expression267,268,360,357. Further, known mechanisms of inducing the
endocytosis of GluR2 in cultured neurons (including bath application of NMDA and
PKC-activating phorbol esters) are inhibited through mutation of the PICK1 PDZ domain
or peptide-mediated interference of GluR2-PICK1 binding361-365.
The interaction of GluR2 with PICK1 is preceded by a number of important
biochemical events that regulate this protein complex. The most critical of these events is
the phosphorylation of the GluR2 P-3 serine residue by protein kinase C alpha (PKCα). As
discussed, the GluR2 c-terminal SVKI PDZ ligand is constitutively bound to GRIP, to
anchor the subunit to the membrane. However, the phosphorylation of the serine residue
within this moiety (serine 880, or S880), by PKCα, is capable of disrupting the GluR2-
GRIP interaction, whilst favouring an intermolecular interaction between GluR2 and
PICK1349,350,362,366-373. Moreover, this phosphorylation of GluR2 is further dependent
upon the trafficking of PKCα to the plasma membrane by PICK1, in a second PDZ
interaction involving the PKCα c-terminal type I QSAV PDZ ligand with the PICK1 PDZ
domain349,360,369,370,372. The binding of PKCα’s PDZ ligand to PICK1 is dependent on
activation of the kinase. This occurs endogenously through the influx of intracellular
calcium, and exogenously with PKC activators such as phorbol esters. Activation of
PKCα exposes the PDZ ligand for binding the PICK1 PDZ domain, by altering its
conformation from folded to linear360. Finally, localization of the PKCα-PICK1 complex
to the plasma membrane is dependent on an interaction between the PICK1 BAR domain
and GRIP, which brings the complex in close structural proximity to GluR2
itself360,367,374. Indeed the steps involved in this GluR2 endocytotic cascade are quite
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Figure 5. Steps involved in the intracellular trafficking of the GluR2 subunit. 1) Influx of
intracellular calcium (usually through the NMDA receptor) activates cytosolic PKCα,
freeing up its PDZ ligand (QSAV) for binding available PDZ domains. 2) Binding of
PKCα’s PDZ ligand to PICK1’s PDZ domain disrupts the auto-inhibitory interaction
between the PICK1 PDZ and BAR domains, exposing the PICK1 BAR domain for
binding other proteins. 3) PICK1 traffics activated PKCα to the GRIP/GluR2 complex,
through the interaction of the PICK1 BAR domain with a 55 amino acid binding region
(Br) sequence in GRIP. The PICK1 PDZ domain is now in close proximity to the GluR2
PDZ ligand SVKI. 4) PICK1 competes with ABP/GRIP for binding the GluR2 SVKI
ligand. 5) PKCα phosphorylates serine 880 in SVKI to SPO4VKI. 6) GluR2 phosphorylated
at serine 880 is no longer able to bind to GRIP, its synaptic anchor. GluR2 binds PICK1
through the SVKI-PDZ domain interaction. 7) GluR2 is internalized from the cell surface.
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Figure 5. Steps involved in the intracellular trafficking of the GluR2 subunit. Modified with permission from Lu and Ziff, 2005, Neuron. “PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking”. 47, 407-421.
69
complicated, and as a result are outlined for clarification in Figure 5 and its
accompanying figure caption.
It is now known that this cascade plays a critical role in the activity-dependent
trafficking of AMPA receptors, as well as in modulation of the GluR2 content of synaptic
AMPARs, thereby controlling critical properties of AMPA receptor biophysics and
initiating synaptic plasticity. Forms of synaptic plasticity including LTD and LTP are
thought of as cellular analogues of learning and memory, with PICK1-mediated
trafficking of GluR2 playing an integral role in these synaptic modifications. Indeed there
is good evidence that PICK1-mediated internalization of GluR2 is one of the mechanisms
through which neurons increase their basal excitability and calcium permeability.
1.5.1.2.4 GluR2 trafficking in synaptic plasticity
Several comprehensive reviews exist on the subject of AMPA receptor trafficking
as a cellular mechanism of synaptic plasticity. Accordingly, the balance of this section
will focus specifically on the evidence supporting an involvement of PICK1-mediated
trafficking of GluR2 in modulating AMPA receptor phenotype.
To understand the role of these proteins in initiating changes to AMPA receptor
biophysics, an effective experimental strategy is transfection, that is, viral-mediated up-
regulation of a protein in a native neuronal population or cell line. Indeed studies of
PICK1 transfection into hippocampal slices have yielded important information on the
role of the PICK1-GluR2 interaction in modulating the properties of surface AMPA
receptors. When expressed exogenously, PICK1 increases AMPA receptor EPSC
amplitude, induces inward rectification of the current-voltage relationship, as well as
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confers a sensitivity of the AMPA receptor population to polyamine antagonism, all
defining characteristics of GluR2-lacking, calcium-permeable AMPA receptors267,268.
Concomitant immunocytochemstry and subcellular fractionation revealed that in fact
surface GluR2 expression (but not total protein) had markedly decreased after PICK1
transfection, without an appreciable change in GluR1 surface levels, arguing against a
role for PICK1 in AMPA receptor exocytosis in the reported increase in EPSC
amplitude267,268. Perhaps more compelling is the requirement for endogenous kinase
activity in regulating these effects, as PICK1-mediated GluR2 removal after PICK1
transfection is abolished through PKC inhibition and NMDA receptor blockade,
clarifying an integral role for a regulated signaling cascade in modifying the AMPA
receptor GluR2 content. Other experiments have corroborated these results. When
PICK1 is transfected into the hippocampus in the presence of GluR2 c-terminal peptides
(acting as dominant negative decoys for PICK1 binding, thereby disrupting endogenous
PDZ interactions), the AMPA receptor phenotype is also unchanged, suggesting a
requirement for PICK1-SVKI binding in GluR2 protein removal267,268. Indeed this
interaction between GluR2 and PICK1 is also associated with endogenous phenotypic
changes to AMPA receptor physiology, specifically during a switch from GluR2-
containing to GluR2-lacking receptors276,373,375. Collectively, these experiments provide a
compelling role for PICK1 in decreasing the GluR2 content of synaptic AMPA receptors,
resulting in an increase in both synaptic strength and calcium permeability through the
increased expression of GluR2-lacking receptors.
The PICK1-mediated switch in AMPA receptor subunit composition is
reminiscent of the previously discussed observations that the AMPA receptor GluR2
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content is reduced following the induction of LTP269,376, resulting in a higher average
single channel conductance and strengthening of synaptic inputs. Therefore, investigators
have examined the role of the PICK1-GluR2 protein interaction in initiating LTP.
Firstly, experiments that replicated the PKC and NMDA receptor-dependent effects of
PICK1 transfection on AMPA receptor function also showed that LTP is occluded after
PICK1 upregulation268. Secondly, acute knockdown of PICK1 expression with the use of
shRNA interferes with the initiation and maintenance of hippocampal LTP268. Thirdly,
expression of PICK1 binding, PDZ-ligand peptides mimicking the GluR2 c-terminus
interfere with the development of LTP268. Finally, LTP is absent in hippocampal slices
taken from PICK1 knockout mice268. These experiments examining the physiological role
of PICK1 demonstrate a clear role for the PICK1-mediated decrease in surface GluR2 in
synaptic strengthening as well as increasing neuronal calcium permeability.
1.5.1.2.5 GluR2 trafficking in TBI
A loss of surface GluR2 protein following injury to the CNS is, by all logical
assumptions, an undesirable situation. The most problematic consequence of reduced
GluR2 surface expression is probably heightened neuronal calcium permeability, which
as discussed, would predispose neurons to excitotoxic cellular injury. Indeed a reduction
in the population of AMPA receptors containing GluR2 would not only impart
susceptibility to elevated extracellular glutamate, but might also impart lethality upon
synaptic concentrations of glutamate that under other circumstances remain innocuous.
Certainly the neuroprotective effects of sustaining surface GluR2 were demonstrated in
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the studies highlighting REST-dependent decreases in GluR2 mRNA following
ischemia280,283, which are likely due to resilience to excitotoxic injury.
There is evidence to suggest that modification of the AMPA receptor GluR2
content might occur following TBI. As highlighted previously, traumatic injury to
neuronal cultures dramatically increases AMPA receptor mediated depolarizations249,250,
supporting a role for receptors with a higher single-channel conductance in neuronal
signaling after TBI. This is further supported by the findings that calcium-permeable
AMPA receptors appear in cortical neurons following in vitro stretch injury and in vivo
spinal cord trauma, studies which indeed demonstrate a GluR2-lacking AMPA receptor
phenotype in traumatically injured neuronal populations252,377. Also, excessive
stimulation of NMDA receptors occurs following trauma378, providing the necessary
NMDA receptor stimulation and calcium influx required for PKC activation during
GluR2 endocytosis. Indeed studies have shown that following TBI, PKC activity
markedly increases, and moreover undergoes a translocation from a constitutively
cytosolic residence to membrane-bound, suggesting PKC-dependent modification of
membrane-embedded substrates379.
Plenty of evidence also exists for the neuroprotective effects of AMPA receptor
antagonism after TBI380-382. Although this does not necessarily suggest that GluR2-
lacking receptors contribute to neuronal physiology, it does highlight the possibility that
pathological events are initiated at AMPAergic synapses, which are known to initiate
substantially more cell death when GluR2-lacking receptors are present (discussed
previously). The cytoprotective effects of AMPA receptor blockade might be due not
only to a reduction of intracellular calcium, but also zinc. It is well recognized that
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AMPA receptors lacking GluR2 are highly zinc permeable189,383, and there exists indeed a
marked cytotoxic elevation in free ionic zinc in neurons following experimental brain
trauma384-387. Free zinc is taken up by mitochondria in an effort to restore zinc
homeostasis but, similar to the effects of mitochondrial calcium uptake, this leads to
potent mitochondrial dysfunction, prolonged loss of mitochondrial membrane potential
and free radical generation.
A reduction of GluR2 protein in the traumatic brain might also predispose
neuronal populations to situations of relative ischemia, by contributing to heightened
metabolic demand at a time where glucose delivery is impaired. Neuronal
hyperexcitability (i.e., an enhancement of constitutive depolarizations induced at
AMPAergic synapses) is a possible consequence of GluR2 loss, reflected by the
previously discussed increases in average AMPA-mediated EPSC amplitude. In the
traumatic brain, this might translate clinically to epileptic discharges and increases in
cerebral metabolic rate of glucose metabolism (CMRG) 388,389. Coupled with post-
traumatic injury to microvasculature (as described, a situation of decreased cerebral
perfusion), hyperexcitable neuronal populations create a situation of relative ischemia,
which is clinically the biggest contributor to secondary injury after TBI 390 .
1.6 Rationale for proposed study Our understanding of traumatic brain injury (TBI) has evolved considerably from
a simple self-limiting physical trauma, to an evolving and progressive biological injury
amenable to meaningful intervention. In order to design a therapeutic approach to the
treatment of secondary injury and neuronal dysfunction following brain trauma, an
74
understanding of the aberrant molecular events occurring at the cellular level is
necessary. Excitotoxicity, a major contributor to secondary injury events after TBI, can
occur through two mechanisms as discussed; either through substantial elevation of
extracellular glutamate (e.g., triggered by uncontrolled vesicle fusion or a dysfunction of
astrocytic glutamate transporters) or alternatively, by changes to the post-synaptic
response to physiologic glutamate that render stimulation of glutamate receptors
cytotoxic.
The present study was undertaken, in a very broad sense, to examine this
alternative hypothesis of excitotoxic cell death following traumatic brain injury; that is,
an appreciable change to glutamate receptor function that confers neuronal injury during
excitotoxicity. Specifically however, since the GluR2 subunit has dramatic control over
AMPA receptor ionic permeability and conductance, the study was performed to
investigate the possibility that a reduction of surface GluR2 protein contributes to
secondary injury following TBI. The study aimed to investigate the involvement of
GluR2 trafficking in TBI through biochemical assays (i.e., western blotting, co-
immunoprecipitation, and immunofluorescence) as well as any associated changes to
AMPA receptor phenotype that result from aberrant GluR2 trafficking with the use of
whole cell and field electrophysiology, calcium imaging, and vulnerability to excitotoxic
injury. A wealth of information exists regarding the involvement of specific protein-
protein interactions in the regulated endocytosis of GluR2 from the plasma membrane,
and this work aimed to examine the activity of these proteins following neuronal trauma,
as well as the cytoprotective efficacy of inhibiting the protein-protein interactions
responsible for GluR2 internalization.
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To examine these pathways in mechanistic detail, and yet with sufficient whole
animal (and therefore clinical) relevance, it was decided to use a multi-system approach,
from a cell-free system in some assays, to a cell culture model, and finally, an in vivo
model of TBI. The cell culture model of TBI used in this thesis involved a mechanical
stretch injury -- which was first established as exhibiting sublethal properties and
therefore suitable for examining the susceptibility of neuronal populations to secondary
injury without the added confound of cell death initiated by mechanical trauma – coupled
with mild excitotoxicity, to include a model that contains the heterogeneous sequelae of
insults (both mechanical and biochemical) faced by injured neurons following TBI. The
excitotoxic injury was a low concentration of NMDA, which was applied to the cultures
immediately following stretch to mimic glutamatergic excitotoxicity, and more
specifically, activation of extrasynaptic NMDA receptors. Excitotoxicity and the
activation of these extrasynaptic receptors are documented pathophysiological
phenomena noted following both fluid percussion injury in rats, and following TBI in
humans. A further elaboration of this model, including the physics of the injury, data on
its initial characterization (including dose response curves for cell death and severity of
the stretch injury) and further explanation of its rationale is found in the next section.
The whole animal model used in this thesis was the well established fluid
percussion injury device (FPI), which involves the extradural injection of a column of
fluid to the rat brain, a model which reproduces many of the clinical consequences of TBI
in humans, including diffuse axonal shearing, contusion, and widespread neuronal injury.
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1.7 Statement of Hypotheses
1.7.1 General Hypotheses In this thesis, the following main hypothesis was tested:
A reduction of surface GluR2 protein contributes to secondary injury after TBI. This hypothesis was fashioned based on the data supporting a role for GluR2
endocytosis in increasing both neuronal calcium permeability as well as single channel
conductance. Given that both early sodium-mediated cell swelling and calcium-
dependent apoptotic cell death contribute to neuronal injury following TBI, we
hypothesized that a reduction of surface GluR2 protein might contribute to these
processes. Previous findings further report that AMPA receptor conductances markedly
increase following trauma and that AMPA receptor antagonism is cytoprotective,
observations which might involve a mechanism of surface GluR2 downregulation.
Collectively, we conjectured that this post-synaptic modification of AMPAergic synapses
might underlie excitotoxic neuronal death after TBI.
1.7.2 Specific Hypotheses In addition to our main hypothesis which proposes that GluR2 endocytosis
contributes to cellular injury after TBI, we sought to investigate the mechanisms
responsible for its internalization. This lead to the following sub-hypotheses:
i) Post-TBI GluR2 endocytosis is mediated by the intracellular machinery responsible for constitutive and activity-dependent trafficking
This sub-hypothesis is based on the extensive literature highlighting the involvement
of specific intracellular cascades in modulating the GluR2 content of surface AMPA
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receptors. Specifically, this study examined the involvement of the NMDA receptor in
mediating GluR2 internalization after trauma, as well as the role of PICK1 PDZ
interactions in injury-induced AMPA receptor modification. The purpose of examining
these cascades was to differentiate between non-specific effects of GluR2 internalization
– for example, those mediated by applying a mechanical force – and the involvement of
regulated intracellular signaling in GluR2 trafficking.
ii) Targeted inhibition of GluR2 endocytosis increases cellular survival after TBI
The purpose of this hypothesis was to examine the relevance of GluR2 trafficking
in neuronal survival after TBI. Although it is possible that GluR2 trafficking occurs
following injury to the CNS, the cytotoxic relevance of this phenomena is unknown.
Accordingly, it is of utmost importance when trying to parse out mechanisms of neuronal
death and dysfunction after injury to identify if various phenomena actually contribute to
cell death or if their effects are tangential (or even endogenously cytoprotective). To
inhibit GluR2 internalization, we designed a custom, cell-permeable peptide inhibitor of
PICK1-PKCα protein binding, which was validated as an inhibitor of this protein-protein
interaction prior to its introduction as a putative cytoprotective compound. Further detail
on the design and testing of the compound is presented in the following chapter.
The primary use of this PICK1-PKCα inhibitor was not in an effort to examine a
novel treatment for TBI. Rather, this compound was used to validate the involvement of a
specific mechanism in GluR2 trafficking. Some of the final experiments did employ this
inhibitor in cell survival assays, but this was more with the purpose of examining the role
of GluR2 endocytosis in conferring vulnerability to neuronal damage.
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1.8 Statement of Objectives In order to address our hypotheses, the following specific aims were defined:
1) To investigate if calcium-permeable AMPA receptors contribute to neuronal physiology after TBI
2) To examine the intracellular mechanisms responsible for the expression of CP-AMPARs
3) To investigate the physiological significance and cytoprotective efficacy of interrupting the expression of CP-AMPARs after TBI.
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Chapter 2 – Model Characterization and General
Methods
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Chapter 2: General Methods
2.1 Preface The following section contains detailed methodologies for the entire repertoire of
methods employed in this thesis. They are subdivided into methods used for the in vitro
model of TBI, as well as the whole animal preparation. As the former paradigm involved
some characterization (e.g., assays of membrane integrity and a dose-response
relationship for cellular injury vs. injury severity), the data on the initial use of the model
is presented as well. Each methodology is accompanied by a supporting rationale for its
use in examining GluR2 trafficking following traumatic brain injury. This section also
contains a list of contributions to the data collected in this thesis.
2.2 In vitro methods
All procedures described here were approved by the Animal Care Committee at St.
Michael’s Hospital and complied with regulations of the Canadian Council on Animal
Care.
