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The Effects of Oxygen Glucose Deprivation and TRPM7 Activity on Slingshot Phosphatase and P-21 Activated Kinase
Activity
by
Ervis Kola
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Cell and Systems Biology
University of Toronto
© Copyright by Ervis Kola (2013)
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The Effects of Oxygen Glucose Deprivation and TRPM7 Activity on Slingshot Phosphatase and P-21 Activated Kinase Activity
Ervis Kola
Master of Science
Department of Cell and Systems Biology
University of Toronto
2013
Abstract
Transient Receptor Potential Melastatin 7 (TRPM7) is a ubiquitously expressed divalent cation
channel implicated as a key regulator of neuronal cell death in stroke. Our research group has
previously shown that TRPM7 dependent cytoskeletal regulation particularly via cofilin mediates
neuronal death in oxygen glucose deprivation (in vitro stroke model). LIMK1 phosphorylation
was also shown to decrease downstream of TRPM7 activation during anoxia. In the present
study we investigated the effects of TRPM7 activation during anoxia, on three regulators of
LIMK and cofilin; Rho-associated kinase 2 (ROCK2), P-21 activated kinase 3 (PAK3) and
Slingshot family phosphatase 1 (SSH1). Our findings suggest that PAK3 activity is
downregulated during OGD through TRPM7 independent mechanisms. However, SSH1 activity
appears to be regulated downstream of TRPM7 in a manner that is consistent with LIMK and
cofilin regulation. Overall, our work suggests that SSH1 is a new link between anoxia-induced
TRPM7activity and cofilin hyperactivation.
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Acknowledgments
I would firstly like to thank my graduate supervisor Dr. Michelle Aarts for giving me the
opportunity to pursue a Master’s Degree at the University of Toronto Scarborough. Dr. Aarts
provided a comfortable and supportive environment and for this I am very grateful. I would also
like to thank my committee members Dr. Zhengping Jia and Dr. Rongmin Zhao for their
constructive criticism and advice throughout my graduate degree.
I am very grateful for current and past members of the Aarts lab for their support and technical
advice including Russel Bent, Colin Seepersad, Suhail Asrar, Jonathan Chan, Nagmeh Lesani,
Anjali Anne Ajit, and Kareem Younis. Special thanks go to Melanie Ratnam, who has been an
amazing friend and source of advice and encouragement especially during challenging times, I
will miss you dearly. I am also indebt to everyone in the surrounding research groups for their
support, conversation and technical expertise. Thanks go to Kristin Lizal, Darren Gigliozzi, Chris
Young-Kee, Sherri Thiele, the entire Brown, Terebiznik and Harrison lab. Finally I would like to
thank my family and my wonderful wife, Orsida Luzati-Kola, for the love and support.
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Table of Contents
Acknowledgments..........................................................................................................................iii
Table of contents.............................................................................................................................iv
Lists of tables................................................................................................................................viii
List of figures................................................................................................................................viii
List of Abbreviations...................................................................................................................ix-x
Chapter 1 – Introduction..................................................................................................................1
1.1 Transient Receptor Potential Channels.....................................................................1 - 5
1.2 TRPM subfamily......................................................................................................6 - 7
1.2.1 TRPM1...............................................................................................................8
1.2.2 TRPM2.........................................................................................................8 - 9
1.2.3 TRPM3.......................................................................................................9 - 10
1.2.4 TRPM4/TRPM5.........................................................................................10-11
1.2.5 TRPM6.............................................................................................................11
1.2.6 TRPM8.............................................................................................................12
1.3 Transient Receptor Melastatin 7 (TRPM7)...........................................................12- 13
1.3.1 TRPM7 structure......................................................................................13 - 15
1.3.2 TRPM7 in divalent cation homeostasis....................................................17 - 19
1.3.3 TRPM7 in ischemic stroke.......................................................................19 - 21
1.3.4 TRPM7 and cytoskeleton.........................................................................22 - 23
1.4 Cofilin – the actin depolymerizing factor.............................................................25 - 26
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1.4.1 Cofilin structure and expression...............................................................26 - 27
1.4.2 Cofilin and the regulation of actin dynamics............................................28 - 30
1.4.3 Regulation of cofilin activity....................................................................30 - 31
1.4.4 Cofilin in neurodegeneration....................................................................31 - 33
1.5 Rho associated kinase (ROCK)............................................................................34 - 35
1.5.1 ROCK protein signalling .........................................................................35 - 36
1.5.2 ROCK protein function............................................................................36 - 37
1.6 P-21 activated kinase (PAK)........................................................................................37
1.6.1 Structure and expression of P-21 activated kinases (PAKs)....................37 - 38
1.6.2 Regulation of PAK activity......................................................................38 - 39
1.6.3 PAK protein function...............................................................................39 – 40
1.7 Slingshot phosphatase (SSH).......................................................................................41
1.7.1 Slingshot phosphatase structure...............................................................41 – 42
1.7.2 Regulation of SSH1 activity.....................................................................42 - 43
1.7.3 SSH1 function...........................................................................................43 - 44
Chapter 2 – Objectives and Hypothesis..................................................................................45 - 46
Chapter 3 – Materials and Methods...............................................................................................47
3.1 Antibodies....................................................................................................................47
3.2 Rat Primary Neuronal Culture..............................................................................48 - 49
3.3 In vitro Stroke Model –Oxygen Glucose Deprivation (OGD).............................49 - 50
3.4 Protein extraction.........................................................................................................50
3.5 DC-Lowry Protein Assay.....................................................................................50 - 51
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3.6 SDS- Polyacrylamide Gel Electrophoresis (SDS-PAGE)...........................................51
3.7 Western Blotting...................................................................................................51 - 52
3.8 Immunofluorescent Analysis and Imaging...........................................................52 - 53
3.9 Cell Death, Viability and Apoptosis Assay..........................................................53 - 54
3.10 Statistical Analysis.....................................................................................................54
Chapter 4 – Results........................................................................................................................55
4.1 Neuronal survival following OGD: cytotoxicity and viability.............................55 - 58
4.2 Necrosis is the dominant form of cell death following OGD exposure while
apoptosis is more prevalent following NMDAR and TRPM7 inhibition...................64 - 65
4.3 The effects of oxygen glucose deprivation on ROCK2 activity..................................67
4.4 Oxygen glucose deprivation attenuates P-21 activated kinase 3 activity.............69 - 70
4.5 Modulation of SSH1 activity by TRPM7 following oxygen glucose
deprivation.........................................................................................................................73
4.6 Immunofluorescent analysis of intracellular protein localization........................76 - 77
Chapter 5 – Discussion...........................................................................................................81 - 82
5.1 Anti-excitotoxic treatment protects neuronal cultures against 2 hours of
oxygen glucose deprivation......................................................................................82 - 84
5.2 Different cell death mechanisms appear to dominate during OGD versus
NMDAR and TRPM7 inhibition..............................................................................84 - 86
5.3 Investigating the effects of ROCK signalling in TRPM7 mediated neuronal cell
death.........................................................................................................................86 - 88
5.4 Ischemic depression of PAK3 activity...............................................................89 - 90
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5.5 A role for SSH1 downstream of TRPM7 mediated activation in oxygen
glucose deprivation..................................................................................................90 - 92
5.6 Model of TRPM7 mediated neuronal cell death................................................93 - 94
5.7 Future studies of TRPM7 mediated cell death...................................................95 - 97
Chapter 6 – Summary............................................................................................................98 - 99
Reference list .....................................................................................................................100 - 120
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List of Tables
Table 1. TRP subfamilies...........................................................................................................5 - 6
Table 2. Proposed regulators and substrates of the mammalian TRPM7 channel..................24- 25
Table 3.Antibodies ........................................................................................................................47
List of Figures
Figure 1. TRPM7 Channel Structure.............................................................................................16
Figure 2. Cytotoxicity following oxygen glucose deprivation...............................................58 - 61
Figure 3.Confirmation of MCN rescue of neuronal death using Hoechst and propidium iodide staining..........................................................................................................................61- 63
Figure 4. Apoptosis is not the predominant cell death mechanism observed in neurons following OGD exposure.......................................................................................................65 - 66
Figure 5. OGD, MCN and Gd3+ do not affect phosphorylation of ROCK within the activation loop........................................................................................................................68 - 69
Figure 6. OGD attenuates PAK3 phosphorylation ................................................................71 - 72
Figure 7. TRPM7 modulates SSH1 activity............................................................................74- 75
Figure 8. LIMK1 and P-PAK3 co-immunostaining in cortical neurons................................77 - 78
Figure 9. LIMK1 and P-SSH1 co-immunostaining in cortical neurons..................................79- 80
Figure 10. New proposed model of TRPM7 mediated cofilin regulation during OGD................94
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List of Abbreviations
ABP – Actin binding protein
AD – Alzheimer’s disease
ADF – Actin depolymerizing factor
AET – Anti-excitotoxic therapy
AID – Auto inhibitory domain
AIP1 – Actin interacting protein 1
BDNF – Brain derived neurotrophic factor
CAP – Cyclase associated protein
CIN – Chronophin phosphatase
DMPK – Myotonic dystrophy protein kinase
eEF2 – Eukaryotic elongation factor 2
eEF2-K - Eukaryotic elongation factor 2 kinase
LIMK – Lin-11, Isl 1, Mec-3 kinase
MHR - TRPM homology region
NLS – Nuclear localization sequence
NUDT9H – TRPM2 nudix hydroxylase domain
PAK – P21 activated kinase
PBD – P21 binding domain
PLC – Phospholipase C
ROCK – Rho coiled-coil kinase or Rho associated kinase
TRP – Transient Receptor Potential
TRPA – Transient Receptor Potential Ankyrin
TRPC – Transient Receptor Potential Canonical
TRPM – Transient Receptor Potential Melastatin
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TRPML – Transient Receptor Potential Mucolipin
TRPN – Transient Receptor Potential Nitric Oxide Mechanopotential
TRPP – Transient Receptor Potential Polycystin
TRPV – Transient Receptor Potential Vanilloid
OGD – Oxygen Glucose Deprivation
PH – Pleckstrin homology
PIP2 – Phosphatidylinositol 4,5 bisphosphate
PDGF – Platelet derived growth factor
RBD – Rho binding domain
ROS – Reactive Oxygen Species
SSH – Slingshot family phosphatase
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Chapter 1
Introduction
Transient receptor potential melastatin 7 (TRPM7), a divalent cation permeable ion
channel, has been implicated as a key regulator of neuronal cell death in stroke (Aarts, 2003;
Aarts 2005). TRPM7 is unique in that it contains both a channel pore domain and a functional
enzyme (alpha-kinase), though the contribution of each domain to TRPM7 signalling is unclear.
According to recent studies TRPM7 is thought to play a role in regulating Mg2+ homeostasis, cell
growth, proliferation, death and cytoskeletal regulation (Ryazanova et al 2010; Aarts 2003;
Zhang et al 2010). Our most recent data shows that TRPM7 dependent cytoskeletal regulation,
particularly via the actin binding protein cofilin, mediates neuronal death in oxygen glucose
deprivation (a model of stroke) (Bent 2011). Cofilin is known to be regulated by LIM kinase
(Amano et al. 2002) and Slingshot family phosphatase 1 (Kurita et al. 2008). Recent findings in
our lab have show that LIMK phosphorylation is also regulated downstream of TRPM7
activation in anoxia. The goal of this thesis project is to extend our current findings by
elucidating potential LIMK and cofilin regulators that operate downstream of TRPM7 activation
during oxygen glucose deprivation. In this thesis I will describe Slingshot phosphatase 1 as a
new potential player that could contributes to TRPM7 mediated ischemic pathology via
regulation of LIMK and cofilin activity.
1.1 Transient Receptor Potential Channels
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TRP (Transient Receptor Potential) channels are a distinct superfamily of cation selective
ion channels. TRP channels were first identified in Drosophila, where photoreceptors carrying
trp gene mutations exhibit a transient voltage response to continuous light stimulation. There are
now 28 identified TRP genes, some of which are expressed in a diverse range of organisms from
yeast, to worm, rodents and humans (Venkatachalam and Montell 2007). In addition to their
widespread prevalence in multicellular organisms, TRP channels display enormous diversity in
ion selectivity and regulatory mechanisms. Unlike most ion channels, which are classified
according to ion selectivity or function, TRP channels are classified based sequence homology
and structural characteristics into Group I and Group II TRPs. (Clapham et al. 2005;
Venkatachalam and Montell, 2007). Group I TRPs consist of 5 subfamilies that bear the greatest
sequence homology to the founding member of the superfamily, the Drosophila trp, while Group
II TRPs consist of two subfamilies that are distantly related to group I (Venkatachalam and
Montell 2007). Group I channels include TRPA(ankyrin), TRPC (canonical/ classical), TRPM
(melastatin), TRPN (Nitric oxide mechanopotential) and TRPV (vanilloid), while Group II
consists of TRPP (polycystin) and TRPML (mucolipin) ( refer to Table1) (Clapham et al.