2.2.1 Isolation and dissociation of cortical cell cultures
For the stretch injury model described below, cortical cultures containing both
neurons and glia were prepared from E16-17 Wistar rats (Charles River Laboratories,
Wilmington, MA). Primary cultures were grown on 6-well BioFlex culture plates
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(FlexCell, Hillsborough, NC). Pregnant animals were anesthetized with isofluorane and
sacrificed via decapitation. Embryos were surgically removed, isolated from the amniotic
sac, and decapitated. Embryo heads were placed in 20 ml 1 x Hank’s Balanced Salt
Solution (HBSS, Invitrogen Corp. Carlsbad, CA). Brains were removed and placed in a
separate dish containing 20 ml supplemented HBSS. Cerebral cortices dissected from
whole brains using microdissection forceps, were incubated in 2 ml of 0.1% trypsin
(Sigma-Aldrich, St.Louis, MO) at 37 ºC for 10 mins, and placed in 2 ml HBSS. Tissue
was triturated by glass pipette 10-20 times, and seeding medium (DMEM/F-12
containing 10% Horse serum, Invitrogen) was added. Cortical cells were centrifuged for
5 mins at 1200 rpm, triturated again, re-centrifuged at 700 rpm for 1 minute, and seeded
in plating medium (Neurobasal medium containing 2% B-27 supplement, 1% Fetal
Bovine Serum, 0.5 mM L-glutamine, 25 μM glutamic acid, Invitrogen) onto poly-L-
lysine (5 μg/ml; Sigma) coated plates at a density of 1 x 106 cells/well. Cell counts were
done by loading PBS, Typan Blue (Sigma-Aldrich) and 50 μl of cell suspension into a
hemocytometer. Ninety-six hours after isolation, cells were fed with fresh maintenance
medium (Neurobasal medium containing 2% B-27 supplement, 0.5 mM L-Glutamine,
Invitrogen) containing 10 μM FDU (5 mM Uridine, 5 mM (+)-5-Fluor-2’-Deoxyuridine,
Invitrogen) and left to incubate for 48 hours to halt the growth of non-neuronal cells.
Cells were fed with maintenance medium every 3-4 days until stretch assays. We used
the cells for experiments 11-14 days after isolation consistent with previous in vitro
stretch assays 243,248,249.
2.2.2 In Vitro Model of TBI
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2.2.2.1 Use of stretch injury models in TBI literature As was previously discussed in the section on the biophysics of traumatic brain
injury, the rotational strain produced by rapid changes to angular velocity that occurs
during many types of TBI is nearly impossible to reproduce in an animal model of TBI,
due to the mass effects of the human brain in axonal and somatic injury. As a result, a
number of models have been developed to study TBI at the cellular level, by reproducing
cellular trauma in an in vitro system. One of these models, which will be discussed here
and is used in this thesis, is an electronically controlled pneumatic device that allows the
study of morphologic, physiologic and biochemical responses of cultured neurons to
trauma.
The device used in this thesis to injure cortical monolayers was the Cell Injury
Controller II (Custom Design and Fabrication, Virginia Commonwealth University,
Richmond, VA, USA, Figure 5). An inlet on this injury controller is connected to a tank
of compressed nitrogen, and the controller regulates the pressure and duration of a pulse
of air that is delivered through a closed tube system to an adapter that fits with an airtight
seal into the top of each tissue culture well.
The exact millisecond duration of the valve opening and the air pressure pulse is
tightly controlled by a valve and timer (1-1000 msec) on the unit’s controls. Moreover, an
external output on the system allows recording of the exact time and duration of the
electrical pulse on an oscilloscope or polygraph. The air pressure pulse is delivered by
pressing a trigger on the controller unit. The air between the unit and the culture plate is
immediately vented into the atmosphere once the air pulse is delivered, allowing a rapid
deformation and subsequent rebound of the membrane in the individual wells. The injury
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process is repeated six times per plate (since each plate contains six wells), and an entire
plate (~ 4-6 million cells) is used as an n of 1, per injury condition evaluated.
To allow for the deformation of the cells, they were cultured on BioFlex’s
SilasticTM tissue culture plates, which consist of a flexible silicone elastomer membrane,
with a total growth area of 57.75cm2 (9.62 cm2 per well). When used in conjunction with
the cell injury controller, the plates allow for uniform radial and circumferential strain.
The model of injury was initially characterized by Ellis et al., in 1995 when they
established a dose response-relationship between pressure intensity and both membrane
deformation and lactate dehydrogenase release as a marker of cell death. Subsequently,
the model has been used by numerous laboratories to characterize mechanisms of
secondary injury in TBI. Notably, this is the same model used in the experiments
highlighting trauma-induced augmentation of AMPA receptor current density.
2.2.2.2 The Stretch + NMDA model
Prior to stretch injury, the culture medium in our studies was replaced with 2 ml
HEPES buffered saline (concentrations in mM: 121 NaCl, 5 KCl, 20 glucose, 10 HEPES
acid, 7 HEPES-Na salt, 3 NaHCO3, 1 Na-pyruvate, 1.8 CaCl2, and 0.01 glycine, adjusted
to pH 7.4 with NaOH). On the basis of data suggesting that forces resulting in tensile
elongation following TBI occur in 50 ms or less, the duration of the stretch injury was set
to 50 ms. To establish an initial dose response relationship between cellular injury and
pressure in our culture system, the applied pressure levels ranged from 2.5 (mild) to 7.5
(severe) pounds per square inch (psi), representative of the rotational
acceleration/deceleration injury resulting from rapid changes to angular velocity (ω), and
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subsequently, momentum (L). According to the formula: Impulse (J) = ΔP = FΔt, (where
P = momentum, F = force in newtons, and t = time) and F = pressure (2.9 psi) x area
(1.49 in2), we calculated that at 2.9 psi (the pressure used in the majority of the
experiments) J(on cells) ≈ 9.6 N·s.
In the intact mammalian brain, tissue peripheral to the necrotic core of trauma
undergoes not only mechanical strain, but is also subject to excitotoxic glutamatergic
spillage 241,391 from dead or dying neurons that are injured during the primary injury
event. This post-trauma excitotoxic environment is largely lost in vitro, but may play an
important role in progressive neuronal injury. Our intention with this model was also to
replicate in culture a similar or equivalent biomechanical loading and biochemical
environment as found in in vivo TBI. Thus, immediately following the mild stretch, 10
μM NMDA was added to the wells for 1 hour to mimic this excitotoxic stimulation, a
combinatorial method that has been used by a number of laboratories to mimic both
mechanical injury and glutamatergic receptor stimulation. Previous studies of this dose of
NMDA in cortical cultures have demonstrated no lethality, and in fact promotion of
neuroprotection against subsequent challenges 392. To block NR2b and NR2a containing
NMDA receptors respectively, 5 µM -[2-(4-Hydroxyphenoxy)ethyl]-4-[(4-
methylphenyl)methyl ]-4-piperidinol (Co101244) hydrochloride and (2R*,4S*)-4-(3-
Phosphonopropyl)-2-piperidinecarboxylic acid (PPPA, 100 nm) (Tocris Biosciences,
Ellisville, MO) was bath applied with NMDA. Ki values of PPPA are 0.13 and 0.47 μm
for NR2A and NR2B respectively, which ensured specificity of our approach.
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Figure 6. The cell injury controller and schematic of experimental paradigm.
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2.2.2.3 Toxicity studies: Dose response characterization of stretch pressures
In order to establish the in vitro model, dose–response (injury–cell death)
experiments were performed on the culture cells. Neurons underwent varying levels of
stretch (2.5–7.5 psi) in 2 ml HEPES buffer as described earlier. Wells were subsequently
loaded with 10 µg/ml PI (warmed in 37°C water bath). The quantitative measurements of
PI fluorescence were used as a determination of the prevalence of cell death using a
Victor3V multiwell plate fluorescence scanner (PerkinElmer, Wellesley, MA, USA)
controlled by Workout software (Dazdaq, Finland). All parameters including the size and
number of scanning area, the duration of scanning, etc. were kept constant by using the
same protocol for all groups. A second dye, fluorescein diacetate (FDA) was used as a
marker of healthy, viable cells, as observed by us and others102,393-395. It has been reported
that damaged membranes lose their capacity to retain FDA, and thus will not fluoresce396.
In brief, immediately following stretch, baseline PI and FDA fluorescence readings were
taken, cells were incubated at 37°C in the absence of CO2 and a subsequent reading was
taken 20 h later. Cell death along the continuum of mechanical deformation was
normalized to unstretched wells exposed to 1 mM glutamate for 1 h (Glu). This exposure
routinely produced nearly 100% cell death in our observations, and that of others119,397-399,
and thus PI fluorescence for each condition was normalized to these wells. Cell death was
calculated according to the formula: Fraction dead = F20 – F0/F20GLU – F0GLU, where F20 =
PI fluorescence 20 h post-stretch, F0 = initial PI fluorescence, F20GLU = PI fluorescence of
cells 20 h post-exposure of 1 mM Glu for 1 h, F0GLU = initial PI fluorescence of 1 mM
Glu exposed wells. Cells exposed to 1 mM Glu were identical cultures from the same
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To validate our stretch paradigm as an experimental model of delayed neuron
death, we first examined cell viability at 20 h post-injury along a continuum of stretch
amplitudes as assessed by uptake of propidium iodide. A robust gradient was observed in
PI uptake from magnitudes ranging between 3.5 and 7.5 psi (e.g. cell death averaged 23.2
± 2.45%, n = 3 for 3.5 psi versus 70.1 ± 6.5%, n = 3 for 7.5 psi, Fig. 7). Each magnitude
tested resulted in significantly greater PI uptake than the lower pressure tested (P < 0.05–
0.001, Fig. 7). However, stretch at 2.5 psi did not alter PI uptake relative to controls (P >
0.05). Both conditions averaged approximately 11.5 (± 0.85% for control, ± 1.73% for
2.5 psi, n = 3 for both conditions) of the PI uptake relative to wells treated with 1 mM
Glu (Fig. 7). Mildly stretched neurons also stained brightly with FDA, whereas severely
injured neurons did not (data not quantified, see Fig. 7). This initial data suggested that
insult at 2.5 psi does not confer delayed cell death on its own. It was thus termed, “sub-
lethal”, allowing us to examine the impact of this injury on vulnerability to secondary
insults.
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Figure 7. Dose-Response Characterization of stretch injury model A) Injury magnitude-propidium iodide (PI) uptake dose-response curve for stretch
pressures ranging from 2.5 to 7.5 psi. The percentage of cell death was normalized to
1mM glutamate exposed cells at 20 h post-stretch. Note the absence of increased cell
death at 20 h in mildly stretched neurons (2.5 psi) as compared to control wells. (B)
Representative FDA (green fluorescence, marker of viability) and PI (red fluorescence,
marker of cell death) micrographs of mildly injured (B1, B2) and severely injured (B3,
B4) neurons. Scale Bars = 200 mm. *P < 0.05, ** P < 0.01, *** P < 0.001. Error bars
represent SEM, and each condition represents an experiment repeated in triplicate (i.e., n
= ~ 3 x 106 cells total).
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Figure 7. Dose-Response Characterization of stretch injury model
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2.2.2.4 Carboxyfluorescein assays of membrane permeability
As discussed in the introduction, it is possible that stretched cells may exhibit
enhanced calcium permeability as a result of changes to membrane permeability. As this
thesis intended to employ assays of calcium imaging as well as whole cell
electrophysiology, it was important to examine the impact of the stretch injury on
membrane integrity prior to proceeding with these experiments to avoid potentially
problematic confounds. Plasma membrane permeability following mechanical stretch was
assessed by evaluating uptake of the ordinarily impermeant fluorescent molecule,
carboxyfluorescein, (CBF, MW = 380 Da, radius = 0.5 nm; Sigma). We adopted this
technique (established by Geddes-Klein et al.,245,400) for use in stretch-induced alterations
to cell permeability. The technique however, has also previously been implemented to
detect permeability changes in electroporated cells401,402. Immediately prior to injury,
cells were treated with 100 µM CBF, and nuclei were stained with Hoechst 33 342 (20
µg/ml; Molecular Probes, Eugene, OR, USA). Neurons were stretched in the presence of
CBF and incubated at 37°C, 5% CO2 for 10 min to maximize diffusion of the dye into
cells245. Cells were then rinsed with buffer to ensure the removal of extracellular CBF.
Sections of membranes were detached (0.75 in.), placed in HEPES buffer, and fluorescent
images were taken from five different areas per section of membrane. Cells positively
stained with CBF were later counted and normalized to the total number of Hoescht-
positive nuclei. This procedure was repeated 3–4 times in each condition, across separate
cell isolations.
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Applications of CBF enabled us to image uptake of an ordinarily impermeable
fluorescent molecule and as a result determine immediate, post-stretch alterations to
plasma membrane permeability in injured neurons. Representative CBF micrographs are
shown in Fig. 8 for control cultures, mildly stretched cultures, and severely stretched
cultures. We observed almost no CBF uptake (denoted by bright green staining) in both
control and mildly stretched cultures (quantified at 6.5 ± 1.31% and 5.6 ± 1.91% CBF
positive neurons, respectively, n = 3 for both conditions, Fig. 8). CBF uptake was
significantly higher in severely stretched cultures ( 33.6 ± 4.03%, n = 3, P < 0.001, Fig.
8A). Thus, CBF uptake was a function of the pressure exerted on cultures, and did not
increase in mildly stretched neurons relative to controls. It should be noted, however, that
changes to permeability that would have occurred more than 10 min post-stretch would
not have been accounted for. However, recent work suggests that plasma membrane
permeability changes are transient and repaired rapidly following stretch, if they occur at
all245.
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Figure 8. Mild injury does not increase non-specific neuronal cell membrane
permeability. A) Representative micrographs of CBF uptake and Hoescht staining in
control, mildly injured and severely injured neurons. Based on this data, mild stretch does
not confer the development of non-specific membrane holes or tears from mechanical
deformation, suggesting the preservation of membrane integrity. As a positive control
however, severe injury does significantly increase CBF uptake (P < 0.001).
B) Quantification of the percentage of carboxyfluorescein (CBF) positive cells
normalized to Hoescht positive nuclei in each condition.
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Figure 8. Mild injury does not increase non-specific neuronal cell membrane permeability.
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Our biochemical investigation into GluR2 trafficking following TBI involved two
primary assay methods; co-immunoprecipitation and western blotting. Co-
immunoprecipitation is an assay method that examines protein-protein interactions,
which we used to measure the progression of the GluR2 endocytotic cascade. The
rationale for this approach was based on the assumption that heightened activity of the
GluR2 endocytotic cascade would yield a more robust interaction between the previously
described PDZ proteins upstream of GluR2 internalization. Along this vein, we examined
the intracellular interaction between PICK1 and PKCα, the phosphorylation of GluR2 at
serine 880 (critical for subunit internalization), the interaction between GluR2 and
PICK1, as well as a novel protein interaction between PKCα and PSD-95, which we
hypothesized might underlie an NMDA receptor dependence of GluR2 trafficking. The
following methods were used during these assays:
2.2.3 Protein extraction and quantification Following in vitro treatment, cells (an entire six well plate, repeated 3 times, for a
total of 18 wells per condition) were washed twice with ice-cold HEPES solution. Lysis
buffer (250 µL per well) containing protease inhibitors (50 mM Tris-HCl, 1% NP-40, 150
mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 1mM NaF) was added and cell suspension was agitated at room temperature
for 20 minutes. Cell lysates were collected and centrifuged at 4°C (10,000 rpm), and the
pellet was discarded. Protein quantification was determined using the modified Lowry
method 403. Following quantification, aliquots of 500 µg protein per condition were
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collected and frozen at -80°C for subsequent immunoprecipitation for proteins of interest.
Similar procedures were used in vivo, using homogenates of cortical tissue.
2.2.4 Co-Immunoprecipitation of GluR2 endocytotic proteins All immunoprecipitation procedures were performed at 4°C or on ice. 5 µg of
polyclonal rabbit anti-PICK1 (Abcam Inc, Cambridge, MA), or 5 µg of polyclonal rabbit
anti-PSD-95 (Chemicon, Billerica, MA) was incubated with 500 µg of cell lysate and
mixed by inversion overnight. Before being added to the antibody-lysate mixture, 50 µl
of Protein A agarose beads were washed 3 times with 500 µl of PBS (each time spun for
30 seconds at 10,000 rpm). After washing, protein A agarose beads were added to the
antibody-lysis complex and incubated overnight to capture the antibody-antigen complex.
As a negative control, we also incubated samples in the absence of primary anti-sera,
with only protein A agarose. The antigen-antibody-bead complex was collected by pulse
centrifugation (centrifuged at 14,000 rpm for 5 seconds). The supernatant was discarded,
and the beads were washed 3 times in ice-cold PBS. Bead complexes were then re-
suspended in 60 µl 2x sample buffer (0.5 M Tris-HCl pH 6.8, 20% glycerol, 10% SDS,
1% bromophenol blue, 5% β-mercaptoethanol), and boiled for 5 minutes. The beads were
pelleted by centrifugation, and SDS-PAGE was performed with the supernatant.
2.2.5 SDS-PAGE
For western blotting of whole cell lysates, 20 µg of boiled sample was loaded into
each lane in 2x sample buffer. For electrophoresis of immunoprecipitated samples, 20 µl
supernatant was loaded per lane. For probing of phospho-serine880ct GluR2 and
phospho-serine845 GluR1, 7% Tris/glycine gels were used, whereas a 12% gel was used
96
to probe for PKCα following the immunoprecipitation of PICK1 and PSD-95. Protein
samples were transferred onto nitrocellulose membranes for immunoblotting.
2.2.6 Immmunoblotting
After transfer, membranes were blocked in 5% blotting grade non-fat dry-milk
(BioRad) in TBS-T (0.01 M Tris, 0.1 M NaCl, 0.05% Tween 20) for 1 hour at RT. To
probe for phosho-serine880ct- GluR2, the immunogen (Chemicon, polyclonal, rabbit,
1:1000, diluted in 5% milk block) was a thyroglobulin-conjugated synthetic peptide
corresponding to amino acids 873-883 of rat GluR2, with a phosphorylated serine at
position 880 (LVYGIE(PO4S)VKIA). Immunoblotting for total GluR2 was performed
with a polyclonal rabbit anti-GluR2 (1:1000, Chemicon) antibody. Phosphorylated GluR1
at serine 845 was detected using a polyclonal antibody to PS845 (Abcam, 1:400).