2005). Of the 7 subfamilies, the classical TRPs (TRPCs) share the greatest sequence homology
with the Drosophila trp, while the polycystin TRPs appear to be the most evolutionarily ancient
as they are expressed in the widest range of organisms from yeast to humans (Vankatachalam
and Montell, 2007).
Despite the large amino acid sequence variation among TRP members, TRP channel
subunits share several structural features. Each TRP channel subunit consists of six
transmembrane segments with a pore forming loop (P-loop) between the fifth and sixth domains,
and an intracellular N- and C-termini (Clapham et al. 2005). TRP subunits assemble into
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tetrameric complexes forming the functional TRP channels (Clapham et al. 2005). The N- and C-
terminal ends vary considerably amongst TRP members both in length and amino acid sequence.
The N- and C- termini contain domains, motifs, and enzymatic regions; these domains are in turn
involved in channel assembly, activity, and downstream signalling. Some of the N- and C-
terminal domains and motifs are broadly expressed amongst the different TRP subfamilies while
others are restricted to a particular few (see Table 1). For instance, TRPA, TRPC, and TRPV
contain N-terminal ankyrin repeats which are involved in a variety of functions from activating
ligands to gating by voltage and temperature (Venkatachalam and Montell, 2007). In contrast,
TRPM2, TRPM6 and TRPM7 are the only TRP channels to contain a C-terminal enzymatic
domain, thereby allowing them to respond to stimuli in a bifunctional manner. To add to the
complexity, closely related TRP channel proteins, such as TRP1 and TRP3, form
heteromultimeric complexes with regulatory and biophysical properties different from their
respective monomers (Lintschinger et al. 2000)
TRP channel activity is modulated by a variety of stimuli such as temperature, pH,
osmolarity, mechanical stretch, ion concentration (e.g. Ca2+ and Mg2+), PIP2, sphingolipids, and
plant compounds (Clapham et al. 2005; Venkatachalam and Montell, 2007). Given that TRP
channel proteins are classified according to sequence similarity, it is difficult to predict the
regulatory mechanisms of a particular TRP channel based on subfamily classification. Indeed
variations in channel regulation are observed within members of the same subfamily. For
instance, within the TRPM subfamily TRPM4 and TRPM5 are activated by intracellular Ca2+
depletion; TRPM2 is activated by reactive oxygen and nitrogen species; whereas, TRPM8 is
activated by cool temperatures (8-28°C) (Venkatachalam and Montell, 2007). However, some
similarities in channel regulation do exist within and outside of particular TRP subfamilies as is
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the case with the phospholipase C (PLC) signalling pathway. All members of the TRPC
subfamily are regulated by pathways coupled to PLC stimulation; furthermore, TRP proteins
from other subfamilies such as TRPM and TRPP are also regulated by PLC pathway signalling
(Venkatachalam and Montell, 2007). TRP channels also exhibit enormous diversity in cation
selectivity with the majority of TRP channels being permeable to divalent cations including Ca2+
and Mg2+ (Clapham et al. 2005; Venkatachalam and Montell. 2007). However, exceptions do
exist as is the case with TRPM4 and TRPM5, the only TRP channels that are permeable
exclusively to monovalent cations (Venkatachalam and Montell. 2007).
Physiologically, TRP channels are involved in sensory reception and transduction of
environmental stimuli including light, touch, temperature, taste, olfaction and pain. TRPV1 and
TRPV2 contribute to warm and hot sensation respectively, while TRPM8 and TRPA1 contribute
to cool and cold sensation respectively (Venkatachalam and Montell. 2007, McKemy et al.
2002). TRP channels also function as cellular sensors of their local environment. TRPC3, for
example, regulates axonal guidance by brain-derived neurotrophic factor (Li et al. 1999). Some
of the physiological roles of TRP proteins have been discerned through genetic mutants and
knockout models. For instance, TRPP channels are important for kidney function as autosomal
dominant polycystic kidney disease is caused by TRPP1 mutations (Wu et al. 1998). Mutations
in the TRPML1 gene are responsible for mucolipidosis, a lysosomal storage disorder
characterized by severe neurodegeneration, mental retardation, achlorhydria and retinal
degeneration (Bargal et al. 2000). Additional pathological roles of TRP channels are being
uncovered and they range from hypomagnesemia and hympocalcemia, to progressive familial
heart block, amyotrophic lateral sclerosis, Parkinsonism, Alzheimer’s, and spinal muscular
atrophy (Wu et al. 2010). However, there is still a lot of work to be done, it is unclear how many
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TRP channels are activated in vivo, as very few known endogenous TRP stimulators and
inhibitors have been discovered, thereby making their study even more challenging.
Table 1. TRP subfamilies
There are six TRP subfamilies organized according to sequence homology. Humans do not
express TRPN; all other subfamilies express at least one member. Below are listed some of the
common physiological roles of the TRP subfamily members (data from Clapham et al. 2005;
Venkatachalam and Montell, 2007; Wu et al. 2010)
Subfamily Members found in Humans
Physical Characteristics
Physiological Roles
TRPA (Ankyrin) 1 N-terminal ankyrin repeats
-Sensitive to plant derived irritants (mustard oils) and possibly to mechanical stretch
TRPC (Canonical) 6 N-terminal ankyrin repeats C-terminal TRP domain C-terminal calmodulin/IP3R binding (CRIB) domain N-terminal and C-terminal coiled coil domain
-Receptor mediated Ca2+-dependent secretion and contraction (TRPC1) - Resistance artery myogenic tone, airway regulation (TRPC3) -Agonist dependent vasorelaxation in vascular-endothelium (TRPC4) - Cation influx in response to PLC activation (TRPC6) and Gq activation (TRPC7)
TRPM (Melastatin)
8 N-terminal TRPM homology region C-terminal TRP domain C-terminal coiled coil domain C-terminal alpha kinase (TRPM6 & TRPM7) C-terminal ADP-ribose hydroxylase domain
-Light sensation ? (TRPM1) -Cellular redox sensor (TRPM2, TRPM7) -Ca2+ absorption in renal collecting tubule (TRPM3) -Renal and gastrointestinal Mg2+ uptake (TRPM6) -Myogenic cerebral artery constriction (TRPM4)
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(TRPM2) -Sweet, bitter, umami taste transduction (TRPM5) - Cellular Mg2+ homeostasis; OGD induced Ca2+ influx and neuronal death (TRPM7) -Cold sensation (TRPM8)
TRPN (Nitric Oxide Mechanopotential)
0 N-terminal ankyrin repeats C-terminal TRP domain
TRPV (Vanilloid) 6 N-term ankyrin repeats C-terminal coiled coil domain C-term TRP domain C-terminal Calmodulin/IP3R binding domain
-Ca2+ entry in response to heat, capsaicin; pain sensation (TRPV1) -Ca2+ entry in thermal pain sensation (TRPV2) -warm sensation (TRPV3) -osmolarity sensing (TRV4)
TRPML (Mucolipin)
3 Long extracellular loop between S1 and S2 Short N- and C-terminus C-terminal EF hand
-Ca2+ dependent lysosomal exocytosis (?) (TRPML1)
TRPP (Polycystin) 3 Long extracellular loop between S1 and S2 C-terminal EF hand C-terminal coiled coil domain
-Kidney and liver cyst formation; mechanosensation in kidney cilia (TRPP1) - kidney and retinal development (TRPP2) - Involved in sorting or transport in late endocytic pathway (TRPP3)
1.2 TRPM subfamily
The TRPM subfamily is named after the TRPM1 protein also known as ‘melastatin’
whose expression has been found to correlate inversely with melanoma tumor progression
(Duncan et al. 1998). TRPM channel proteins, like other TRP proteins, contain 6 transmembrane
segments with a pore forming loop between S5-S6, and intracellular N-terminal and C-terminal
ends (Clapham et al. 2005; Venkatachalam and Montell, 2007). Similar to TRPC, TRPV and
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TRPP channels, TRPM proteins contain a C-terminal coiled-coil domain, which is important for
channel assembly, multimerization, and function (Latorre et al. 2009). The presence of an N-
terminal TRPM homology region (MHR) is unique to the TRPM channel subfamily; however, its
functional role is largely unknown (Wu et al, 2010). Another distinct feature of TRPM family
members is the presence of a C-terminal enzymatic domain in TRPM2, TRPM6 and TRPM7
(Clapham et al. 2005; Venkatachalam and Montell, 2007). Tissue distribution varies among
TRPM subfamily member since some TRPMs, such as TRPM2, TRPM4 and TRPM7, exhibit
broad expression throughout multiple tissues whereas others, including TRPM1 and TRPM6 and
TRPM8, are restricted to a small subset of tissues (Fonfria et al. 2006).
TRPM subfamily members have the most divergent biophysical properties and regulation
compared to the other subfamilies such as TRPCs and TRPVs. For instance, TRPM2 channels
have a high permeability for Ca2+, analogous to other TRPs, whereas TRPM7 channels are more
permeable to trace metals and Mg2+ than Ca2+ (Clapham et al.2005; Monteilh-Zoller et al. 2002).
Furthermore, other TRPs, such as TRPM4 and TRPM5, are exclusively permeable to
monovalent cations (Guinamard et al. 2011). TRPM subfamily members also vary in the modes
of regulation, as some are modulated by reactive oxygen species, low pH, intracellular Mg2+ and
Ca2+ levels, and others are modulated by external stimuli such as light and temperature. These
differences in structural organization and regulation of channel activity, in conjunction with the
varying tissue distribution contribute to a diverse range of physiological functions including:
neural tube and embryonic development; maintenance of Ca2+ and Mg2+ homeostasis; sensation
of light, temperature, and taste (Venkatachalam and Montell, 2007).
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1.2.1 TRPM1
TRPM1, the founding member of the TRPM subfamily, serves as a prognostic marker of
melanoma metastasis (Duncan et al. 1998). Despite its discovery15 years ago, very little progress
was made in identifying the molecular properties and physiological functions of TRPM1.
TRPM1 expression is observed in the brain, heart, melanocytes and retina with a predominant
localization in intracellular vesicles (Fonfria et al. 2006; Oancea et al. 2011). This intracellular
localization appears to be regulated by a direct interaction between TRPM1S, a short splice
variant, and TRPM1 full length protein, which inhibits TRPM1 translocation to the plasma
membrane (Xu et al. 2001). Physiologically, TRPM1 is involved in the light sensation; the
TRPM1 mediated cationic current is critical in mediating ON bipolar cell depolarization
following exposure to light (Oancea et al. 2011). In addition, TRPM1 may be involved in
melanin production since TRPM1 expression positively correlates with melanin content in
human melanocytes, while TRPM1 knockdown significantly reduced melanin content in these
cells (Oacea et al. 2009). Despite some recent progress much work needs to be done in
identifying the role TRPM1 plays in melanocytes and in melanoma tumor progression.
1.2.2 TRPM2
Unlike TRPM1, TRPM2 channels are ubiquitously expressed throughout many of the
body tissues with the exception of cartilage and bone (Fonfria et al. 2006). TRPM2 is a non-
selective cation channel that is permeable to both monovalent and divalent cations including
Ca2+, Mg2+ and Na+ (Wu et al. 2010). A unique feature of TRPM2 channels is the presence of a
C-terminal nudix hydroxylase domain (NUDT9-H), which is homologous to the NUDT9 of
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ADP-ribose hydroxylase (Perraud et al. 2001). Cyclic ADP-ribose, arachidonic acid, and
intracellular Ca2+ positively modulate TRPM2 activity (Hara et al. 2002). This is particularly
important since TRPM2 functions as a cellular sensor of oxidative stress. Reactive oxygen and
reactive nitrogen species activate the TRPM2 channel through the generation of ADP-ribose
from mitochondria; the consequent Ca2+ influx ultimately leads to cell death (Perraud et al. 2005;
Kaneko et al. 2006). In addition, inhibition of the ROS mediated TRPM2 current is protective in
both heterologous expression systems and primary neurons (Perraud et al, 2005; Kaneko et al.
2006). Besides acting as a cellular redox sensor, TRPM2 is involved in the regulation of insulin
secretion by pancreatic β-cells as well as in ROS induced chemokine production by immune cells
(Togashi et al. 2006; Yamamoto et al. 2008). Thus, the development of TRPM2 specific
inhibitors could help alleviate ROS mediated pathologies.
1.2.3 TRPM3
TRPM3 is one of the least characterized members of the TRPM subfamily. In humans
TRPM3 is primarily expressed in the kidney, with lower expression also found in brain, testis
and spinal cord (Lee et al. 2003). Heterologously expressed TRPM3 channels mediate a
constitutive Ca2+ influx, which is enhanced by a hypotonic environment and blocked by Gd3+
(100µM) treatment (Grimm et al. 2003; Lee et al. 2003). Treatment with D-erythro-sphingosine
selectively enhances the TRPM3 mediated Ca2+ current; however, whether this interaction is
physiologically relevant remains unknown (Grimm et al. 2005). A characteristic feature of
TRPM3 channels is the presence of multiple splice variants, some of which have been shown to
exhibit clearly distinct biophysical properties. Using two splice variants that only differed at a
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splice site forming the pore region of the TRPM3 channel, Oberwinkler and colleagues (2005)
revealed that one isoform was exclusively permeable to monovalent cations while the other was
highly permeable to divalent cations. This finding further complicates the interpretation of
TRPM3 biophysical data as investigators have to take into consideration the splice variants
expressed in their experimental paradigm.