Following immunoprecipitation with PICK1, we probed for PKCα using a mouse,
monoclonal anti- PKCα antibody (1:350, Upstate Biotechnology, Lake Placid, NY). We
sought to verify this interaction using both the aforementioned monoclonal antibody
(1:350) and a separate rabbit anti- PKCα (1:350, Abcam) antibody. Hence, the
immunoblots presented using the latter antibody contain a heavy chain IgG band at 55
kDa (because both the immunoprecipitating and immunoblotting antibody were
polyclonals hosted in rabbit), whereas the blots using the monoclonal PKCα antibody do
not contain the heavy chain IgG band. All primary antibodies were incubated overnight at
4°C. After washing in TBS-T, HRP-conjugated goat anti-rabbit IgG (1:3000, Chemicon)
or HRP-conjugated goat anti-mouse (1:3000, Chemicon) was added for 1 hour at RT. We
visualized immunoreactivty using an ECL western blotting detection kit (Perkin-Elmer).
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To verify equal protein loading in whole cell blotting, membranes were re-probed with
mouse anti-beta actin (1:2000, Sigma), mouse anti-ERK (1:40,000, Sigma), and HRP-
conjugated goat anti-mouse (1:3000). For immunoprecipitation, membranes were re-
probed for the immunoprecipitating protein (PICK1, 1:500 or PSD-95, 1;1000), and
results were normalized to the amount of IP protein per lane. All immunoblotting and
immunoprecipitation experiments were repeated in triplicate, with densitometry
performed within the linear range of analysis. Densitometry analysis was performed
using Gel-Pro Analyzer software (Media Cybernetics, San Diego, CA). Integrated optical
density of PKCα in both immunoprecipitation conditions was expressed as a ratio of
PKCα:PICK1 or PKCα:PSD-95. All results are normalized to control cultures, which are
assumed to represent 100% expression.
2.2.7 Acid Strip Immunofluorescence
In addition to examining the protein-protein interactions that underlie GluR2
trafficking, we also wanted to visualize GluR2 protein inside our cultured neurons. To
visualize the internalization of GluR2, 2 µg/mL monoclonal anti-GluR2 (Chemicon)
recognizing the extracellular N-terminus was bath applied to live cultures in medium.
Cells were incubated at 37°C for 10 minutes, and washed with warm HEPES containing
buffer to remove unbound antibody. Where appropriate, cells where then subjected to our
model of injury. To examine the effect of blocking NR2b-containing NMDA receptors on
AMPAR internalization, 5 µM Co101244 hydrocholoride was bath applied with NMDA.
Following injury, cells were incubated for 1 hour at 37°C, and washed with ice-cold
HEPES buffered saline to stop endocytosis. After the wash, cells underwent a 4 minute
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acid strip using ice cold solution of 0.2 M acetic acid, and 0.5 M NaCl, pH 2.8. Cells
were thoroughly washed in buffer again, and fixed for 15 minutes in 4%
paraformaldehyde. After fixation, cells were permeabilized with 0.1% Triton-X (or not
permeabilized as a negative control), and anti-rabbit Alexa 488 secondary antibody was
applied (1:1000, diluted in 4% NGS in PBS) for 1 hour at RT. As a second negative
control, live cells were fixed, permeabilized, and incubated with anti-rabbit Alexa 488
secondary antibody alone (i.e., no primary antisera). To visualize fluorescence, images
were acquired on a Leica DMIRE2 confocal microscope using a 20X objective, digitally
magnified 16X on dendritic spines. Image capture settings were standardized for all
images. A Z-series projection of 3–4 images at 0.5 μm step intervals was used for each
image capture and settings were always in the linear range of signal intensity.
To quantify dendritic immunofluorescent staining, we examined 1-2 distal
dendrites per cell which contained distinct protruding spines and did not exceed 50 μm in
length or 3 μm in width of dendritic shaft. Using ImageProPlus software (Silver Spring,
MD) we calculated the area occupied by fluorescent puncta for each process, and divided
this by the total area of the process. We collected data for 10 cells per condition per trial,
and repeated this in triplicate across separate cultures. In each condition, cells were
selected under bright field optics, and the investigator was blind to the treatment
condition.
Spine sizes were quantified by measuring the diameter of the spine head (after
fixation) using Image J. A line was manually drawn in image J across the head of the
spine, which was then converted from pixels to micrometers using the scale bars of the
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image. For the various types of dendritic spines, we measured the maximum diameter
(i.e., the head of a mushroom and thin spine, the base of a stubby spine).
2.2.8 [Ca2+] Measurement
Our hypothesis was that GluR2 internalization would deregulate intracellular
calcium homeostasis, and so it was necessary to visualize intracellular calcium dynamics.
Cortical cells were incubated with 5 µM Fura-PE3 AM (a calcium-binding dye, Teflabs,
Texas) for 40 min at 37ºC, and then washed three times with HEPES buffered saline and
left to incubate further for another 40 minutes to maximize dye hydrolysis. In our model
of mild injury, neurons were incubated with the dye for 40 minutes along with 20 μM
Naspm (1-Napthylacetyl Spermine, Sigma, to selectively block GluR2-lacking
AMPARs). Cells were washed, injured, and allowed to incubate for 60 minutes, to
remain temporally consistent with previous assays. Circular selections of membranes
(0.75’’ diameter) were then removed from the well using a membrane sectioner, and
perfused in HEPES buffer at room temperature. After collecting 150 seconds (30 epochs
of 5 seconds each) of stable baseline data, cells were perfused with HEPES containing
100 µM AMPA and 50 µM cyclothiazide (CTZ, for allosteric regulation of
desensitization) for 45 epochs, and then returned to control HEPES. Cells were excited
for 500 ms alternately at 340 and 380 nm at 5 second intervals, and an image from each
excitation wavelength was captured using a High Performance cooled CCD camera
(Sensicam, Cooke, Eugene OR). Volumetric flow rate of both HEPES buffer and AMPA
+ CTZ containing buffer was 1.2 mL/minute. The emission intensity at 340 nm was
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divided by the intensity at 380 nm, to calculate increases in cytosolic calcium. Figure 5a
provides a temporal schematic of our calcium imaging experiments.
To analyze regions of interest, cells were selected using SlidebookTM software
(Intelligent Imaging Innovations Inc, Denver, CO), with three parameters monitored by
the experimenter: emission at 340 nm, emission at 380 nm, and the ratio of the two
values. Calcium imaging was done in triplicate for control cells, and quadruplicate in the
injury condition, across separate cell culture isolations. Calcium imaging of injury +
Naspm treated cells was also repeated in triplicate. We quantified three data parameters:
i) the amount of time between peak emission and return to baseline, ii) integration of the
area under each calcium curve as an estimate of the relative quantity of excess cytosolic
calcium, and iii) compared values of peak emission during AMPA + CTZ perfusion
(normalized to baseline ratios).
2.2.9 Secondary AMPA Toxicity
We further sought to examine the vulnerability to excitotoxicity of neurons that
had exhibited an internalization of GluR2 protein. Cortical cells underwent stretch alone,
stretch + 10 uM NMDA, or 10 uM NMDA alone in 2 ml HEPES buffer as described
above, and left to incubate for 1 hour at 37ºC. Wells were subsequently loaded with 10
μg/ml propidium iodide (PI) warmed in 37 ºC water bath. The quantitative measurements
of PI fluorescence were used as a determination of the prevalence of cell death using a
Victor3V multi-well plate scanner (PerkinElmer, Wellesley, MA) controlled by Workout
software (Dazdaq, Finland). All parameters including the size and number of scanning
area and the duration of scanning were kept constant by using the same protocol for all
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groups. One hour following stretch, 10 μM NMDA, or the dual insult, baseline PI
readings were taken. Cells were subsequently exposed to 30 μM AMPA and further
incubated at 37 ºC in the absence of CO2 for 1 hour. Cells were washed with buffer, and
subsequent readings were taken 20 hours later. Cell death in each condition was
compared to unstretched wells exposed to 1 mM glutamate (Glu) for 1 hour, which
routinely produced a nearly 100% increase in cell death. Cell death was calculated
according to the formula: % increase in cell death = F20/F0, where F20 = PI fluorescence
20 hours post insult and F0 = Initial PI fluorescence. Cell death was thus normalized to
baseline readings. Cells exposed to 1 mM Glu were identical cultures from the same
dissection, in the same plate.
2.2.10 Whole cell electrophysiology
Whole-cell patch-clamp recording was performed at room temperature in cultured
control neurons, as well as at one hour following injury, to examine any phenotypic
changes to AMPA receptor physiology (e.g., sensitivity to polyamines or changes to
whole cell current amplitude or frequency). The extracellular solution during recording
was comprised of (concentrations in mM): 128 NaCl, 5 KCl, 1.8 CaCl2, 1 Na-Pyruvate,
17 HEPES acid, 20 D-Glucose, 3 NaHCO3, 1 MgSO4, 0.001 tetrodotoxin, 0.01 AP-5.
Intracellular solution was comprised of (concentration in mM): 128 CsOH, 111 gluconic
acid, 4 NaOH, 10 CsCl, 2 MgCl, 10 HEPES acid, 4 Na2ATP, 0.4 Na3GTP, 30 Sucrose,
0.1 1-napthylacetyl spermine (Naspm), pH 7.3, 299 mOsm. Extracellular solution during
sodium-free recordings consisted of: 128 Choline Chloride, 5 KCl, 1.8 CaCl2, 17 HEPES
acid, 20 D-Glucose, 1 MgSO4. Holding potential was maintained at -70 mV, and
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AMPAR-mediated mEPSCs were recorded and filtered at 2 kHz using Clampex software
(Axon Instruments, Union City, CA). mEPSC amplitude was assayed using MiniAnalysis
software (Synaptosoft, Decatur, GA). Event threshold was set to 5 pA, and each mEPSC
was analyzed individually. In examining the effects of 10 μM Tat-QSAV and AAAA,
neurons were treated post-injury, and the peptide remained in the wells until recording.
2.3 TAT peptides Molecular cloning of the proteins involved in GluR2 trafficking has yielded a
tremendous amount of insight into potential methods of perturbing subunit endocytosis.
A widely used method of interfering with protein-protein binding is the exogenous
introduction of a primary amino acid sequence mimicking the binding moiety of one of
the proteins involved in the interaction. By introducing one of these dominant-negative
decoy peptides, the experimenter can effectively bind their protein of interest, thereby
perturbing the endogenous protein interaction. For example, if one is interested in
interfering with a PDZ interaction, a well-accepted experimental strategy involves
occupying the endogenous PDZ domain involved in the interaction with the use of a
peptide mimicking the PDZ ligand of the associated protein.
Although in theory this is an effective strategy, peptides in general are not cell-
permeable, as the plasma membrane of cells presents a tight barrier to the passage of
foreign hydrophilic extracellular cargoes. To apply peptides intracellularly, they can be
introduced via electroporation, single cell microinjection, or via fusion to a virus,
although these methods have a number of shortcomings, including lack of clinical
applicability and a massive immune response in the case of viral injection. An alternative
approach is the introduction of a molecular chaperone protein that is capable of crossing
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the plasma membrane, carrying with it the amino acid cargo that will be used in
perturbing the protein interaction of interest. One of these chaperones is a protein
transduction domain (PTD) produced by the human immunodeficiency virus type 1
(HIV-1) transacting activator of transcription (TAT) protein.
2.3.1 The HIV-1 TAT protein transduction domain
The first example of this type of protein transduction was observed when the full
length HIV Tat protein was found to be capable of entering mammalian cells and
activating transcription from an HIV long terminal repeat promoter construct.
Subsequently, studies defined the specific region of the protein that mediated cellular
uptake, which was identified as an 11 amino acid arginine rich and therefore highly
cationic sequence. This sequence (YGRKKRRQRRR), when fused to other peptides or
oligonucleotides, demonstrated membrane transduction properties on its own and allowed
fused cargoes to carry out intracellular functions ranging from cytoskeletal reorganization
to recombination of genomic DNA. Indeed Tat peptides can also transfer much larger
molecules, including 45 nm iron beads, lambda phage, adenovirus, lipsosomes
complexed with plasmid DNA, and nanoparticles.
The mechanisms of protein transduction have been largely mapped out. It is
thought that the cationic charged amino acids present on the Tat PTD allow the peptide to
form tight and rapid interactions with ubiquitous extracellular glycosaminoglycans,
including heparin sulfate, heparin, and chondroitin sulfate B located on lipid rafts. This
hypothesis originally grew out of the observation that externally added heparin or
heparinase III inhibits cellular uptake of Tat PTD, as does the interference with lipid-raft
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dependent macropinocytosis, a specialized form of fluid phase endocytosis. The binding
of the Tat PTD to anionic extracellular glycoproteins and phospholipids is therefore
thought to be the primary step of protein translocation because it is so electrostatically
favourable. Following the stimulation of macropinocytosis and trafficking of the
macropinosome, a drop in pH is thought to mediate the release of cargoes into the cytosol
or nucleus from enclosed vesicles. This escape from macropinosomes is widely accepted
as the rate limiting step of Tat-mediated protein transduction, with a number of
experimental strategies seeking to enhance cargo release through photoacceleration
strategies and the development of molecules capable of destabilizing macropinosome
lipid bilayers, such as the influenza HA2 pH sensitive fusion domain, which enhances Tat
peptide release.
2.3.2 Design of PICK1 inhibitory TAT peptides
The technique of coupling a transduction domain to a small PDZ-ligand has
shown to be extremely effective both in transducing into cortical neurons, and in
perturbing protein-protein interactions both in vivo and in vitro119,334. We chose to adopt
this technique to perturb the interaction between PKCα and PICK1, thereby interfering
with the protein interactions responsible for GluR2 endocytosis. The structural PDZ
interaction between PICK1 and PKCα is well established, and as discussed it is known
that the extreme C-terminus of PKCα, upon activation by calcium, binds the PICK1 PDZ-
domain via its unique –QSAV sequence 360,404-407. Thus, we chose to create a 15 amino
acid peptide made up of the transduction domain of the HIV-1 transacting activator of
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transcription (TAT) protein and the unique PKCα PDZ-ligand QSAV, for a final
sequence (synthesized by CPC Scientific, San Jose CA) of:
Tat:QSAV: Tyr-gly-arg-lys-lys-arg-arg-glu-arg-arg-arg-glu-ser-ala-val
By introducing this exogenous PKC sequence into our cultured neurons (and
later into the whole animal brain), it was our intention to bind the PDZ domain of
PICK1, thereby inhibiting the PDZ interactions necessary to carry out the
internalization of GluR2. Peptides were tagged with a dansyl chloride moiety for
visualization of transduction. Control peptides (Tat-AAAA) had an alanine quadruplet in
place of the QSAV sequence, which served as a negative control for non-specific effects
of peptide transduction on GluR2 trafficking. Indeed this AAAA sequence does not
represent a functional binding domain for any known proteins.
Peptides were made using solid phase Fmoc chemistry, where the first amino acid
was covalently linked to a solid support with the alpha amino group protected by an
Fmoc (9-fluorenylmethyloxycabonyl) moiety, as described by 408. Using piperidine (a
deprotection agent) the alpha amino group was freed in preparation for coupling the next
amino acid in the sequence. Stepwise addition continued until the desired peptide length
was obtained. After the last amino acid was added, one additional deprotection step was
performed to remove the last moiety on the N terminal amino acid. Peptides were
removed from the solid support by adding trifluoroacetic acid (TFA).
To first identify if our peptides could transduce cultured cortical neurons, peptides
were bath-applied for 30 minutes at a concentration of 10 µM in HEPES buffer. Cultures
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were washed thoroughly to remove un-transduced peptide. Subsequently, sections of
SilasticTM membranes were cut, removed from the plate, and placed in HEPES buffer.
Live-cell fluorescence was visualized by fluorescence microscopy, and fixed cells were
imaged using confocal microscopy. All parameters for image capture were kept constant
among images (aperture, gain, black level, number of passes for Kallman integration).
For confocal imaging, cells were fixed 30 minutes after peptide application, mounted on
slides, and imaged (see below for confocal imaging details). We observed that Tat
peptides rapidly transduced our cortical cultures, represented by marked dansyl
fluorescence in both the soma and dendrites (Figure 10). We further observed that Tat
peptides accumulated in coronal brain slices, confirming that our compound was able to
transduce the membrane of neurons in vivo (Figure 10). However, unlabeled peptides
were used for all experiments not involving visualization to eliminate the possible effects
of the conjugate on the actions of the drug.
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Figure 9. Design of PICK1-inhibitory Tat peptides and mechanisms of Tat-peptide
uptake. A) Dansyl chloride was conjugated to both the Tat protein transduction domain as
well as the PKCα QSAV PDZ ligand. This QSAV sequence binds the PICK1 PDZ
domain. Negative control peptides contained an AAAA moiety instead of the active PDZ
ligand. B) Mechanism of Tat peptide internalization. Tat-mediated transduction occurs by
macropinocytosis. Cationic peptides bind to cell surface proteoglycans on lipid rafts,
stimulating the formation of a macropinosome. Macropinosomes decrease their pH, and
the membrane of the macropinosome vesicle destabilizes, releasing intracellular cargoes.
Peptide release can be enhanced with the addition of membrane-destabilizing agents.
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Figure 9. Design of PICK1-inhibitory Tat peptides and mechanisms of Tat-peptide uptake.
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Figure 10. Transduction of dansyl-Tat-QSAV into cultured cortical neurons and brain
slices in vivo. A) Dansyl chloride-conjugated TAT peptides (10 μM) successfully
transduce live cortical cultures (fixed images taken at 20 min after peptide application).
Scale bars: 10 mm low magnification, 2 mm high magnification. B) Dansyl-Tat-QSAV
also accumulates in native brain slices (live images taken at 40x), indicating successful
transduction of the peptide in vivo. Note that some neurons contain visible cytosolic
accumulation, while others demonstrate marked accumulation of the peptide around the
plasma membrane, presumably from different stages of peptide pinocytosis.
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A)
B)
Figure 10. Transduction of dansyl-Tat-QSAV into cultured cortical neurons and brain
slices (cortex) in vivo.
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2.4 In vivo Methods
2.4.1 Fluid percussion trauma
The fluid percussion injury (FPI) model has been extensively characterized in the
rat model of TBI409. In brief, male Wistar rats (280-350 g) were anesthetized with 2.0–
2.5% halothane delivered in compressed air. Temperature was maintained by a thermal
heating blanket at 37°C. A craniotomy (~ 2–3 mm diameter) was performed in the right
lateral hemisphere, such that the medial edge of the craniotomy was approximately 2 mm
from the midline suture, midway between bregma and lambda. A polyethylene tube was
fixed to the opening with cyanoacrylate adhesive and dental acrylic, filled with 0.9%
isotonic saline and attached to the FPI device. Rats were subject to a 2.0 atmosphere
extradural fluid percussion impact. Bone wax was used to close the hole in the skull, and
scalps were sutured prior to recovery in a temperature-controlled chamber. Tat peptides
(dissolved in ddH2O) were administered via intraperitoneal or intravenous injection at 1-3
mg/kg after closure of the head incision (i.e., approximately 10 minutes after the impact).
2.4.2 Slice Electrophysiology Similar to our in vitro recordings, we sought to examine any changes to the
primarily AMPA receptor-mediated hippocampal field responses following fluid
percussion trauma. All rat slice recordings were performed between 3 and 6 hours after
fluid percussion trauma. Stimulation (0.1 ms in duration) was delivered using a bipolar
tungsten electrode over a range of 40-90 μA generated by a Grass S88 stimulator (Grass
Instrument, West Warwick, RI) and delivered through a PSIU6 isolation unit. Recording
electrodes were pulled from filamented borosilicate glass capillary tubes with a P-97
Flaming/Brown micropipette (Sutter Instruments Co.). Pipettes with resistances of 2–
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3 MΩ were backfilled with 150 mM NaCl. Signals were digitally recorded using an
Axopatch 200B amplifier (Axon Instruments, Foster City, CA). All recordings were
performed at room temperature and analyzed by pCLAMP software (Axon Instruments).
Extracellular solution (perfused at a rate of 7 ml/min) during all recordings consisted of
(concentration in mM): 126 NaCl, 3 KCl, 1.4 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26
NaHCO3, 20 glucose, and, when necessary, 0.02 Naspm, bubbled with carbogen (95%
O2, 5% CO2), 285 ± 5 mOsm. One hour after FPI, animals were decapitated, and the
brains were extracted in ice-cold aCSF. Recordings were performed on 450 µm
transverse hippocampal slices. Slices were acclimated to room temperature for a
minimum of 60 minutes prior to recording. Recording electrodes were placed in the CA1
stratum pyramidale, with stimulation electrodes placed in the schaffer collateral tract. For
sensitivity to the synthetic polyamine 1-naphthylacetyl spermine, 5 minutes of perfusion
with control aCSF was followed by a 7.5 minute perfusion with aCSF + 20 µM Naspm.
Slices were returned to control aCSF after Naspm treatment. For electrophysiological
recordings, Tat peptides were administered to animals at a concentration of 3 mg/kg I.V
(in a 1 mL volume of saline as vehicle) following FPI but prior to decapitation and brain
slicing.
2.4.3 TUNEL staining
To examine the prevalence of apoptotic cell death, we performed terminal
deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), which
labels DNA strand breaks initiated by cleavage of genomic DNA during programmed cell
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death. The strand breaks, or “nicks”, can be identified by labeling the free 3’-OH
terminals with modified nucleotides in an enzymatic reaction.
Anesthetized rats were transcardially perfused with 0.9% isotonic saline followed
by 4% paraformaldehyde. The brain was postfixed overnight in 4% paraformaldehyde 0.5
M acetate solution before paraffin embedding. Coronal brain slices were sectioned at 10-
µm thickness. After deparaffinization of sections, slides were treated with proteinase K,
and the TUNEL labeling procedure was carried out according to the manufacturer’s
protocol. For quantification of TUNEL labeling, three areas of the cortex were taken,
each with a field of view 820 x 650 μm (533 mm2). Areas corresponded to medial, core,
and lateral to the fluid percussion impact site. Sections were taken from Bregma – 4.3
mm, according to Paxinos and Watson (1998). Data was normalized to the total number
of cells (labeled with a Hoescht counterstain) identified in the field of view; this
translated to a sampling of approximately 2500-3000 cells per animal.
2.5 Contributions The experimental data presented in this thesis was collected and analyzed entirely
by the author. This included isolation and dissociation of cell cultures, protein lysis and
quantification, western blotting, co-immunoprecipitation, single cell and slice
electrophysiology, calcium imaging, toxicity assays, peptide design and testing, and
immunofluorescence. The one exception is the TUNEL staining, which was performed
by Ms. Elaine Liu. Quantification for this assay was performed by the author. Technical
assistance for molecular biology and electrophysiology, as well as manuscript editing for
publication of the findings was provided by Dr Eugene Park. Dr Jinglu Ai, Dr Loren
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Martin, and Mr. Zikai Zhou, also generously provided their knowledge in
electrophysiology to help set up the experiments.
2.6 Statistics
All in vitro data are representative of trials repeated at least three times across
separate cell culture isolations unless otherwise indicated. In vivo data was collected with
an n of 4-6 animals, unless otherwise indicated. Data are presented as mean ± SEM.
One-way ANOVAs with post-hoc Tukey tests, or Dunn tests (in cases where tests of
normality failed) were used to identify significant differences between treatment
conditions in all assays.
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Chapter 3: GluR2 trafficking in modeled brain trauma
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3.1 Preface This section of data comprises the molecular biology and biochemistry employed
in this thesis. Here, we examined the cell signaling involved in GluR2 trafficking in two
models of brain trauma: in vitro cortical injury, as well as in the lateral fluid percussion
injury preparation. The assays examined the various critical endpoints in GluR2
endocytosis, and accordingly, the involvement of certain proteins in mediating post-
traumatic internalization of GluR2. The first component of this section involves
presentation of the in vitro findings; western blotting of GluR2 phosphorylation, co-
precipitation of GluR2 trafficking proteins, and acid strip immunofluorescence on
dendritic spines of cortical cultures. The second component examines these phenomena at
an acute time point following whole animal trauma.
3.2 Phosphorylation of GluR2 serine 880 following in vitro trauma correlates with susceptibility to AMPA toxicity
In our first assay, we sought to examine the prevalence of phosphorylated GluR2
in traumatized cortical cultures. Indeed the plethora of experimental evidence in the
literature that suggests GluR2 endocytosis is preceded by PKCα-dependent serine 880
phosphorylation. 349,369 led us to investigate this post-transcriptional modification, as we
hypothesized this might influence the synaptic composition of GluR2. We examined this
protein modification and its role in delayed cell death in our previously established model
of sublethal mechanical trauma followed by mild excitotoxicity. Relative to control
cultures, our model of TBI produced a rapid increase in detectable levels of serine 880
phosphorylated GluR2 [GluR2 phosphorylation = 164 ± 10.3% of control, p < 0.01,
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Figure 11A and 11B, but see also quantification in Figure 12]. However, because our
injury employed two distinct insults to the cultures to mimic TBI (mechanical strain as
well as application of NMDA), we further examined the impact of these individual
injuries on GluR2 phosphorylation in an attempt to parse out which component might be
responsible for the reported effect. Notably, neither stretch injury alone nor application of
10 μM NMDA for 1 hour had a significant effect on GluR2 phosphorylation [GluR2
phosphorylation = 109 ± 2.3% of control, p = 0.12 for 10 μM NMDA vs. control; GluR2
phosphorylation = 91 ± 8.3% of control, p = 0.16 for stretch vs. control, Figure 11A and
11B), suggesting a synergistic co-operation between mechanical trauma and stimulation
of the NMDA receptor in increasing the phosphorylation of GluR2. Co-operation
between stretch injury and stimulation of the NMDA receptor is indeed a phenomenon
reported throughout the literature by a number of different investigators, although
discussion of the mechanism responsible for this synergy will occur later in this chapter.
With the development of a model that increased GluR2 phosphorylation, we
sought to investigate the possibility of an increased vulnerability of the injured cultures to
AMPA receptor-mediated excitotoxicity. Indeed our hypothesis was that phosphorylation
of GluR2 would lead to a reduction of surface protein expression, thereby increasing the
inherent cytotoxicity of AMPA receptor stimulation. In line with this hypothesis, our in
vitro TBI model of stretch + NMDA resulted in an increased vulnerability of cortical
cells to a one hour challenge of 30 μM AMPA (24.98 ± 4.8% increase in cell death, p <
0.05, Figure 1D and 1E), evidenced through markedly greater cellular uptake of
propidium iodide at 24 hours following early post-injury AMPA receptor stimulation.
Accordingly, conditions that did not enhance the expression of phosphorylated GluR2 did
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not result in delayed cell death following exposure to 30 µM AMPA challenge (stretch +
AMPA = 0.97 ± 2.8% increase in cell death; NMDA + AMPA = 5.7 ± 2.5%, p > 0.05, n
= 3 cultures, Figure 1D). Stretch + NMDA without a secondary AMPA treatment also
did not result in an increase in delayed cell death (4.12 ± 1.4% increase, p > 0.05).
Representative micrographs of PI uptake are presented in Figure 11C. Collectively, these
initial results suggested that stretch injury coupled with NMDAR stimulation resulting in
GluR2 phosphorylation conferred heightened sensitivity to excitotoxic challenge of a
dose of AMPA that remained innocuous in normal conditions. This has immediate
implications in the pathophysiology of secondary excitotoxic injury after TBI which will
be discussed later.
3.2.1 NMDA receptor dependence of GluR2 phosphorylation
The synergistic effect of NMDA and stretch injury on GluR2 phosphorylation
suggests perhaps a trauma-induced modification of the NMDA receptor that increases
receptor calcium influx. However, to further understand the downstream effectors
responsible for GluR2 phosphorylation after NMDA application to traumatized cultures,
we sought to understand which subpopulation of NMDA receptors might be mediating
this effect. As discussed in the introduction, the two predominant subtypes of the NMDA
receptor are NR1/NR2A, as well as NR1/NR2B, the former which is primarily synaptic,
the latter extrasynaptic. We treated cultures with antagonists of both NMDA receptor
subtypes. Significant differences in GluR2 phosphorylation were indeed detected among
treatment groups (p < 0.001, F = 26.197). Post-hoc analysis revealed attenuation of
GluR2 phosphorylation by selective antagonism of NR2B-containing NMDA receptors
(32.7 ± 6.1% of control, p < 0.001, Figure 1F), while antagonism of NR2A-containing
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NMDARs was ineffective (Figure 11E). Thus, this suggests the likely possibility that
extrasynaptic NMDA receptor stimulation is primarily responsible for trauma-induced
GluR2 phosphorylation. Data for GluR2 phosphorylation is quantified in Figure 12.
3.3 In vitro trauma increases PICK1-PKCa binding
The data suggesting that GluR2 phosphorylation increases in this model of trauma
raises the possibility that the endogenous cellular machinery responsible for GluR2
trafficking is activated post-injury. To examine the involvement of GluR2 endocytotic
proteins in mediating this post-traumatic phosphorylation, we next incubated cultures
with our 15 amino acid TAT peptide (Tat-QSAV) that mimics the extreme c-terminus
PDZ-binding motif of PKCα, thereby designed to inhibit the PICK1-PKCα protein
interaction (discussed previously as a critical interaction during GluR2 internalization
from the cell surface). After confirming successful transduction of the peptide (Figure
10), we examined the effects of Tat-QSAV on the protein interaction between PKCα and
PICK1 in the in vitro injury paradigm. First, we observed that Stretch + NMDA
significantly augmented PKCα-PICK1 binding (169 ± 5.6% of control; p < 0.01, Figure
12A), an effect similarly attenuated by NR2B-containing NMDA receptor antagonism
(65.6 ± 9.6% of control levels, p < 0.01, Figure 12A). Because this interaction is
dependent on activation of PKCα, this result suggested an NMDA receptor dependent
activation of the kinase, leading to increased PICK1 binding. We further examined the
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Figure 11. Stretch + NMDA increases S880 phosphorylation of GluR2 and vulnerability
to secondary AMPA toxicity. A) Western blot of GluR2 phosphorylation at c-terminus
serine residue 880. Stretch + NMDA (but neither condition alone) markedly increased
phosphorylation. Membranes were stripped and re-probed for β-actin as a loading
control. (B) Data represented in (A), quantified as integrated optical density (IOD)
normalized to control values, which are assumed to represent 100% expression. (C)
Representative micrographs of propidium iodide (PI) fluorimetry after stretch + AMPA,
NMDA + AMPA, stretch + NMDA or stretch + NMDA + AMPA. Scale bars = 75 μm.
(D) Plate scanner quantification of PI uptake in all toxicity studies performed. Treatments
that do not enhance the expression of GluR2S880ct (stretch alone, or 10 μM NMDA
alone) do not increase the vulnerability of cortical cells to subsequent challenge of 30 μM
AMPA. (E) Antagonizing NR2b-contaning NMDA receptors attenuates the injury-
induced increase in GluR2 phosphorylation. (F) NR2A antagonism does not reduce
GluR2 phosphorylation. ERK1,2 was used as a loading control. **P < 0.01.
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Figure 11. Stretch + NMDA increases S880 phosphorylation of GluR2 and vulnerability
to secondary AMPA toxicity
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effects of Tat-QSAV on perturbing this increase in PKCα-PICK1 binding, and found that
the compound, when administered pre-stretch, successfully disrupted the interaction
between PKCα and PICK1 following injury (68.3 ± 16.7% of control, p < 0.01, Figure
12A). Tat-AAAA (our other peptide lacking an intact PDZ binding motif) however, was
ineffective (162.9 ± 6.5% of control, Figure 12A) suggesting that the interference with
PKCα-PICK1 binding did result from non-specific effects of Tat peptide transduction.
Data for these co-precipitation experiments are quantified in Figure 12B. Tat-QSAV, but
not Tat-AAAA, also interfered with trauma-induced phosphorylation of GluR2
(respectively, 90.5 ± 18.3% of control, p < 0.05, vs. 161.8 ± 11.1% of control, p = 0.43
vs. stretch + NMDA, Figure 12C), suggesting further that this PKCα-PICK1 increase was
potentially involved in the post-traumatic phosphorylation of GluR2. We also assayed
for total GluR2 protein expression, which we found did not differ in any of the treatment
conditions (p = 0.67, Figure 12C). Thus, the increased phosphorylation of GluR2 did not
translate to a reduction of total cellular protein, in contrast to the epigenetic silencing of
GluR2 described in cerebral ischemia. Data of total and phosphorylated GluR2 is
quantified in Figure 12D.
3.4 PKCa is embedded in the NMDAR complex:
PKCa co-precipitates with PSD-95
Our previous data demonstrated that in vitro trauma confers the association of
PICK1 with PKCα. It is known that PKCα activation increases its binding with PICK110
and that PKCα activation can occur endogenously via binding of intracellular calcium18.
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Figure 12. Stretch + NMDA confers association of PKCa with PICK1. A) As assayed
through co-immunoprecipitation at one hour following trauma, in vitro injury promotes
an NR2B-dependent association of PKCa with PICK1. TaT-QSAV, relative to TAT-
AAAA and injured (untreated) cultures, markedly diminishes bound levels of PKCa to
PICK1, suggesting this compound can successfully compete for the endogenous PICK1
PDZ domain. Membranes were stripped and re-probed for PICK1. (B) Quantification of
data presented in (A). Data are expressed as the ratio of PKCa/PICK1, and each condition
is normalized to control levels. (C) TaT-QSAV, but not TaT-AAAA, also reduces post-
traumatic S880 phosphorylation of GluR2, highlighting the potential involvement of the
PICK1-PKCa increase in downstream phosphorylation of GluR2. (D) Total GluR2 does
not change in any of the treatment conditions. (E) Quantification of total and
phosphorylated GluR2 across conditions.
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Figure 12. Stretch + NMDA confers association of PKCa with PICK1.
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Given the NR2b-dependence of the PKCα:PICK1 increase, we sought to understand
further the mechanism behind increased activation of PKCα in this preparation.
PKCα’s type I PDZ ligand (QSAV) has the potential to form a stable PDZ
interaction with another protein, PSD-95, which contains three PDZ domains, and is also
structurally connected to the NMDA receptor. PSD-95 is a membrane-associated
guanylate kinase (MAGUK) scaffolding protein that plays an important role in linking
calcium derived from the NMDA receptor (particularly NR2B-containing receptors) to
activation of downstream substrates, including for example neuronal nitric oxide synthase
(nNOS), a protein activated by calcium influx following ligand-mediated opening of the
NMDAR channel. We hypothesized that since the increased association between PKCα
and PICK1 was also NMDA receptor dependent (i.e., the binding between these proteins
was perturbed by an NR2B antagonist) that a similar scaffold was provided by PSD-95 to
PKCα activation. Thus, we attempted to co-precipitate PKCα with PSD-95. We first
observed co-immunoprecipitation of PSD-95 with PKCα (Figure 13A and 13B), with
PICK1 pull-down, whole cell lysates and bound nNOS (a known binding partner of PSD-
95) as positive controls (Figure 2H). This association was a novel finding, the first to
demonstrate that PKCα might be physically embedded within the NMDA receptor
complex.
Subsequently, we observed that the PKCα-PSD-95 interaction was also markedly
increased after Stretch + NMDA (168 ± 30.3% of control levels, p < 0.05, Figures 13C
and D). Both NR2b-antagonism (74 ± 28.1% of control, p < 0.01 compared to stretch +
NMDA) as well as Tat-QSAV (123 ± 9.2% of control, p < 0.05 compared to stretch +
NMDA) attenuated the increase in PKCα-PSD-95 binding (Figure 2I, J and K). Tat-
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AAAA had no observable effect (171 ± 22.4% of control, p < 0.01 relative to control,
Figure 2J and K). Indeed this data confirmed that the association between PKCα and
PSD-95 was likely occurring via the PKC PDZ-ligand, as mimicking this sequence
attenuated the interaction. This structural association between PKCa and the NMDAR
complex sheds further mechanistic light on how signaling at NR2b-containing NMDARs
might lead to post-traumatic GluR2 phosphorylation, and ultimately, endocytosis.