1.2.4 TRPM4/TRPM5
TRPM4 and TRPM5 share many structural and functional features, and thus will be
described together. In humans TRPM4 is ubiquitously expressed throughout all tissues
examined, with highest expression found in the intestine and prostate (Fonfria et al. 2006).
TRPM5 however, is predominately found in taste cells, with some expression in the pituitary,
kidney, intestine, pancreas, and prostate (Perez et al. 2002; Fonfria et al. 2006). TRPM4 and
TRPM5 channels are permeable to monovalent cations, a feature unique to these two among the
entire TRP superfamily (Guinamard et al. 2011). The presence of specific amino acid residues
within the channel loop region, located between transmembrane segment 5 and 6, are deemed
responsible for the monovalent cation selectivity (Nilius et al. 2005). TRPM4 and TRPM5 are
activated by a rise in intracellular Ca2+; their activity is also enhanced by membrane
depolarization (voltage sensitive), and PIP2treatment (Nilius et al. 2005; Guinamard et al. 2011).
Interestingly, intracellular ATP inhibits TRPM4 but has no effect on TRPM5 activity (Nilius et
al. 2005). The presence of an ATP binding domain on TRPM4 appears to be critical for TRPM4
inhibition, as mutations in the ATP binding domain abolish this effect (Nilius et al. 2005).
Another compound that takes advantage of the ATP binding site is decavanadate, which has been
11
shown to enhance TRPM4 activity without affecting TRPM5 (Nilius et al. 2004). In this
situation decavanadate is likely competing with ATP for the ATP binding site (Nilius et al.
2004). These structural and functional differences between TRPM4 and TRPM5 channels could
prove useful in isolating the currents of each channel. Physiologically TRPM5 is involved in
taste transduction whereas TRPM4, given its widespread tissue distribution, has been implicated
in numerous processes including: modulation of insulin secretion, immune response, constriction
of cerebral arteries, inspiratory neuron firing rate, and cardiac activity (Guinamard et al. 2011).
1.2.5 TRPM6
TRPM6 is a non-selective divalent cation permeable channel that is expressed in the
brain, kidney, and intestine; with the latter two showing the greatest expression (Fonfria et al.
2006). TRPM6 and its closest homologue TRPM7 have similar protein structure and biophysical
properties. Both proteins have a C-terminal alpha kinase and a channel pore permeable to
divalent cations (Wu et al. 2010). TRPM6 mediates a small Ca2+ and Mg2+ inward current under
physiological conditions (Wu et al. 2010). This current in inhibited by intracellular Mg2+ and
potentiated by acidic extracellular pH (Voets et al. 2004; Jiang et al. 2005). TRPM6 mutants
have been linked to hypomagnesaemia and secondary hypocalcaemia, suggestive of a role for
TRPM6 in Mg2+ and Ca2+ uptake in the intestine and kidney (Schlingmann et al. 2002; Voets et
al. 2004). Furthermore, TRPM6 may play are role in neural tube closure in development since
TRPM6 knockout animals have neural tube defects and die by embryonic day 12.5 (Walder et al.
2009).
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1.2.6 TRPM8
The TRPM8 channel is best characterized for its involvement in cold sensation. Initially
identified in the prostate, TRPM8 expression has been observed in many other tissues including
liver, dorsal root ganglia, trigeminal ganglia, bladder, male genital tract, tongue, gastric fundus,
vascular smooth muscle, lung, spinal cord and brain (Fonfria et al. 2006; Liu and Qin, 2011).
TRPM8 can be activated by multiple types of stimuli from innocuous cool to noxious cold
temperatures (McKemy et al. 2002). In addition, cooling agents such as menthol and icilin
induce a response similar to thermal cold by activating TRPM8 (McKemy et al. 2002). TRPM8
activity is modulated by a variety of factors including PIP2, α2- adrenoreceptor signalling,
polyphosphates and lipid rafts (Liu and Qin, 2011). Recently TRPM8 has been suggested as a
potential therapeutic target for chronic pain relief and prostate cancer (Liu and Qin, 2011).
1.3 Transient Receptor Melastatin 7 (TRPM7)
Transient Receptor Melastatin 7 (TRPM7) is a ubiquitously expressed non-selective
divalent cation channel with murine expression greatest in the heart, kidney and cerebrum (Keil
et al. 2006). In humans the greatest expression is seen in the heart, adipose tissue and bone
(Fonfria et al. 2006). TRPM7 channels are permeable to divalent cations such as Ca2+ and Mg2+
as well as trace metals (Monteilh-Zoller et al. 2002). Normally, under physiological conditions
the TRPM7 currents are inhibited, that is they exhibit a very small inward current. However,
TRPM7 channels have the potential to drive a large inward Ca2+ current during conditions of
anoxia (OGD), oxidative stress, and low extracellular Ca2+ (Aarts et al. 2003; Xiong et al. 2002).
TRPM7 channel activity can be blocked through a variety of non-specific inhibitors including:
13
Gd3+ (Aarts et al. 2003); 2-aminoethoxydiphenyl borate (2-APB) (Peppiatt et al. 2003); 5-
lipoxygenase inhibitors namely, NDGA, MK886 and AA861 (Chen et al. 2010); as well as
intracellular Mg2+ ([Mg2+]i) and Mg2+-ATP ([Mg2+-ATP]i) (Nadler et al. 2001). Investigating the
function and regulation TRPM7 channels, in vivo, has been difficult particularly due to the
embryonic lethality of TRPM7 mutants and the absence of specific TRPM7 activators and
inhibitors. However, some advancement has been made through targeted knockdown using viral
vectors.
1.3.1 TRPM7 structure
The structure of TRPM7 is similar to other TRPM subfamily members but it does contain
some distinct features (see Figure 1). Similar to TRPM members, TRPM7 contains 6
transmembrane domains that are flanked by an intracellular N-terminal and C-terminal end
(Clapham et al. 2005; Wu et al. 2010). A loop region between segment 5 and segment 6 makes
up the channel pore of TRPM7 (Clapham et al. 2005; Wu et al. 2010). The N-terminus contains
a large TRPM homology region (MHR) that is conserved amongst TRPM subfamily members. A
study by Phelps and Gaudet (2007) shows that MHR is essential for TRPM8 channel localization
since deletions within the MHR region impair TRPM8 localization to the plasma membrane.
Whether, the MHR region plays a similar role for TRPM7 and other TRPM family members
remains to be determined. TRPM7 also contains an N-terminal snapin binding domain important
for vesicle fusion and neurotransmitter release (Krapivinsky et al. 2006; Brauchi et al. 2007).