3.5 Traumatic injury increases GluR2 endocytosis
Though we had biochemical data suggesting that GluR2 endocytosis might be
occurring (and had identified a possible mechanism of the NMDA receptor dependence
of the effect), we sought to have direct evidence for GluR2 internalization from the cell
surface. To this end, we employed a protocol known as acid-strip immunofluorescence, a
technique which labels surface receptors, allows for endocytosis to proceed, and
subsequently strips away any remaining staining on the surface of the neuron with an
acidic solution that destabilizes the antibody-antigen complex. When the cells are
permeabilized, the assay detects internalized protein that was initially present on the
surface of the cell.
One hour after our traumatic injury, this acid-strip immunofluorescence revealed
significant internalization of GluR2 (ratio of internal GluR2:dendrite area = 0.038 ± .003
relative to 0.012 ± .001 in control cultures, p < 0.001, Figure 14A and 14B). This
provided us with evidence that surface GluR2 protein was being internalized into the
cytosol. However, since bath application of NMDA can cause GluR2 endocytosis,
controls were run with both 10 and 50 μM NMDA alone (the latter as a positive control).
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Figure 13. PKCa co-precipitates with PSD-95: A potential link from the NMDAR to
GluR2 endocytosis. A-B) PKCa co-immunoprecipitates with PSD-95 in cortical cell
lysates. PICK1 I.P was used as a positive control for the PKCa immunoblot in (A), and
blotting for nNOS was used as a positive control for the PSD-95 I.P in (B). Note the lack
of nNOS in the PICK1 I.P. (C) PKCa and PSD-95 exhibit a stronger interaction after
Stretch + NMDA. Antagonism of NR2b-containing NMDA receptors with Co101244
attenuates this increase. Membranes were stripped and re-probed for PSD-95. (D)
Identical co-immunoprecipitation experiments as outlined in (C), using a polyclonal
antibody to PKCa. This antibody also recognized higher levels of bound PKCa to PSD-95
in conditions in which GluR2 phosphorylation was increased. TAT-QSAV attenuates the
injury-induced increase in PKCa–PSD-95 co-immunoprecipitation (far right lane), but
TAT-AAAA is ineffective. (E) Quantification of co-precipitated PKCa with PSD-95,
expressed as the ratio of PKCa/PSD-95, and normalized to control values. # P < 0.05
versus control; ## P < 0.01 versus control **P < 0.01; ***P < 0.001; *P < 0.05.
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Figure 13. PKCa co-precipitates with PSD-95: A potential link from the NMDAR to
GluR2 endocytosis
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10 μM NMDA on its own did not increase internalized GluR2 (ratio = 0.009 ± .002, 10
μM NMDA vs. 0.012 ± .001, control, p > 0.05, Figure 3A and B), a stark contrast to the
effect of this dose of NMDA when combined with stretch injury. As expected, 50 μM
NMDA did cause a significant increase in internalized GluR2 (ratio = 0.029 ± .005, p <
0.01 vs control, p > 0.05 vs stretch + 10 μM NMDA). Thus, we observed that stretch
injury significantly augmented the GluR2 endocytotic response of a low dose of NMDA,
with a synergistic effect of the mechanical injury and the excitotoxin similar to what was
observed in the assays of GluR2 phosphorylation.
We employed similar antagonistic approaches to what was used in our assays of
phosphorylated GluR2. NR2b-antagonism significantly reduced GluR2 internalization
(ratio = 0.023 ± .0004, p < 0.05 relative to stretch + NMDA), as did Tat-QSAV (ratio =
0.022 ± .004, p < 0.05). The compounds did not differ significantly in their levels of
attenuation (p = 0.47). Importantly, both of our negative controls (non-permeabilized
cells and cells incubated only with secondary antibody, Figure 14i) exhibited only diffuse
background staining, indicating the efficacy of our acid-strip protocol in eliminating the
binding of our primary antibody to surface receptors, as well as the specificity of our
staining for GluR2. Thus, the data was in line with our hypothesis that NMDA and
PICK1-mediated GluR2 phosphorylation leads to subunit endocytosis.
In addition to examining the impact of the stretch injury on GluR2 endocytosis,
we also examined cytoarchitechtural changes to the injured neurons. Relative to control
cultures (0.37 ± .01 spines per μm, mean spine head diameter = 0.76 ± .04 μm, n = 21
cells) stretch + NMDA also had the incidental effect of decreasing dendritic spine density
and increasing the mean diameter of remaining spine heads (0.26 ± .02 spines per μm,
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mean spine head diameter = 1.14 ± .06 μm, n = 28 cells, p < 0.001 relative to control for
spine density and diameter, Figure 3D and 3E). We hypothesized that GluR2 endocytosis
was contributing to this morphological damage since surface GluR2 stabilizes dendritic
spines through an extracellular interaction between the GluR2 N-terminus and pre-
synaptic N-cadherin 410. Indeed Tat-QSAV preserved dendritic spine density and reduced
average spine size in injured neurons (0.38 ± .01 spines per μm, mean spine head
diameter = 0.76 ± .02 μm, n = 19 cells, p < 0.001 relative to injured (untreated) for spine
density and mean diameter, Figure 3C, 3D and 3E). There was no statistical difference
between injured cultures treated with Tat-QSAV and uninjured cultures (p = 0.40 for
spine density, p = 0.49 for spine diameter). NR2b antagonism resulted in a mean spine
diameter similar to controls (0.83 ± .01 μm, n = 24 cells, p < 0.001 vs injured (untreated),
p = 0.11 vs control, Figure 3E) but did not rescue dendritic spine density (0.29 ± .01
spines per μm, p = 0.45, Figure 3D] suggesting that NMDAR blockade was less effective
in restoring normal dendrite morphology relative to the Tat-QSAV peptide. These results
suggest that preventing GluR2 endocytosis also helps preserve neuronal morphology after
traumatic injury, and corroborates the evidence that GluR2 protein was in fact
internalized.
3.6 PICK1-mediated endocytosis of GluR2 following fluid percussion trauma
Our in vitro findings raised the possibility that traumatic injury to a population of
neurons is capable of inducing the trafficking and internalization of GluR2 protein, an
AMPA receptor modification that might impart vulnerability to secondary excitotoxicity.
To validate this hypothesis, we next assayed cortical and hippocampal GluR2
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Figure 14. Stretch + NMDA increases GluR2 endocytosis. (a) Inverted confocal phase
contrast images of cortical dendritic spines were overlayed with staining of internalized
GluR2 after acid stripping. Stretch +10 μM NMDA conferred distinct GluR2-positive
puncta in dendritic spines, whereas control neurons did not (far left panel). NR2b
antagonism (Co101244) and TAT-QSAV significantly decrease GluR2 internalization
after Stretch + NMDA, but GluR2 endocytosis was still higher than controls. In all
conditions, arrows indicate spines that stained positively for internalized GluR2; 50 μM
NMDA was used as a positive control, and resulted in intense staining along the dendrite
of internalized GluR2. (ai) Negative controls of non-permeabilized cells, and cultures
treated only with secondary antibody. (b) Quantification of immunofluorescent data
expressed as the ratio of internalized GluR2/area of dendrite. *P < 0.05 versus control, #
P<0.05 versus injured, **P < 0.01 versus control; Scale bars = 2 μm. (c) Representative
confocal images of dendritic morphology in injured, untreated neurons (left panel) and in
injured neurons treated with 10 μM TAT-QSAV. Overlay represents internalized GluR2.
Note that in the absence of internal GluR2 staining, dendritic spine density is increased.
Scale bars = 2 μm. (d) Quantification of dendritic spine density as spines per mm. (e)
Quantification of mean dendritic spine head diameter (μm). ***P < 0.001 versus control;
**P < 0.01, ###P < 0.001 versus injured.
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Figure 14. Stretch + NMDA increases GluR2 endocytosis
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phosphorylation in a whole animal preparation of neuronal trauma, employing the fluid
percussion injury (FPI) device. Phosphorylated GluR2 was markedly increased in the
cortex (247.2 ± 31.2% of control animals, n = 6, p < 0.01) and hippocampus (251.5 ±
43.1% of control, n = 5, p < 0.01) of injured animals (Figure 15A, cortical blots shown).
We also observed significantly more GluR2 (141 ± 11.8% of control, p < 0.05, Figure
15C) bound to PICK1 in cortical lysates taken post FPI (Figure 15B, and 15E), a
biochemical indication of subunit internalization early after trauma. After demonstrating
successful perturbation of the PICK1-PKCα protein interaction with Tat-QSAV, but not
with Tat-AAAA (Figure 15D), we observed that intraperitoneal injection (1 mg/kg) of
Tat-QSAV significantly inhibited the association of GluR2 with PICK1 after FPI (Figure
15E and 15F, n = 5, p < 0.01), suggesting this peptide successfully interferes with post-
traumatic mechanisms of GluR2 internalization in vivo. Intravenously administered Tat-
AAAA at 3 mg/kg (triple the dose of Tat-QSAV), did not influence the characteristic
increase in the GluR2-PICK1 interaction after FPI (145.6 ± 24.1%, Figure 15E&F, p <
0.05 vs QSAV and sham, p > 0.05 vs FPI).
3.7 Summary of results Our biochemical data employing two models of experimental TBI revealed that
neuronal trauma promotes the endocytosis of GluR2 surface protein. We observed in our
cortical injury model that GluR2 is phosphorylated at serine 880, internalized from
dendritic spines, and that subunit trafficking can be interrupted by perturbing the binding
between PICK1 and PKCα. We further identified a likely mechanism of the NMDA
receptor dependence of GluR2 phosphorylation, highlighting a novel protein interaction
between PKC and PSD-95, the NMDAR-bound scaffolding protein that links
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Figure 15. In vivo traumatic brain injury (TBI) promotes GluR2 phosphorylation and
association with PICK1. (a) Representative immunoblot of PS880 GluR2 in injured
cortex 1 h after 2 atmosphere fluid percussion injury. ERK 1/2 is used as a loading
control. (b) Representative coimmunoprecipitation of PICK1 with GluR2 and PKCa after
forebrain trauma, showing GluR2 endocytosis 1 h after the injury (c) Quantification of all
GluR2/PICK1 coprecipitation experiments. (d) TAT-QSAV, but not a control peptide,
can perturb PICK1–PKCa protein interactions in vivo. (e) Animals treated after trauma
with 1 mg/kg TAT-QSAV show significantly less co-precipitation of GluR2 with PICK1
1 h after injury, suggesting this peptide can effectively prevent GluR2 endcocytosis in
injured animals. TAT-AAAA has no effect on the injury-induced increase in
GluR2/PICK1 (f) Quantification of GluR2/PICK1 co-precipitation with or without
injection of TAT peptides.
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Figure 15. In vivo traumatic brain injury (TBI) promotes GluR2 phosphorylation and association with PICK1
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glutamatergic calcium influx to downstream effector proteins. Upon investigation of
these effects in vivo, we observed a similar post-traumatic GluR2 phosphorylation. We
further reported an upregulation in the association between GluR2 and PICK1, a
biochemical indication that the subunit was being internalized from the cell surface.
Finally, exogenous interference with the PICK1-PKC interaction following intravenous
peptide injection prevented the association of GluR2 with PICK1, suggesting that post-
traumatic GluR2 endocytosis in vivo is also dependent on the trafficking of PKC to the
plasma membrane by PICK1.
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Chapter 4: Phenotypic AMPAR changes in modeled brain trauma
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4.1 Preface The previous chapter described, at the molecular level, changes to the trafficking
of the GluR2 subunit that were observed in our two models of traumatic brain injury.
The biochemical and immunocytochemical data provided evidence that traumatic injury
imparted the endocytosis of GluR2. However, there were no observations made with
respect to any phenotypic changes to AMPA receptor behaviour that occurred following
this reduction of surface GluR2 protein. As discussed in the introduction, there is
compelling basic science evidence that PICK1-mediated endocytosis of GluR2 confers
the increased expression of calcium-permeable, GluR2-lacking AMPA receptors, and that
these receptors impart neuronal vulnerability to cell death and damage. In this section of
the thesis, we examined the effects of GluR2 endocytosis on AMPA receptor-mediated
electrophysiology, calcium influx, and neuronal death. This section employed an analysis
of post-injury AMPA receptor whole-cell miniature excitatory post-synaptic events, free
calcium concentrations, AMPA-receptor mediated field potentials, and finally, the
influence of interfering with GluR2 endocytosis on delayed cellular death and apoptosis
in both our in vitro injury paradigm and our whole animal TBI preparation.
4.2 AMPAR-mediated mEPSC activity following in vitro traumatic injury Occluding GluR2 endocytosis reduces AMPAR mEPSC amplitude
To examine the contribution of GluR2-lacking AMPA receptors to neuronal
physiology following traumatic injury, we took advantage of a number of the
characteristics of calcium-permeable AMPA receptors. As was discussed in detail in the
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introduction, it is known that GluR2-lacking AMPA receptors have a higher single
channel conductance than receptors containing GluR2189 and are sensitive to polyamine
antagonism. To investigate if these changes occurred to the AMPA receptors native to
our neuronal population, we performed whole cell patch clamp of neurons at one hour
following the traumatic injury, and isolated AMPA receptor mediated responses by
antagonizing voltage-gated sodium channels and NMDA receptors.
After stretch + NMDA, AMPAR-mediated mEPSCs indeed exhibited
significantly larger amplitudes than control neurons (26.76 ± 1.62 pA vs.18.33 ± 0.69 pA,
p < 0.01, Figure 4B), as well as a 36.4 ± 5.4 % reduction in amplitude following
application of 1-naphthylacetyl spermine (Naspm), a polyamine antagonist of GluR2-
lacking but not GluR2-containing AMPARs (Figure 16C and D). Control mEPSCs did
not demonstrate Naspm sensitivity (control + Naspm = 18.38 ± 0.81 pA), consistent with
the presence of predominantly GluR2-containing AMPARs in control cortical neurons.
Naspm treatment did not significantly alter the frequency of mEPSCs, which were also
unchanged between control and injured cultures [control + Naspm = 0.36 ± .04 Hz;
control alone = 0.43 ± .01 Hz; injury = 0.31 ± .06 Hz; injury + Naspm = 0.45 ± .01 Hz,
Figure 16E].
There are a number of mechanisms through which AMPA receptor whole cell
currents might increase, including phosphorylation of the channel, increased agonist
potency, or a reduction of desensitization. To directly measure whether GluR2 trafficking
was contributing to the increased whole cell currents we incubated injured cultures with
Tat-QSAV prior to patch. Notably, Tat-QSAV reduced mEPSC amplitudes in injured
cultures to 14.72 ± 0.95 pA. Tat-AAAA treatment reduced amplitudes to 22.13 ± 0.58
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pA. Both treatment amplitudes were significantly lower than injury levels (Figure 4E, and
4F). However, mEPSCs were significantly reduced in QSAV treated cultures relative to
AAAA treated cultures (p < 0.05), suggesting a significant effect of PICK1 inhibition
independent of any effects that peptide transduction alone may have on excitability
(Figure 4F). The mechanisms through which Tat peptide transduction might have non-
specific effects on glutamatergic receptor physiology are discussed in the next section.
4.3 AMPA receptor-mediated calcium influx following in vitro trauma: Polyamine antagonism of GluR2-lacking AMPARs lowers cytosolic Ca2+ load
The cytotoxicity of AMPA receptor stimulation that occurs following a reduction
of surface GluR2 protein is largely dependent on the excessive influx of calcium through
calcium-permeable AMPA receptors. Indeed the expression of these receptors is
innocuous when extracellular calcium is chelated or removed from the bath. Thus, we
sought to visualize post-injury intracellular calcium dynamics following perfusion with
AMPA (schematic in Figure 17A), to examine if GluR2 endocytosis augmented cytosolic
Ca2+ loads.
Prior to stimulation of the cells with AMPA, we recorded baseline calcium
following the in vitro injury, to first measure the impact of our model on intracellular
calcium. Baseline calcium of control neurons was significantly lower than in neurons
exposed to stretch + NMDA (0.11 ± .01 vs. 0.19 ± .01, respectively, p < 0.01, Figure 5B
and 5C), indicating the insult affected cytosolic Ca2+ levels prior to AMPAR stimulation.
Indeed this observation was critical to our hypothesis, as our work suggested calcium-
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Figure 16. Traumatic injury in vitro increases AMPAR mEPSC amplitude and sensitivity
to intracellular polyamines. A) Representative AMPAR-mediated mEPSC traces of
injured and control neurons (one hour after insult) showing an increase in average
mEPSC amplitude. Ai) Average mEPSC traces overlaid. Black trace = Control neurons,
Red trace = Injured neurons. B) Representative traces showing that the amplitude of
AMPAR-mediated mEPSCs is not influenced by inclusion of polyamines (Naspm) in the
patch pipette. C) Following trauma however, AMPAR mEPSCs demonstrate sensitivity
to Naspm, an antagonist of GluR2-lacking receptors. D-E) Quantification of mEPSC
amplitude and frequency in the two treatment conditions. F) Post-injury co-precipitation
of PICK1 and PKCα in the presence of Tat-QSAV and Tat-AAAA and resultant mEPSC
activity. QSAV-treated neurons exhibited a significant reduction from AAAA treated
cells in mEPSC amplitude and bound PKCα:PICK1. G) Quantification of mEPSC
amplitudes in all conditions. Neurons were held at -70 mV. ** p < 0.01 vs control; * p <
0.05 vs control; ## p < 0.01 vs injured; # p < 0.05; † p < 0.01 vs Tat-AAAA.
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Figure 16. Traumatic injury in vitro increases AMPAR mEPSC amplitude and sensitivity to intracellular polyamines.
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dependent activation of PKCα initially after the injury. We further observed the effects of
AMPA receptor activation on intracellular calcium at one hour after trauma. After
applying AMPA, peak emissions normalized to baseline ratios did not differ between
control and injured neurons (2.07 ± 0.45 x baseline vs. 1.84 ± 0.12 x baseline,
respectively, p = 0.19) (Figure 5B and 5E). However, injured neurons exhibited
significantly longer calcium extrusion times (5.93 ± 1.59 minutes vs. 1.65 ± 0.51 minutes
respectively, Figure 5B and 5D p < 0.01). Integration for the area under the curve as a
surrogate indicator of intracellular calcium levels indicated a 2.11 fold larger area relative
to control neurons (62.37 ratio·epochs vs. 29.62 ratio·epochs, Figure 5F).