The proximal C-terminal end of TRPM7 contains a TRP domain, which interacts with
phosphatidylinositol 4,5 bisphosphate (PIP2) a positive regulator of some TRP channels (Rohacs
14
et al. 2005; Bae et al. 2011). The TRP domain is followed by a coiled-coil domain, which is
essential for TRP channel protein assembly and tetramerization (Tsuruda et al. 2006).
What sets TRPM7 apart from most TRP channels is the presence of an α-kinase in the
distal end of the C-terminus, a feature that it only shares with TRPM6 (Clapham et al. 2005; Wu
et al. 2010). Alpha kinases are relatively rare enzymes that share no sequence homology with
conventional eukaryotic Ser/Thr kinases (Ryazanov et al. 2002). Unlike conventional kinases
which phosphorylate their substrates within loops, turns or regions with irregular structure; alpha
kinases phosphorylate their substrates within alpha-helices, hence their name (Ryazanov et al.
1999). Interestingly, the structure of the TRPM7 catalytic domain is similar to that of
conventional protein kinases despite the amino acid sequence differences (Ryazanov et al. 2002),
however; different sets of residues are engaged in contacts with the base and sugar moieties of
ATP making the TRPM7 alpha-kinase an ideal target for drug development (Li et al. 2011).
Since the discovery of the TRPM7 alpha-kinase domain, there has been much interest and
controversy in identifying the regulatory mechanisms and binding partners of this channel with
kinase activity (chanzyme). Runnels and colleagues (2002) were among the first to report a
model of TRPM7 regulation via the PLC signalling pathway. The authors suggested that PIP2 is a
key modulator of TRPM7 function, as receptor mediated activation of PLC and the consequent
hydrolysis of PIP2 inhibited TRPM7 currents (Runnels et al. 2002). In contrast, a study by
Takezawa et al (2004) showed that receptor mediated activation of PLC had no effect on
TRPM7 current, thereby suggesting that PIP2 depletion is not the mechanism contributing to
TRPM7 inhibition. Rather, they argue that receptor mediated regulation of TRPM7 activity
occurs at the level of adenylyl cyclase, as enhanced cyclic AMP levels enhance TRPM7 channel
activity (Takezawa et al. 2004). Another study by Langeslag et al (2007) was in complete
15
disagreement with Runnels and colleagues (2002) showing that receptor mediated activation of
PLC and the consequent PIP2 depletion led to enhanced TRPM7 channel activity(Langeslag et
al. 2007). Furthermore, the authors showed that cAMP levels did not affect the TRPM7 currents
(Langeslag et al. 2007). Some of these differences could be accounted for by variations in the
expression models used, the PLC receptor agonists employed, as well the electrophysiological
recording methods. However, the findings of Langeslag et al (2007) appear to be consistent with
a physiological regulation of endogenously expressed TRPM7.
Controversy also extends to investigations into the role of the TRPM7 alpha kinase
domain. It was originally suggested that the TRPM7 alpha kinase was required for channel
activity (Runnels et al. 2001). However, it was later shown that kinase dead mutants still form
functional channels and that the kinase domain is not essential for channel activation (Schmitz et
al. 2003). Instead, the alpha-kinase appears to play modulatory role by reducing the sensitivity of
TRPM7 channel to [Mg2+]i mediated suppression, which in turn will feedback to modulate kinase
activity via Mg2+ influx (Schmitz et al. 2003). The TRPM7 alpha-kinase phosphorylates a
number of proteins including Annexin I (Dorovkov and Ryazanov 2004), Myosin IIA (Clark et
al. 2006) and eEF2-K (Perraud et al. 2011) implicating TRPM7 in Ca2+ signal transduction,
cytoskeletal organization and regulation of translation respectively. The significance of the above
mentioned interactions and the various regulatory roles of TRPM7 will be discussed in the
sections below.
16
Figure 1. TRPM7 Channel Structure.
The TRPM7 channel is unique in that it contains both a channel domain and an alpha-kinase
domain hence it’s often called a ‘chanzyme’. TRPM7 channels consist of six transmembrane
subunits flanked by an intracellular N-terminus and C-terminus. The channel pore resides
between S5-S6, and is permeable to various divalent cations including Ca2+ and Mg2+, whereas
the kinase domain resides in the C-terminal end. Both the channel domain and the alpha-kinase
domain have been implicated in numerous signalling pathways, and have shown to influence
each other’s activity.
Snapin = snapin binding protein; TRP = conserved TRP box domain; CC = coiled coil domain;
Ser/Thr kinase = apha-kinase domain
17
1.3.2 TRPM7 in divalent cation homeostasis
TRPM7 channels are inherently permeable to a wide range of divalent cations with the
largest permeability for Zn2+ and Ni2+ followed in order by Ba2+, Co2+, Mg2+, Mn2+, Sr2+, Cd2+
and Ca2+ (Monteilh-Zoller et al. 2002). The above observation also points out that, in contrast to
other TRPs, TRPM7 is more permeable for Mg2+ than Ca2+, consistent with its involvement in
Mg2+ homeostasis (Monteilh-Zoller et al. 2002). In divalent cation free extracellular solution
large K+ and Na+ currents develop (Runnels et al. 2001; Schmitz et al. 2003), whereas in the
presence of trivalent cations, such as La3+ and Gd3+, TRPM7 currents are blocked (Runnels et al.
2001; Monteilh-Zoller et al. 2002; Aarts et al. 2003). Heterologous expression systems have
shown that TRPM7 channels are constitutively active and that their activity is regulated by
intracellular Mg2+ and Mg2+-ATP. Patch clamp experiments reveal a rapid induction of TRPM7
currents within minutes following depletion of intracellular Mg2+; addition of intracellular Mg2+
or Mg2+-ATP ( ≥1mM) strongly inhibited the TRPM7 channel currents (Nadler et al. 2001;
Schmitz et al. 2003). Initially it was believed that Mg2+-ATP directly contributed to inhibition of
TRPM7 channel activity through an ATP-mediated mechanism (Nadler et al. 2001); however,
follow up studies have showed that Mg2+-ATP inhibits TRPM7 currents by functioning as a
source of Mg2+ (Kozak and Cahalan 2003). Given that under physiological conditions TRPM7
channels exhibit only 10% of their maximal activity (Nadler et al. 2001), the above findings are
indicative of a feedback mechanism involved in maintaining cellular Mg2+ homeostasis.