We tested the efficacy of Naspm (an antagonist of GluR2-lacking receptors) in
reducing peak Ca2+ in injured neurons and in improving calcium extrusion. Baseline
calcium of Naspm-treated injured cells was comparable to injured (untreated) cells (0.21
± 0.01 vs. 0.19 ± .01, respectively, p = 0.13, Figure 5B, and 5C), suggesting that GluR2-
lacking AMPARs were not responsible for the initial trauma-induced elevation of
baseline emission ratios. However, after AMPA application, peak calcium was
significantly lower in Naspm-treated injured cells relative to injury (untreated) (1.52 ±
.04 x baseline, p < 0.01 vs. values for injury, Figure 5E, and 5G), suggesting a
contribution of calcium-permeable AMPARs in the initial rise of Ca2+ in injured neurons
during perfusion of AMPA. We have previously shown that Naspm does not impact Ca2+
influx in control neurons 251. Further, the time from peak to extrusion in Naspm-treated
injured neurons was 0.88 ± 0.21 minutes, a significant reduction from that of injured
(untreated) cells (p < 0.05) but not of control cells (p = 0.12, Figure 5B, and 5D).
Integration of the Naspm-treated calcium curve yielded a value of 28.29 ratio · epochs, a
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value similar to that obtained from control cultures (29.62 ratio · epochs). Thus, our
calcium imaging data suggested not only that GluR2-lacking AMPA receptors mediate
calcium influx following in vitro injury, but also that their expression protracts calcium
extrusion.
4.4 Interfering with GluR2 endocytosis is cytoprotective in vitro Tat-QSAV and Naspm reduce excitotoxicity
There is clear evidence for the involvement of elevated cytosolic calcium in
mediating neuronal death during excitotoxicity. However, we had no direct evidence at
this point that suggested the expression of calcium-permeable AMPA receptors were
necessarily involved in the cytotoxicity of AMPA in this preparation. To test this, it was
necessary to examine the cytoprotective efficacy of both calcium-permeable AMPA
receptor antagonism as well as interfering with GluR2 trafficking. We repeated the
previous toxicity assays of stretch + NMDA followed one hour later by a 30 μM AMPA
challenge. Post-injury treatments included 20 μM Tat-QSAV, 20 μM Tat-AAAA, and
100 μM Naspm. Stretch + NMDA again resulted in a marked susceptibility to secondary
AMPA toxicity (23.3 ± 5.9% increase in cell death, n = 3 cultures, Figure 6B). However,
Tat-QSAV applied with stretch + NMDA afforded significant cytoprotection against
AMPA excitotoxicity 20 hours after injury [9.58 ± 2.9% increase in cell death, n = 4
cultures, p < 0.05, Figure 6B]. Naspm also demonstrated a trend towards cytoprotection
against cell death conferred by AMPA [1.78 ± 5.7% increase in cell death, n = 3 cultures,
p = 0.055, Figure 6B].
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Figure 17. Stretch + NMDA promotes calcium influx through calcium-permeable
AMPARs. A) Temporal schematic of calcium imaging experiments B) Fura PE3 data
over the entire recording period. Baseline ratios of neurons exposed to Stretch + NMDA
are significantly higher than those of control neurons. As well, after perfusion of 100 μM
AMPA and 50 μM CTZ the duration of excess cytosolic Ca2+ is prolonged. Selective
antagonism of GluR2-lacking AMPARs (100 μM Naspm) lowers peak AMPA-induced
Ca2+ and mitigates the prolonged elevation in intracellular calcium. C) Quantification of
baseline Fura ratios (340/380 nm). D) Quantification of Δt of peak calcium levels to
return to baseline E) Quantification of peak ratio normalized to baseline. 100 μM Naspm
reduces peak calcium. F) Integration of the calcium curves shown in (B) reveals a 2.11-
fold increase in the area under Stretch + NMDA curve relative to control neurons. There
are no error bars in this graph because these are the integrals of the mean calcium curves.
G) Representative Fura-PE3 micrographs of baseline (left column) and peak (right
column) emission in control neurons (top row), Stretch + NMDA (middle row) and
Stretch + NMDA + 100 μM Naspm (bottom row). Scale bars = 40 μm.
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Figure 17. Stretch + NMDA promotes calcium influx through calcium-permeable AMPARs.
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Tat-AAAA demonstrated no attenuation of AMPA-induced cell death (29.2 ± 3.9%
increase in cell death, n = 3 cultures, Figure 6B). Importantly, there was no significant
difference in cell death between groups at 1 hour after the insult. These results suggest
that a portion of the delayed (i.e., secondary) cell death that occurs in this model of
trauma could be prevented through preservation of surface GluR2 or antagonizing
GluR2-lacking AMPARs.
4.5 Hippocampal CA1 is hyperexcitable following fluid percussion trauma: Excitability is lowered with TAT-QSAV application To ascertain a measure of AMPA receptor phenotype in the injured whole animal,
we performed CA1 field recordings following Schaffer collateral stimulation, a well-
established glutamatergic synapse which we demonstrated to be an almost entirely
AMPA receptor mediated response after complete rundown following 6-cyano-7-
nitroquinoxaline-2,3-dione (CNQX) application (Figure 19B). We first measured the
gross amplitude of the CA1 AMPA-receptor mediated evoked population spike over a
range of 12 stimulation amplitudes (ranging from 10-120 μA). Statistically, we ran a
two-way repeated measure ANOVA, with independent variables of stimulation amplitude
and treatment (i.e., Ctrl, FPI, and FPI + 3 mg/kg Tat-QSAV I.V). Significant differences
were detected among our treatment groups (P = 0.002). To follow up the two-way
ANOVA and parse out where the differences lay, one way ANOVA followed by Student-
Neuman Keuls tests were performed to identify differences between groups at each
individual stimulation amplitude. Over a range of 10-80 μA, we observed that fluid
percussion trauma markedly increased the CA1 evoked response, (P < 0.05, Figure 19C).
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Figure 18. Inhibiting GluR2 endocytosis is neuroprotective. A) Top row: representative
propidium iodide fluorimetry 20 hours after exposure of cortical neurons to Stretch +
NMDA + AMPA, with or without the presence of polyamines or inhibitory peptides.
AMPA was applied for 1 hour, with or without peptide/polyamine treatment, at 1 hour
following Stretch + NMDA. Bottom row: brightfield images of the corresponding field
represented in top row. B) Quantification of normalized PI fluorimetry by plate scanning
at 1 hour and 20 hours after AMPA treatment. Scale bar 200 μm. # p < 0.05 vs control; *
p < 0.05 vs injured.
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Figure 18. Inhibiting GluR2 endocytosis is neuroprotective
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However, when FPI animals were treated intravenously with 3 mg/kg Tat-QSAV, the
evoked response was significantly lower across all stimulation amplitudes, suggesting
potentially the involvement of PICK1-dependent processes in mediating this synaptic
potentiation. At higher stimulation amplitudes (80-120 μA), a marked depression of the
population spike was maintained in Tat-QSAV treated animals (P < 0.05 for all
stimulation amplitudes), despite a lack of a significant difference between control and
injured animals over these treatment points (P > 0.05 for all). Notably, we were able to
achieve a similar potentiation of the CA1 population spike with exogenous PKC
activation, which was performed via application of 1 μM phorbol 12-myristate 13-acetate
(phorbol ester, PMA, Figure 19D). Collectively, these results suggest a partial
enhancement of CA1 population spike amplitude by FPI that could be attenuated by
interfering with PICK1-dependent protein interactions or mimicked by activation of
protein kinase C, two proteins which play a key role in the removal of surface GluR2
protein. Given that GluR2-lacking AMPA receptors have a higher single channel
conductance per receptor complex, and that Tat-QSAV reduced CA1 evoked responses,
we next hypothesized that perhaps AMPA receptors devoid of the GluR2 subunit were
contributing to the elevation of CA1 population spike amplitude.
4.6 Hippocampal CA1 Naspm sensitivity increases after FPI Occlusion of CA1 Naspm sensitivity is achieved through interference with GluR2 endocytosis.
Having observed CA1 hyperexcitability that was attenuated by PICK1 inhibition,
we next investigated whether a traumatically injured hippocampus demonstrated an
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Figure 19. Post-injury CA1 hyperexcitability is attenuated by Tat-QSAV treatment. A)
Schematic of recording procedure in sagittal hippocampal slices. Recording electrodes
were placed in the stratum pyramidale of area CA1, while stimulation occurred at the
axons of the schaffer collateral tracts originating in area CA3. DG = dentate gyrus. B)
CA1 population spike amplitude is nearly completely abolished during perfusion of the
slice with 20μM CNQX, an indication that this synapse is an appropriate measure of
AMPA receptor-mediated evoked responses. C) CA1 excitability is markedly increased
3-6 hours following fluid percussion injury, an effect that is attenuated by intravenous
treatment of animals with 3 mg/kg Tat-QSAV. The effect is particularly noticeable at
lower stimulation amplitudes (10-80 μA). D) Potentiation of the CA1 evoked response
can be achieved via perfusion with phorbol esters (PMA), exogenous activators of PKC,
which stimulate the endocytosis of GluR2.
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Figure 19. Post-injury CA1 hyperexcitability is attenuated by Tat-QSAV treatment
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increased expression of calcium permeable AMPARs, as these receptors have a higher
single channel conductance, and as has been discussed, contribute to progressive
excitotoxic cell death and dysfunction251,252,276,279,280,287,411. We found during recordings
from FPI rats that CA1 population spikes exhibited a Naspm-induced rundown to 58.9 ±
1.7% of baseline, a significantly greater inhibition than sham animals (78.9 ± 0.79%, p <
0.05, Figure 20A). This increased sensitivity of CA1 physiology to antagonists of
calcium-permeable AMPA receptors suggests that these receptors contribute more
significantly to synaptic transmission in injured animals relative to controls. However,
injecting animals intravenously with Tat-QSAV (3 mg/kg) following the traumatic injury
occluded Naspm-induced rundown of CA1 population spike amplitude (88.2 ± 5.6 %,
Figure 20B), providing evidence that GluR2 trafficking is integral in the expression of
calcium-permeable AMPARs. Notably, Naspm sensitivity was preserved in Tat-AAAA
injected animals (59.3 ± 8.3% of baseline (p < 0.01 vs sham and QSAV, p > 0.05 vs FPI,
Figure 20B). As a final positive control, we also treated animals (3 mg/kg) with a GluR2
c-terminal PICK1 binding peptide that has been used throughout the literature to interfere
with AMPA receptor trafficking, Tat-SVKI. This peptide similarly mimics a PICK1 PDZ
binding motif by replicating the GluR2 c-terminal PDZ ligand. Naspm-induced
population spike rundown was occluded (97.2 ± 14.1% of baseline, p < 0.05 vs FPI) in
Tat-SVKI treated animals in a similar fashion to those animals treated with Tat-QSAV
(Figure 20B), providing further evidence that PICK1-mediated GluR2 endocytosis was
involved in the post-traumatic expression of calcium-permeable AMPARs. Collectively,
our biochemical and electrophysiological data suggested that calcium-permeable AMPA
receptors were expressed via GluR2 endocytosis following whole animal trauma.
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Figure 20. CA1 hippocampal physiology is sensitive to antagonists of calcium-
permeable AMPA receptors after TBI. A) Naspm-induced rundown of CA1 population
spike amplitude was significantly greater in injured animals, supporting the in vitro
findings that these molecular modifications lead to incorporation of phenotypically
different channels. Representative traces illustrating rundown of population spike
amplitude during the recording period appear above the graph. B) Prevention of GluR2
endocytosis with Tat-QSAV or Tat-SVKI, both PICK1 binding peptides, significantly
reduces CA1 naspm sensitivity. Tat-AAAA was ineffective in occluding naspm
sensitivity. * p < 0.05. ** p < 0.05.
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Figure 20. CA1 hippocampal physiology is sensitive to antagonists of calcium-permeable AMPA receptors after TBI
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4.7 Occluding GluR2 endocytosis reduces apoptotic cell death: Post-traumatic DNA fragmentation is reduced by interfering with GluR2 trafficking Biochemically, our data suggested that the GluR2 subunit was internalized
following fluid percussion trauma. Electrophysiologically, we identified a contribution
for GluR2-lacking receptors to hippocampal physiology. However, our hypothesis was
that the expression of these receptors played a significant role in the susceptibility of
neurons to secondary injury following brain trauma. Thus, it was necessary to ultimately
examine the cytoprotective efficacy of Tat-QSAV, and thereby delineate whether the
aberrant trafficking of GluR2 has any cytotoxic implications.
We performed TUNEL staining of coronal brain slices at 24 hours following fluid
percussion injury, quantifying the prevalence of DNA fragmentation with a sampling of
approximately 3000 cortical cells per animal. TUNEL staining was accompanied by a
nuclear counter-stain for Hoescht 33342, allowing us to quantify data as the percentage of
cells identified as TUNEL positive (thereby normalizing the data to cell density).
Quantification of slices was performed completely blind. Following fluid percussion
trauma, 6.08 ± 1.49% of cells were identified as TUNEL positive (n = 6). However, there
was a marked reduction of TUNEL positive cells in slices obtained from animals treated
intravenously with Tat-QSAV (1.47 ± 0.6% TUNEL positive, n = 6 animals, P < 0.05), as
well as a reduction of chromatin condensation (Figure 21D, arrowheads), suggesting that
interference with PICK1-dependent protein interactions reduces cortical DNA
fragmentation following experimental TBI (quantification in Figure 21E).
One problem with the use of TUNEL staining is the tendency of the method to
identify DNA strand breaks that occur from cytotoxic processes other than apoptosis
(e.g., fragmentation produced by reactive oxygen species such as peroxynitrite). To
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confirm that the TUNEL staining was corroborated by other assays of apoptotic cell
death, we performed co-precipitation experiments with cytochrome c and apoptotic
peptidase activating factor 1 (APAF-1). The cyt-c-APAF-1 complex is recognized as an
important initiator of apoptotic cell death, which binds and cleaves procaspase-9,
releasing the mature and activated form of the cysteine protease. In turn, caspase-9
cleaves and activates the effector caspases 3 and 7, which carry out the execution phase
of programmed cell death. As evidenced in Figure 21F, TUNEL staining was
accompanied by an observable interaction between cytochrome-c and APAF-1, an
interaction that was only present in injured tissue and was confirmed by both positive and
negative controls (Figure 21F). Thus, this biochemical data provided further evidence
that apoptotic cell death indeed was occurring following FPI.
4.8 Summary
These data provide evidence for an increased contribution of GluR2-lacking
AMPA receptors to neuronal physiology following TBI. We observed in both our whole
cell patch clamp and CA1 field electrophysiological assays that the sensitivity of the
AMPA receptor response to a selective antagonist of GluR2-lacking receptors (Naspm)
was significantly increased. We also observed that AMPAR-mediated calcium influx
was augmented following trauma, and could be similarly attenuated by Naspm
application. Further evidence for the involvement of higher-conductance GluR2-lacking
receptors in neuronal signaling post-TBI comes from our observation that AMPAR
mEPSC amplitudes are increased, as is the basal excitability of hippocampal CA1, an
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electrophysiological response which we showed to be almost entirely AMPA receptor
mediated. To ascertain the potential involvement of GluR2 trafficking in the subsequent
expression of GluR2-lacking receptors, we treated both neurons and cultures with our
peptide inhibitor of the PICK1-PKC protein interaction. We found not only that this
peptide inhibitor occluded the expression of calcium-permeable receptors and dampened
AMPA receptor-mediated electrophysiological responses, but also that the compound,
when administered post-injury, provided cytoprotection against apoptotic cell death in
vivo. Collectively, our results provide evidence for a cascade of GluR2 endocytosis which
promotes the cytotoxic expression of calcium-permeable AMPA receptors.
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Figure 21. Perturbing GluR2 endocytosis affords cytoprotection from apoptosis at 24
hours following fluid percussion trauma. A) Sham tissue is non-reactive for TUNEL
staining, a marker of endonuclease-mediated DNA overhang initiated during
programmed cell death. Top panel, Hoescht 33342 nuclear stain, a non-specific marker of
cellular nuclei. B) Top row: TUNEL staining is markedly increased following fluid
percussion trauma. Bottom row: intravenously administered Tat-QSAV reduces the
prevalence of TUNEL positive cells. C) Contralateral tissue is non-reactive for TUNEL
staining in both treatment conditions. D) 40 x magnification of cortical cells revealing
co-localization of TUNEL positive neurons with condensed chromatin (arrowheads), two
hallmarks of the terminal stages of apoptosis. Nuclei of Tat-QSAV treated animals are
less condensed, and do not co-localize with TUNEL staining to the same extent. E)
Quantification of TUNEL positive neurons normalized to the total number of cells in the
sampling area. F) Co-precipitation experiments with pull-down of cytochrome c and
blotting for bound APAF-1 reveal a cyt-c-APAF1 complex only in injured tissue. Far left
lane: positive control of APAF-1 (whole cell lysate). Negative controls and sham tissue
do not demonstrate binding between cytochrome c and APAF-1. Cyt-c IgG appears only
in lanes where co-precipitation was performed.
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Figure 21. Perturbing GluR2 endocytosis affords cytoprotection from apoptosis at 24
hours following fluid percussion trauma.
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Chapter 5: Discussion, Limitations and Future Directions
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5.1 Preface
The specific aims of this thesis were directed at investigating the hypothesis that a
reduction of surface GluR2 protein contributes to neuronal vulnerability to secondary
injury following TBI, by increasing the population of calcium-permeable AMPA
receptors. In two experimental models, in vitro and in vivo TBI, we described molecular
and phenotypic alterations to AMPA receptor trafficking and physiology that had
profound effects on neuronal viability. At the cellular level, we found that the
endocytosis of surface GluR2 protein after trauma contributes to the expression of
GluR2-lacking AMPARs, and the susceptibility of neurons to excitotoxicity (see figure
22). Accordingly, our data employing the use of Tat peptides intended to disrupt GluR2
trafficking suggest that GluR2 internalization is an aberrant event occurring in
traumatized neurons that contributes to delayed neuronal death and calcium overload.
5.2 Corroborating studies
Consistent with the observations presented in this work, several other studies have
proposed that a reduction of surface GluR2 contributes to secondary injury and neuronal
death after CNS insult. Firstly, ischemic incorporation of GluR2-lacking AMPARs and
association of GluR2 with PICK1 was reported in cultured hippocampal neurons279. In
that study, internalization of GluR2 was associated with a similar polyamine-sensitive
increase in mEPSC amplitude. Over the course of our investigation (i.e., simultaneous to
our study), an independent lab investigated the mechanism of GluR2 internalization in
this ischemic model and found an identical cascade to what is reported in this thesis.
Indeed it was shown in mid 2009 that activation of NMDA receptors following ischemia
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leads to a PICK1-dependent switch in AMPA receptor subunit composition from GluR2-
containing to GluR2-lacking. Moreover, the investigation showed that peptides that
interfere with the GluR2 c-terminal PDZ interaction with PICK1 occlude the expression
of GluR2-lacking AMPARs and provide cytoprotection in hippocampal neurons exposed
to OGD375. This finding further supports our hypothesis that NMDA receptor activation
following TBI might lead to an identical reduction of surface GluR2 via the PDZ
interactions responsible for subunit trafficking.
Concomitant to the undertaking of our study, other experimental paradigms of
CNS injury reported aberrant GluR2 trafficking in conditions involving neuronal
hyperexcitability and calcium overload. GluR2 S880 phosphorylation, GluR1 S845
phosphorylation (discussed next in Future directions), and enhanced AMPAR mEPSCs
were reported during neonatal epilepsy, a condition also marked by neuronal
hyperexcitability276. Indeed that study suggested that the simultaneous removal of GluR2
coupled with the delivery of GluR1 was capable of remodeling the AMPAergic synapse
to become profoundly more calcium-permeable. Neuronal hyperexcitability in the spinal
dorsal horn also contributes to the pathophysiology of chronic pain, another condition
that has shown to involve aberrant NMDAR and PKC-dependent GluR2 trafficking. In a
study that employed an animal model of peripheral inflammation, it was demonstrated
that nociceptive hypersensitivity induces synaptic GluR2 internalization in dorsal horn
neurons, an effect mediated by serine 880 phosphorylation, and activation of PKC
downstream of NMDA receptors.
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Figure 22. Schematic demonstrating proposed signaling involved in post-traumatic
internalization of GluR2 and subsequent expression of GluR2-lacking AMPARs. A)
After TBI, intracellular calcium coming through the NMDA receptor activates PKCα via
its association with PSD-95 in the NMDAR complex. Activated PKCα binds PICK1, and
is trafficked to the membrane where it phosphorylates GluR2 at serine 880. GluR2
associates with PICK1 and is internalized from the cell surface, enhancing the expression
of GluR2-lacking AMPARs. B) Proposed mechanism of cytoprotection. Antagonism of
GluR2-lacking AMPA receptors with Naspm, or occluding the binding of PKCα with
PICK1 and/or PSD-95 via Tat-QSAV reduces GluR2 internalization and expression of
calcium-permeable AMPARs after TBI.
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Figure 22. Schematic demonstrating proposed signaling involved in post-traumatic internalization of GluR2 and subsequent expression of GluR2-lacking AMPARs.
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Perhaps most compelling however was the observed switch from GluR2-
containing AMPARs to GluR2-lacking AMPARs reported after a more severe
mechanical stretch injury - characterized by marked inward-rectification of the AMPA
receptor current-voltage relationship - 252, in a study also demonstrating neuroprotective
effects of Naspm antagonism. Collectively, this work has built a growing body of
evidence suggesting that the loss of surface GluR2 protein is an important contributing
factor to neuronal dysfunction and cell death in excitotoxic CNS disease. In order to
prevent the loss of surface GluR2, the intracellular mechanisms responsible for its
aberrant endocytosis need to be mapped out, a problem which this study begins to
address.
The finding that prevention of GluR2 endocytosis reduces secondary injury after
TBI is supported by many investigations that have recapitulated the result that injured
neurons are dramatically more susceptible to glutamatergic stimulation than healthy cells.
A number of cell culture models using mechanical injury devices have shown increased
excitotoxin lethality following trauma119,120,251,412. However, the more compelling
evidence comes from whole animal models. One such study used the fluid percussion
device followed by microdialysis of glutamate to investigate a possible co-operation
between trauma and excitatory amino acids in mediating neuronal damage after TBI. This
study was undertaken on the basis of the observations that cerebral glutamate levels
measured in patients by microdialysis (16-350 μM) are sufficient to kill neurons in
culture, but not in the intact brain of the normal rat413. The authors therefore sought to
identify a synergistic effect between excitatory amino acid–mediated damage and other
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posttrauma mechanisms. Following central FPI, the authors reported that glutamate
perfusion produced a lesion significantly larger than both FPI + mock CSF perfusion, and
glutamate perfusion alone. Furthermore, the lesion volume of the FPI + glutamate group
exceeded the summed mean volumes from the FPI + mock CSF, and glutamate alone
groups. This highlights a clear susceptibility of injured tissue to glutamate receptor
stimulation. Coupled with the observations reported by us and others that CA1
hippocampal glutamatergic transmission is significantly augmented following trauma,
this data supports the theory that trauma induces a post-synaptic modification of
glutamate receptor functioning, which might include the type of AMPA receptor
remodeling reported in this thesis.
5.3 Co-operation of Stretch + NMDA
In our in vitro model, we observed that 10 μM NMDA did not result in the
internalization or phosphorylation of GluR2 unless it was combined with stretch injury.
Notably, we also observed that while stretch + NMDA on its own was not immediately
cytotoxic, it imposed a marked vulnerability to secondary AMPA insult. There are a
number of possibilities to explain the cooperative effects of stretch injury and NMDA on
both AMPA receptor trafficking and neuronal susceptibility to secondary injury.
Mechanical trauma reduces the magnesium block of the NMDA receptor243 potentially
allowing a previously innocuous dose of NMDA to initiate substantially more calcium
influx in injured neurons vs controls. Indeed NMDA is markedly more lethal to stretched
neurons than uninjured cultures119 and initiates larger calcium transients245 after sublethal
stretch. These findings help in the understanding of how the two insults might cooperate
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in calcium-dependent PKC-activation and GluR2 phosphorylation. Also, mechanical
trauma elevates intracellular superoxide levels in cortical neurons119,120. Superoxide plays
an important role in PKC activation via thiol oxidation414, including the regulation of
kinase activity during LTP415,416 when PKC is active in the post-synaptic density and
plays a role in GluR2 removal268. It is possible that oxidative modification causes
preferential binding of PKC to various substrates, and it would be worthwhile to
investigate the hypothesis that superoxide is responsible for the post-injury PKCa-PSD95
association. In this scenario, PKCa – structurally connected to PSD-95 and embedded in
the NMDAR protein complex after stretch – would be primed for activation from
subsequent NMDAR stimulation.
5.4 Limitations of the current study
5.4.1 Non-specific Tat peptide interactions
It is important to recognize the possibility that occupying the PDZ-domains of
PICK1 and/or PSD-95 with Tat-QSAV might be cytoprotective in a more non-specific
fashion than inhibiting PKCα binding. The PDZ-domain of PICK1 interacts with at least
45 other known PDZ-ligands. Occupying this domain with a -QSAV peptide could
conceivably interfere with other PICK1 protein interactions. Further, there might
conceivably exist other intracellular PDZ targets of the –QSAV sequence present on our
peptide. Thus, while we demonstrate the successful perturbation of the PICK1-PKCa and
PSD-95-PKCa association, we cannot definitively exclude the possibility that the
cytoprotective effect of the compound is mediated elsewhere. Future work will include
knocking down the expression of PKCα and/or PICK1 and investigating if the
cytoprotective effects of the peptide are occluded.
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Prior to the knockdown experiments however, proteomics can be used to identify
the PDZ domains with which Tat-QSAV interacts. Studies have been carried out which
have employed cloning of the ~ 470 human PDZ proteins in the SMART database,
followed by fusion of the proteins to GST. By coating plates with anti-GST antibody,
these cloned PDZ proteins can be immobilized and probed with potential binding
partners. Incubation of individual wells with labeled Tat-QSAV would be a high-
throughput method of identifying the interacting partners of our peptide. Moreover,
immobilization of GST-PICK1 followed by probing with purified PKCα in the presence
of varying concentrations (e.g., 0.001-100 μM) of our tat peptide inhibitor could be done
to identify the IC50 of the peptide. This would provide valuable information on the
intracellular concentration of the peptide necessary to achieve sufficient inhibition of the
protein-protein interaction.
5.4.2 Non-specific effects of Tat peptide transduction In our whole cell patch clamp electrophysiological assays, we observed a
significant non-specific effect of tat peptide transduction on miniature AMPA receptor-
mediated EPSCs. Indeed incubation of our injured cultures with Tat-AAAA, a non-
functional negative control peptide significantly reduced the amplitude of injured events.
Though our active PDZ-ligand (QSAV) induced a further and significant reduction from
our inactive control (providing a role for GluR2 trafficking in the increased event
amplitude), the mechanism by which our Tat-AAAA peptide decreased AMPA-mediated
mEPSCs is at present unknown. The simplest explanation is that the peptide may have
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non-specifically blocked AMPA channels. However, based on current insight into tat
peptide transduction, there are other likely explanations.
Tat peptides, as discussed in the introduction, enter cells through lipid-raft
dependent fluid phase macropinocytosis. The interaction of the cationic Tat PTD with
lipid rafts – enriched in cholesterol and anionic sphingolipids – is an electrostatic
interaction followed by endocytosis of the raft along with the extracellular peptide
cargoes417. The link between this mechanism and its effect on the functioning of AMPA
receptors can be made via the studies describing the effects of lipid raft depletion on
AMPA receptor surface expression and electrophysiology. It is now known that AMPA
receptors are associated in detergent-resistant membranes in dendritic spines with the
cholesterol and sphingolipids present on lipid rafts, and that raft depletion reduces the
density of AMPA receptors found on dendritic spines418,419. It is therefore conceivable
that the macropinocytosis following Tat peptide transduction might be accompanied by a
loss of AMPA receptor surface expression, translating unsurprisingly to a decrease in the
amplitude of AMPA receptor mediated events. To parse this out, an appropriate further
experiment would include bath application of tat-peptides to our cortical cultures,
followed by immunocytochemical GluR1 N-terminal surface labeling to examine the
density of AMPA receptors following Tat-mediated protein transduction.
5.4.3 Co-precipitation: What does it mean? Interestingly, in the present study, the reduction of surface GluR2 and subsequent
AMPA receptor potentiation was NMDA receptor dependent. The simplest explanation
is that the NMDAR dependence arises because of the structural link between PKCα and
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PSD-95. The -QSAV sequence on PKCα’s extreme c-terminus is a type I PDZ ligand
with the potential to form a stable interaction with two of PSD-95’s PDZ domains333.
However, our co-precipitation data does not rule out the possibility that PKCα is
indirectly associated with PSD-95, via a binding partner that is able to bind both the
kinase’s PDZ-ligand and one of PSD-95’s PDZ domains.
Indeed this problem is the hallmark shortcoming of using co-immunoprecipitation
as an assay method. While co-immunoprecipitation can demonstrate that two proteins are
found in the same cellular complex, the assay does not prove that the two proteins are
physically touching one another, that is, directly associated. Since all of our co-
immunoprecipitation experiments were performed using cell lysates, it is possible that
two co-immunoprecipitating proteins in our experiments were linked together by a third
protein that acts as a scaffold. This possibility is more likely in certain scenarios than
others. The PKC-PICK1 as well as GluR2-PICK1 protein interactions have been
extensively defined through yeast-two hybrid screening and direct recombinant protein
pull-down assays. However, an appropriate further experiment to fully validate the
interaction between PSD-95 and PKCα that we report would involve purification of both
proteins, followed by dot blotting. Indeed immobilization of purified GST-labeled PSD-
95 onto a nitrocellulose membrane, followed by incubation with myc-tagged purified
PKCα would definitively identify that the proteins are capable of a direct interaction. It
was our intention to perform this experiment, however, a lack of expertise in protein
cloning (and therefore an inability to secure purified PSD-95 protein) in our laboratory
prevented us from doing so. Nonetheless, this assay is necessary to verify that the kinase
is a direct PDZ binding partner of PSD-95.
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5.4.4 TNFα-induced AMPA receptor trafficking: An alternative mechanism of calcium-permeable AMPA receptor expression An alternative mechanism for the neuronal phenotype observed in this work (i.e.,
an increased expression of calcium-permeable AMPA receptors) involves a pro-
inflammatory cytokine that is central to the inflammatory response that occurs after
cerebral trauma. Tumor necrosis factor alpha (TNFα), released from neighbouring
glial cells during CNS inflammation, was recently shown to increase AMPA
receptor surface expression ex vivo, and specifically, to increase the expression of
calcium-permeable AMPA receptors420. Indeed brain slices incubated with TNFα
exhibit a marked increase in naspm sensitivity during whole-cell AMPAR patch
clamp, and also exhibit marked elevations in AMPAR-derived free calcium421,422.
The evidence that this process might contribute to excitotoxicity following CNS
trauma is compelling. For example, co-injection of TNFα with low doses of kainic
acid produces marked neuronal death in vivo that far exceeds injection of the
glutamate agonist alone423. Secondly, it was recently shown during spinal cord
injury that TNFα induces the surface trafficking of GluR2-lacking AMPA
receptors, thereby imparting a vulnerability to secondary excitotoxic injury
mediated at AMPAergic synapses. Interestingly, in this paradigm, a soluble TNFα
receptor mitigated these effects, providing convincing evidence that this cytokine
can remodel the glutamatergic synapse during CNS injury to include calcium-
permeable AMPA receptors377.
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In our experimental paradigm, there is likely to be a significant increase in
parenchymal TNFα levels. It has been shown that fluid percussion trauma induces
marked elevations in TNFα levels424-427, and it is also known that injured neurons will
release TNFα themselves428, something which may have occurred during our in vitro
stretch injury protocol. Thus it is possible that this mechanism contributed to the
increased naspm sensitivity that we observed after trauma. However, our observations
that peptide-mediated PICK1 inhibition attenuated the expression of calcium-permeable
receptors suggest that if TNFα-mediated AMPA receptor trafficking occurs after
experimental TBI, it is likely in parallel to the effects that we observed.
5.5 Future Directions The data presented in this thesis raise some compelling questions that remain to
be answered. For example, we have yet to identify the intracellular events that follow the
internalization of GluR2 protein (e.g., protein degradation or recycling). We have also not
investigated the trafficking of GluR1, another AMPA receptor subunit whose surface
delivery, rather than internalization, might mimic the phenotype we observed in many of
our experiments. Moreover, it is unknown at present what impact the application of
GluR2 endocytotic inhibitory peptides has on physiologic synaptic plasticity, given that
the PICK1-dependent expression of GluR2-lacking AMPARs is a mechanism critical to
the development of LTP. In the interest of pursuing these questions, we have collected
preliminary data that begins to address these issues.
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5.5.1 Total GluR2 levels are reduced by 24 hours following trauma One intriguing difference between our work and other work that has studied the
influence of GluR2 expression on cell survival during CNS injury is that our work was
focused on local protein trafficking, as opposed to global regulation of protein
transcription. Indeed the work studying REST-dependent epigenetic silencing of GluR2
expression in cerebral ischemia identified that by silencing RNA transcription, total
GluR2 protein levels were reduced by 24-48 hours following global ischemia. Whether or
not our acute experiments highlighting protein endocytosis (performed within hours of
the injury) translated to a down-regulation of total protein at a more delayed time point is
largely unknown. Accordingly, we investigated total GluR2 expression at 24 hours
following FPI in a small sample of four animals. Notably, we observed that total GluR2
expression was down-regulated by 24 hours after FPI (67.3 ± 11.1% of control in
ipsilateral cortex, 54.4 ± 7.2% in contralateral cortex, n = 4, P < 0.01, figure 23), a
finding that has been reported by other investigators in both experimental TBI and spinal
cord injury429-431, particularly in apoptotic neurons101.
It remains to be seen if the early endocytosis that we report is the mechanism
responsible for the delayed down-regulation of GluR2, although there is evidence that
GluR2 endocytosis leads to lysosomal degradation, and subsequently, the expression of
calcium permeable AMPA receptors. Indeed one particularly relevant investigation
reported that the endosomal protein NEEP21 associates with the PDZ scaffolding
molecule GRIP1 and GluR2; and that when the NEEP21-GRIP interaction is lost, GluR2
surface expression decreases, causing GluR2 accumulation in early endosomes and
lysosomes, and inward rectification of AMPAR EPSCs (a property of GluR2-lacking
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AMPARs)432. Thus, it is conceivable that PICK1 targets internalized GluR2 to acid
hydrolase-filled lysosomes, where a reduction of total protein would occur. Appropriate
experiments to parse out whether post-traumatic GluR2 endocytosis leads to protein loss
might include a) examination of total GluR2 levels with an without Tat-QSAV, to
examine if interfering with GluR2 trafficking influences total protein expression at 24
hours, and b) to co-precipitate GluR2 with known late endosomal or lysosomal proteins
(e.g., RAB7 and RAB9 and mannose 6-phosphate receptors) after TBI, to examine if this
is the mechanism of protein loss.
5.5.2 GluR1 trafficking may increase following trauma: NO-mediated GluR1 serine 845 phosphorylation occurs following traumatic injury
An alternative mechanism of an increased population of GluR2-lacking AMPA
receptors might be through increased exocytotic delivery of GluR1, thereby allowing for
the incorporation of GluR1 homomeric channels. One way through which GluR1 delivery
occurs is through nitric oxide-mediated phosphorylation of a critical GluR1 serine reside
(845), that allows for binding of the subunit with cyclic GMP-dependent kinase II and
delivery to the plasma membrane433. Indeed, mild mechanical trauma coupled with
NMDA receptor activation produces high levels of nitric oxide (NO) through the NR2b-
PSD-95-nNOS cascade in cortical neurons119. Accordingly, we performed western blots
for GluR1 phosphorylation at S845. Following stretch + NMDA, phosphorylated GluR1
increased to 302 ± 47.6% of control levels (p < 0.05, Figure 24A). Notably, cells treated
with an NR2b antagonist did not exhibit a significant increase in phosphorylated GluR1
relative to control cultures (p = 0.15, Figure 23A), suggesting the mechanism of this
phosphorylation was NR2b-dependent, likely because of the structural scaffold between
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Figure 23. Total GluR2 protein levels are reduced at 24 hours following FPI. A) Western
blot of total GluR2 protein in ipsilateral and contralateral cortex. ERK 1,2 was used as a
loading control. B) Quantification of total GluR2 protein levels normalized to sham
animals.