The role of TRPM7 in ion homeostasis is ultimately linked with cell death since deletion
of TRPM7 from B-lymphocytes leads to proliferative arrest, cell senescence and eventually cell
death (Schmitz et al. 2003). Supplementation with excess Mg2+ or expression of a TRPM7
mutant lacking kinase activity corrected the Mg2+ deficiency and led to functional recovery of
18
the TRPM7 knockout cells suggesting that TRPM7 channel function alone is sufficient for Mg2+
homeostasis (Schmitz et al. 2003). This effect was specific to Mg2+, since only Mg2+ containing
salts complemented the TRPM7 deficiency (Schmitz et al. 2003). In addition, TRPM7 function
is essential in development as TRPM7 knockout mice exhibit embryonic lethality by gestation
day 7.5 (Jin et al. 2008). Surprisingly, the alpha-kinase domain of TRPM7 appears to play an
essential role since deletion of the TRPM7 kinase domain was sufficient to generate early
embryonic lethality in mice (Ryazanova et al. 2010). Furthermore, mice heterozygous for the
kinase deletion were viable but had defects in intestinal Mg2+ absorption; leading the authors to
suggest that TRPM7 is essential for both cellular and whole body Mg2+ homeostasis (Ryazanova
et al. 2010).
Calcium is a ubiquitous signalling molecule involved in numerous cellular processes
including growth, differentiation, migration, synaptic activity, and cell death. Given its
contribution to numerous signaling pathways intracellular Ca2+ levels are tightly regulated
through a combination of controlling Ca2+ influx, efflux and storage. Dysregulation of Ca2+
homeostasis results in the activation of signalling cascades that are otherwise silent or operating
at a low level ultimately leading to a pathological state (Arundine and Tymianski, 2003). In
addition to its role in Mg2+ homeostasis, TRPM7 has been implicated in a number of Ca2+
mediated physiological processes such as cytoskeletal regulation, cell adhesion and cell
migration (Su et al. 2006; Wei et al. 2009). Furthermore, TRPM7 Ca2+ currents have been shown
to have the most profound effects in various pathologies including ischemia and cancer (Aarts et
al. 2003; Chen et al. 2010; Middelbeek et al. 2012). The TRPM7 channel has been recently
identified as a mechanical sensor, conducting localized Ca2+ currents in the leading edge of
migrating cells (Wei et al. 2009). This localized Ca2+ entry from TRPM7 is amplified by Ca2+
19
release from the ER through IP3R thereby generating the calcium microdomains that are required
for guidance and directional migration (Wei et al. 2009). TRPM7 Ca2+ currents have also shown
to regulate cell adhesion by controlling the activity of m-calpain, a Ca2+ dependent protease (Su
et al. 2006). A pro-migratory role for TRPM7 has been identified in human nasopharyngeal
carcinoma whereby inhibition of the TRPM7 Ca2+ current, or TRPM7 knockdown, impaired
migratory potential (Chen et al. 2010). On the other hand, enhancement of TRPM7 current, or
TRPM7 overexpression, increased the migratory potential of human nasopharyngeal carcinoma
cells (Chen et al. 2010). In addition to its role in nasopharyngeal carcinoma, TRPM7 channel
function has been shown to be essential in breast tumor cell metastasis, and can be used as a
prognostic marker of poor patient outcome (Middelbeek et al. 2012). TRPM7 Ca2+ currents also
contribute to anoxic neuronal cell death, the specifics of which will be discussed in the following
section.
1.3.3 TRPM7 in ischemic stroke
Ischemic stroke is a consequence of transient or permanent reduction in cerebral blood
flow in a restricted vascular region due to vessel occlusion or hemorrhage. Neuronal damage
results from a combination of excitotoxicity, calcium overload, and inflammation that ultimately
results in cell death (Aarts and Tymianski 2005). Generally, focal impairment prevents the
transport of oxygen, glucose and other substrates to the affected tissue. This state of oxygen and
glucose deprivation (OGD) interrupts oxidative phosphorylation by the mitochondria, which
reduces ATP production resulting in a rapid depletion of ATP levels (Katsura et al. 1994). The
profound loss of ion gradients and membrane depolarization that follow (Katsura et al. 1994)
20
cause an excess of excitatory amino acids (EAAs) to be released in extracellular space (Arundine
and Tymianski, 2003). These supraphysiological glutamate levels stimulate surrounding neurons
thereby initiating a positive feedback loop that results in even more glutamate release. The
prolonged exposure to EAA stimulation and the consequent dysregulation of Ca2+ homeostasis
can lead to the activation of signalling events that ultimately result in cell death, hence the term
excitotoxicity (Arundine and Tymianski, 2003).
It has been established that virtually all glutamate receptor subtypes contribute to
excitotoxicity through a Ca2+ mediated process including ionotropic N-methyl-D-aspartate
receptor(NMDAR), 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) receptor(AMPAR), and
kainite receptors (Choi 1985,1987; Arundine and Tymianski, 2003). Treatment of primary
cortical neurons with glutamate receptor antagonists is neuroprotective for up to 1 hour of OGD
(Aarts et al. 2003). Blockage of NMDA subtype glutamate channels in animal models of stroke
has also shown to be neuroprotective and prevent excitotoxicity but only when administered
prior to or immediately following stroke onset(Aarts and Tymianski 2005). Despite the strong
role of for excitotoxicity in ischemic brain damage human clinical trials using anti-excitotoxic
therapies (AET) failed to show any significant neuroprotection, and were associated with many
adverse side effects (Cheng et al. 2004). In retrospect is not that surprising that global inhibition
of glutamate signalling would have profound physiological effects given glutamate’s
involvement in many cognitive functions including learning and memory.
The failure of AET indicated that although glutamate receptor signalling contributes to
neurotoxicity, other non-excitotoxic mechanisms are also involved. A study by Aarts and
colleagues (2003) identified a non-excitotoxic mechanism of neurotoxicity involving TRPM7
mediated cation currents. The authors used OGD or NaCN treatment as their in vitro model of
21
ischemia on primary cortical neurons. AET treatment was able to prevent the ensuing Ca2+
influx normally observed following OGD, for up to 1 hour; however, by 2 hours of OGD
intracellular Ca3+ levels were similar to the untreated OGD cells, suggesting that AET is not
sufficient to prevent Ca2+ dysregulation following prolonged ischemia (Aarts et al. 2003).
Furthermore, AET unmasked a TRPM7 mediated cation current with high permeability for Ca2+.
Heterologous TRPM7 expression enhanced the current whereas Gd3+ treatment or TRPM7
knockdown blocked it (Aarts et al. 2003). In addition, TRPM7 inhibition was neuroprotective for
up to 3 hours of OGD, even in the absence of AET, indicating thatTRPM7 activity contributes to
neuronal death independently of glutamate signalling (Aarts et al. 2003). Reactive oxygen
species (ROS) potentiated both the TRPM7 current and neuronal cell death, whereas antioxidant
treatment inhibited both the TRPM7 current and neuronal death (Aarts and Tymianski; 2003).