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Figure 23. Total GluR2 protein levels are reduced at 24 hours following FPI.
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NR2B and nNOS activation. We also probed simultaneously for total GluR1 levels, of
which there was no significant difference between treatments (p = 0.71, Figure 23A).
We also identified another nitric oxide dependent modification of the AMPA
receptor through whole-cell electrophysiology. We observed that injured cortical neurons
displayed markedly increased activity (qualitatively) relative to control neurons when
sodium-free extracellular solution was perfused (Figure 24B). Given that the dominant
cation in these experiments was choline, the most likely-explanation for this activity
would be calcium-mediated currents. Notably, when we inhibited nitric oxide synthase
activity with L-NG-Nitroarginine methyl ester (L-Name), we were unable to replicate the
sodium-free firing of the neurons (Figure 24B). This preliminary western blotting and
electrophysiological data suggest that perhaps nitric oxide dependent delivery of GluR1
accompanies GluR2 trafficking in the expression of calcium-permeable AMPA receptors.
5.5.3 Tat-QSAV treatment does not occlude induced synaptic plasticity: Hippocampal LTP is preserved with PICK1 inhibition
Our initial hypotheses were crafted based on the physiological role of PICK1 in
remodeling the AMPAergic synapse during synaptic plasticity. Indeed a major cellular
mechanism underlying activity-dependent plasticity of glutamatergic transmission is the
regulated trafficking of AMPARs, particularly the trafficking of GluR2. As discussed in
the introduction, PICK1-mediated control of GluR2 surface levels is a key mechanism in
the induction of LTP in hippocampal CA1. The removal of GluR2, and therefore the
incorporation of GluR2-lacking, higher conductance channels, is thought to underlie a
lasting increase in synaptic efficacy during the induction of learning and memory. Thus
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Figure 24. Stretch + NMDA increases NO-dependent GluR1 S845 phosphorylation. A)
GluR1 S845 increases after Stretch + NMDA and is mitigated by NR2b antagonism
(quantification on right). B) Removal of extracellular sodium abolishes AMPAR-
mediated mEPSCs in cortical neurons held at -70 mV. C) AMPAR mEPSCs persist in the
absence of extracellular sodium in cortical neurons 1 hour following Stretch + NMDA.
D) Addition of 100 μM L-NAME to inhibit nNOS and GluR1 phosphorylation largely
attenuates the sodium-free firing in injured cortical neurons.
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Figure 24. Stretch + NMDA increases GluR1 S845 phosphorylation
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we had a final interest in examining the impact of Tat-QSAV on the induction of LTP in
the hippocampus, to investigate the physiological significance of inhibiting GluR2
trafficking. We further sought to identify the impact of FPI, an injury known to induce
GluR2 internalization, on the induction of LTP.
Given that we observed GluR2 internalization and associated hyperexcitability in
the hippocampus, we conjectured that perhaps LTP in this area would be occluded.
Indeed it is known that LTP is impaired in the hippocampus after FPI, and we thought
that an increase in basal excitability due to remodeling of the AMPAergic response might
underlie this impairment. However, contrary to this hypothesis, we observed that there
was no impairment in LTP induction in injured animals. In slices from control animals (n
= 4), population spike amplitude was maintained at 155.3 ± 13.2% of baseline (30th epoch
used for analysis). Following FPI (n = 7, 3-6 hours after the injury), population spike
amplitude increased to 151.1 ± 13.1% of baseline, an insignificant difference from
uninjured animals (figure 25, P > 0.05). Similarly, intravenous injection of Tat-QSAV (3
mg/kg, n = 4) was without effect on hippocampal population spike LTP, with baseline
levels increasing to 153.1 ± 18.2%. Thus, these results suggest one of two possibilities: 1)
that GluR2 trafficking is not involved in the induction of LTP in hippocampal CA1, or 2)
that following injury, mechanisms outside of AMPA receptor trafficking are responsible
for LTP induction.
5.5.4 – Does inhibition of the PICK1 PDZ domain represent a future anti-excitotoxic therapy? The concept that PICK1-mediated protein interactions might underlie
neurological dysfunction in a number of disorders is beginning to gain considerable
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Figure 25. Hippocampal LTP is unaffected following FPI, and uninfluenced by Tat-
QSAV treatment. 100 Hz theta burst stimulation of the schaffer collateral tract was
applied to induce LTP of the population spike in stratum pyramidale of the CA1 cell
layer. Top panel: LTP of the population spike amplitude is successfully induced in both
control and injured animals. Bottom panel: Treatment of injured animals with Tat-QSAV
is without effect on CA1 LTP. Slices were stimulated 3-6 hours following FPI.
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Figure 25. Hippocampal LTP is unaffected following FPI, and uninfluenced by Tat-QSAV treatment.
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attention in neurobiology literature. In general, PICK1 serves as an attractive molecular
target because of its nature as a PDZ domain, which has been identified as a putative drug
target across a variety of different diseases. For example, blocking the PDZ interaction
between the NMDA receptor and PSD-95 with membrane-permeable peptides results in
selective inhibition of neuronal nitric oxide synthase (nNOS) activation and a dramatic
reduction of ischemic injury following experimental stroke334. In cancer, recent evidence
suggests that blocking the PDZ domains of Na+/H+ exchanger regulatory factor 1
(NHERF-1), dishevelled, or AF-6 might have tumor suppressing potential434. Finally,
our data here is the first to show that inhibition of the PICK1 PDZ domain reduces cell
death following CNS injury involving excitotoxicity.
Inhibition of the PICK1 PDZ domain has evolved from a conceptual idea to a
reality in recent months based largely on the emerging data reporting that PICK1-
medaited protein interactions contribute to neuropathic pain, excitotoxicity, and drug
addiction272,435. At the forefront of this effort is an investigation that screened
approximately 44,000 compounds as small-molecule inhibitors of the PICK1 PDZ
domain436. Remarkably, a non-peptide small molecular inhibitor of the PICK1 PDZ
domain was identified (known as FSC231) which has an affinity for the domain similar to
that of the endogenous peptide ligand (Ki ~10 μM). Physiologically, FRET and
coimmunoprecipitation experiments demonstrated that FSC231 crossed the plasma
membrane and inhibited the interaction between GluR2 and PICK1 in cultured neurons.
Moreover, FSC231 interfered with GluR2 trafficking, which is consistent with inhibiting
PICK1’s involvement in GluR2 endocytosis. Finally, FSC231 inhibited both LTD and
LTP expression in CA1 hippocampal neurons, consistent with inhibition of PICK1’s
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bidirectional effect on synaptic plasticity. The work in this thesis has laid the foundation
for the evaluation of FSC231 as a putative therapeutic against secondary cell death after
TBI.
5.6 Significance of Findings The data presented in this thesis contribute to a novel understanding of the
neuronal mechanisms responsible for excitotoxic cell death following traumatic injury to
the brain. Much of the current literature on glutamate receptor-mediated cellular injury
following trauma highlights increased extracellular glutamate as the initiating event in
excitotoxicity; however, our data introduces the possibility that trauma-induced post-
synaptic receptor modification can impart lethality upon otherwise innocuous glutamate
levels. As previously discussed, these data are corroborated by a number of investigations
describing similar trafficking of the GluR2 subunit under pathological conditions, and
provide a plausible mechanism responsible for the previous observations detailing the
cytotoxicity of physiological glutamate in traumatized neurons.
In addition to presenting a different conceptual understanding of excitotoxicity,
these data also provide a mechanism through which these changes can occur. Our
peptide-mediated interventional approach has highlighted PICK1 as the major contributor
to post-traumatic GluR2 endocytosis, and our data has further elucidated a potential
NMDA receptor-dependent mechanism through which GluR2 internalization is initiated.
Hopefully, this will translate in the future to a more targeted therapeutic approach to
excitotoxicity that circumvents the shortcomings and potential non-specific effects of
global glutamate receptor antagonism after TBI. The development of FSC231 as a small
molecule inhibitor of the PICK1 PDZ domain is an example of such efforts, and as stated
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by the authors of that study, was inspired in part by the data presented in this thesis and
its associated publications.
5.7 Conclusions
1) Neuronal trauma confers the endocytosis of the AMPA receptor GluR2 subunit,
evidenced by subunit phosphorylation, internalization, and a physical association
with its major trafficking proteins at early time-points after traumatic injury in
vivo and in vitro.
2) The trafficking of GluR2 protein increases the expression of calcium-permeable
AMPA receptors, evidenced through whole cell and field electrophysiology and
imaging of cellular calcium dynamics. Importantly, perturbation of GluR2
endocytosis reduces the expression of calcium-permeable AMPA receptors.
3) Interruption of GluR2 trafficking confers cytoprotection against excitotoxic injury
in two experimental paradigms -- in vitro stretch injury, where PICK1-binding
peptides protected against AMPA toxicity, and fluid percussion trauma, where
these same peptides reduced apoptotic cell death 24 hours after trauma.
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Reflective Appendix
This brief appendix was written in the days following the final oral defense of the
work, and reflects some of the more compelling and clinically relevant issues that were
raised during that meeting.
Relevance of GluR2 endocytosis to white matter injury:
Over the course of the discussion, it was postulated that the mechanisms
described in this thesis might also contribute to white matter injury, a prominent
pathophysiological feature underlying severe functional impairment post TBI. This
connection was made based on certain work demonstrating a marked susceptibility of
oligodendrocyte cell cultures to AMPA receptor-mediated neuronal injury. Indeed if a
post-traumatic modification of the GluR2 content occurred in oligos in a fashion similar
to what was seen in our neuronal population, this might lead to eventual oligo cell injury,
axonal demylenation, and a withdrawn trophic support for neurons in our whole animal
preparation. Notably, McDonald et al., (1998) showed in their Nature Medicine paper
that rodent oligodendrocytes are highly susceptible to AMPAR-mediated excitotoxicity,
both in culture and following stereotaxic injection of AMPAR agonists. This is a
compelling finding demonstrating the sensitivity of a non-neuronal cell type to AMPA-
mediated cell death with immediate relevance to axonal viability.
However, a follow up study examining the clinical applicability of this
phenomenon described the salient observation that rodent and human oligodendrocytes
differ vastly in their levels of AMPA receptor expression, leading to a cautionary
interpretation of McDonald et al.’s findings. In 2004, Wosik et al demonstrated that in
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fact human oligos express low levels of ionotropic glutamate receptors in vitro and are
resistant to high and sustained doses of AMPA/Kainate, even in the presence of reduced
receptor desensitization, which would exacerbate stimulation of the receptor. In the same
investigation, the group performed identical experiments in rodent oligos, demonstrating
a marked vulnerability to identical doses of the AMPAR activators. This observation
raises important questions about the clinical relevance of the pre-clinical work supporting
AMPAR-mediated cell death in oligos as a critical mechanism of white matter injury in
CNS disease.
Model descriptors: Why the stretch injury is described as an impulse:
Another relevant concept mentioned during the final defense was an inquiry into
our decision to describe the stretch model in terms of the impulse (J) inflicted on the
neurons. We chose to offer an approximation of the impulse experienced by the neurons
to help in the replication of the model by others. Because impulse is equal to FΔt, and
most models have strict control over the duration of injury (ie. it is relatively easy to keep
this variable constant from lab to lab because most systems have control over the duration
of valve opening), the largest variable in this equation would be the force, which we
thought easier to standardize than other variables, such as pressure (P) strain (e) or the
stretch ratio (λ). Force, being equal to pressure·area, can be calculated by labs lacking the
necessary equipment to measure strain/stretch. For example, our lab lacks a high-speed
camera necessary to make accurate strain measurements experienced by the cultures (this
would require measurement of axon length before and during the stretch injury). Thus we
have found it difficult to recapitulate the models which describe neurons as having
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undergone a certain level of strain (eg., 130% of initial axon length). Though this appears
to be the standard in the literature, we imagine that other labs have similar difficulties in
designing a system to replicate a specific level of strain.
In the thesis and its publications, we also described the pressure exerted on the
cells for all experiments (i.e., between 2.5 and 2.9 psi). However, in the literature this can
be confusing. The initial papers characterizing the stretch injury model described a dose
response of pressure-cell death that ranged over pressures from 10-70 psi (Ellis et al.,
1995). However, these were the regulator pressure readings, not the pressure measured
by the transducer following rebound of the silastic membrane. Also, because not all labs
have tissue culture wells of the same size (ie., 35 mm as in our study), the force exerted
on the neurons will change if the pressure is the only variable that is standardized. Indeed
we have found this to be the case with our fluid percussion injury device. By
standardizing force (and time) labs can adjust the pressure exerted on the cells given a
certain well size, such that the force (pressure x area) is constant (and therefore impulse is
too, if time is also a constant).
It has been reported that an internal chamber pressure during stretch injury from
between 5-7 psi correlates to a strain on axons of 0.58-0.77, or 58-77% beyond its initial
length (Smith et al, JNeurosci, 1999). Given the linear relationship between pressure and
tissue deformation described in this model (Ellis et al, 1995), we can approximate that
our tissue strain would measure close to half of the lower end of this approximation, or
approximately 30%. Notably, this level of strain for a mild injury model is highly
comparable to other labs measurements of strain during mild injury (Arundine et al.,
2004, Lau et al., 2006, JNeurosci). The next best approximation we can make with
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respect to comparing our injury with other labs because of this shortcoming in measuring
tissue strain is with biological/biochemical outcome. Our model is sub-lethal, and does
not appear to alter membrane integrity. These are endpoints that we used to compare to
other models in the literature and establish the differences between mild/moderate/severe.
We believe that impulse offers some approximation of the in vivo situation. As
mentioned in the thesis on page 24, the forces that result in this tensile elongation during
TBI are thought to occur in 50 ms, the duration of stretch applied in our model (and
accounted for in our calculation of impulse). The situation becomes more complicated
when looking at the force applied to the neurons and how this compares to the intact
brain. One shortcoming of the model is that it is two dimensional, and thus difficult to
approximate how this type of stretching corresponds to a three dimensional environment
found in the intact brain. In terms of biological outcome, we believe our model is
analogous to an in vivo mild trauma based on the following: 1) there is no cell death after
the injury, 2) there are no observable changes to membrane permeability, 3) there is no
evidence of axotomy, and 4) there is no accumulation of cytoskeletal swellings. Indeed in
a moderate injury, these are salient pathophysiological features of more severe axonal
injury models and accordingly, more severe in vivo models and clinical TBI. Further we
believe that what does occur in this model is analogous to a mild injury in vivo, as we
observed GluR2 phosphorylation in both a mild FPI and our stretch model, as well as
electrophysiological hyperexcitability.
191
Can we be sure that NR2B containing NMDARs are extrasynaptic?
In this thesis, we found that GluR2 internalization was an NR2B-dependent
phenomenon. There is a plethora of literature suggesting that NR2B is primarily an
extrasynaptic protein. Indeed early papers described a reduction of ifenprodil sensitivity
(an indication of NR2B subunit expression) of synaptic activity as neurons developed in
culture, promoting the hypothesis that the proportion of synaptic NR2B was lessened in
favor of NR2A expression, and that NR2B containing receptors are extrasynaptic.
However, experiments in the last 5 years have shown that in the presence of MK-801, an
antagonist of synaptic NMDARs, the eletrophysiological response of NMDARs is not
abolished completely by ifenprodil, suggesting the presence of NR2A subunits at
extrasynaptic sites (see for example Thomas et al., 2006). We did not perform any
experiments examining the distribution of these subunits, and thus it cannot be
definitively concluded that the NR2B-containing receptors mediating GluR2
phosphorylation are exclusively extrasynaptic. Future experiments will include parsing
this out in greater detail.
How else might we examine the impact of non-specific Tat peptide transduction on AMPAR mEPSCs?
One of the findings that were slightly problematic in this thesis was that Tat-
AAAA dampened AMPAR-mediated events in a non-specific fashion. We thought it
pertinent to address this point in slightly greater detail, and to discuss experimental
approaches that might help us further understand this observation. One way to identify if
the peptide was non-specifically blocking channels or if AMPARs were removed from
the surface following peptide transduction would be to mutate the Tat sequence to a non-
192
functional moiety that contains an equal number of positive charges. In this scenario, one
would need to confirm that the peptide was not being taken up into cells by similarly
tagging it with dansyl chloride. If the neurons stained negatively for the dansyl but
exhibited blockade of AMPAR mediated currents, this would suggest that the non-
specific antagonistic action of the peptide (and therefore any peptide) was extracellular
(ie fitting into the channel pore, blocking the binding site, allosteric modulation etc).
However, if the currents were unaffected, this would suggest the peptide needs to be
intracellular to exert its effects and would support a role for endocytosis in the
antagonism of AMPAR mEPSCs.
One might also stimulate macropinocytosis of other molecules (e.g., eosinophil
cationic proteins) to see if there is a similar depression of AMPAR-mediated events. If
so, one could suggest that endocytosis of any cargo perturbs the surface expression of
AMPARs. One might also apply the tat peptide in the presence of inhibitors of
endocytosis (cyclodextran or chlorpromazine) to ensure that this process is critical to the
blockade.
Final Thoughts:
One last consideration in this thesis is that of the relevance of GluR2 endocytosis
in mediating secondary neuronal cell injury after TBI in the context of all other sequelae
present in traumatized brain tissue. In TBI research, it is frequently difficult to contribute
more than a minor “piece of the puzzle” to the vast array of knowledge surrounding
mechanisms of cell death after trauma. Thus any one observation can seem diminished,
as it will likely only address a minor contributor to cell injury. Whether the effects that
193
we observed are a dominant contributor to post-TBI neuronal injury or dysfunction is
unknown, particularly in the clinical context. However, the data supporting a
cytoprotective role for PICK1 inhibition combined with the corroborating studies that
were undertaken at the same time as ours, suggest that this is unlikely to be an
epiphenomenon without relevance to neuronal survival. Thus, we think further pursuit of
this mechanism is warranted.
194
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