The above findings led the authors to propose a mechanism whereby ROS production during
ischemia activates TRPM7 channels leading to Ca2+ influx, which in turn generates more ROS
thereby starting a positive feedback loop that leads to Ca2+ overload and neuronal death (Aarts et
al, 2003). A follow up study by Sun and colleagues (2009), using an in vivo rat model of global
ischemia, revealed the involvement of TRPM7 channels in hippocampal neuronal injury.
Suppression of TRPM7 expression in CA1 neurons via intrahippocampal injection of viral
vectors containing shRNA specific to TRPM7 had no adverse effects on animal survival,
neuronal morphology, neuronal excitability or synaptic plasticity (Sun et al, 2009). However,
TRPM7 knockdown was neuroprotective against ischemia and preserved neuronal morphology
and function. Behavioural assays also revealed that TRPM7 suppression preserved hippocampal
dependent memory following ischemia (Sun et al. 2009).
22
1.3.4 TRPM7 and cytoskeleton
The cytoskeleton forms the structural framework of a cell and regulates many aspects of
cellular physiology including cell shape, migration, signalling, division, differentiation, and
death. TRPM7 is a bifunctional protein that influences the cytoskeleton through channel
conductance and/or the alpha-kinase domain. A study by Nadler and colleagues (2001) was the
first to provide evidence regarding the involvement of TRPM7 in cytoskeletal regulation. The
authors reported that overexpression of TRPM7 in HEK293 cells resulted in a loss of cell
adhesion, cell rounding, and ultimately cell death (Nadler et al. 2001). Su et al (2006) extended
these findings by showing that localized TRPM7 currents in peripheral adhesion complexes
contribute to loss of cell adhesion via activation of a Ca2+-dependent protease, m-calpain. It was
later discovered that TRPM7 channel activity causes nitrosative and oxidative stress, which
induces cell rounding through p38 MAPK/JNK dependent activation of m-calpain (Su et al.
2010). TRPM7 currents also contribute to directional cell migration through the generation of
high calcium microdomains called ‘Ca2+-flickers’ (Wei et al. 2009). Platelet derived growth
factor (PDGF) treatment increased the frequency of the Ca2+-flickers and induced directional cell
migration of fibroblasts; whereas suppression of TRPM7 expression decreased the frequency of
Ca2+-flickers and impaired the turning of fibroblasts in response to PDGF (Wei et al. 2009). The
above studies reveal a mechanism of actomyosin cytoskeleton regulation through a TRPM7
mediated Ca2+ current. However, it was recently shown that a TRPM7 mediated Mg2+ current
also regulates the actomyosin cytoskeleton in fibroblasts (Su et al. 2011). TRPM7 knockdown
disrupted the actomyosin cytoskeleton, focal adhesions, cell polarity and directional migration all
of which could be rescued with the expression of a kinase dead TRPM7 channel (Su et al 2011).
23
The TRPM7 alpha-kinase is also involved in cytoskeleton regulation. Annexin I is the
first identified substrate of the TRPM7 alpha kinase (see Table 2)(Dorovkov and Ryazanov
2004). Annexin 1 is a Ca2+ and phospholipid binding protein that can promote Ca2+ dependent
membrane fusion, as well as regulating cell growth, differentiation and apoptosis
(Monastyrskaya et al. 2009). TRPM7 phosphorylates annexin 1 at a conserved Ser5 residue in the
N-terminal region, in a Ca2+ dependent manner (Dorvkov and Ryazanov, 2004). Heterologous
TRPM7 expression in HEK-293 cells enhances annexin I phosphorylation. Given that the N-
terminal region of annexin proteins governs their interactions, Ser5 phosphorylation via TRPM7
may modulate annexin I function (Dorovkov and Ryazanov, 2004).Myosin IIA is another
substrate of the TRPM7 alpha-kinase (see Table 2). Clark et al (2006) have shown that the
TRPM7 alpha kinase directly interacts with myosin IIA heavy chain in a Ca2+ dependent manner.
Furthermore, this interaction results in myosin IIA heavy chain phosphorylation consistent with a
role for TRPM7 in regulating actomyosin contractility (Clark et al. 2006). TRPM7 can also bind
to actin; however this does not result in actin phosphorylation (Clark et al. 2006). The
bifunctional nature of TRPM7 allows it to regulate the actin cytoskeleton predominately through
divalent cation influx but also through the direct phosphorylation of cytoskeletal proteins. Given
that TRPM7 channel currents modulate kinase activity and the kinase domain in turn modulates
channel activity is indicative of an additional layer of regulation that could serve to ensure an
appropriate response by TRPM7.
24
Table 2: Proposed regulators and substrates of the mammalian TRPM7 channel
Region of TRPM7 interaction
Proposed interacting protein
Proposed function Notes Reference
C-terminal kinase domain
Phospholipase C (PLC) isoforms
Receptor mediated activation of PLC leads to inactivation of TRPM7 current
Inactivation appears to be due to a direct interaction with PIP2 and not its downstream signalling molecules
Runnels et al. 2002
TRP consensus domain
PLC, PIP2 Receptor mediated activation of PLC causes PIP2 breakdown leading to TRPM7 activation
TRPM7-dependent Ca2+ increase correlated with PLC activation. Similar results as above were observed when [Mg2+]i was below physiological levels (1-2mM)
Langeslag et al. 2007
C-terminal kinase domain
cAMP via PKA
TRPM7 activity is regulated through its endogenous kinase domain responding to changes in cAMP levels brought forth by Gi/Gs regulation of adenylyl cyclase.
PLC activation has no effect on TRPM7 current
Takezawa et al. 2004
Channel pore Mg2+, Mg2+-ATP
TRPM7 regulates Mg2+ homeostasis and divalent cation fluxes based on metabolic state of the cell
Increased intracellular [Mg2+] strongly suppress the TRPM7 mediated current
Nadler et al. 2001
Direct/indirect ?
Beta actin TRPM7 binds to actomyosin cytoskeleton via beta actin
IP: beta-actin was present in a complex with TRPM7
Clark et al. 2006
Alpha kinase domain
Myosin IIA TRPM7 α-kinase phosphorylates Myosin IIA in a Ca2+
dependent manner. This interaction affects actomyosin cytoskeleton.
TRPM7 mediated Ca2+ influx enhances TRPM7/Myosin IIA interaction via active kinase domain. Active kinase is required as in kinase dead TRPM7
Clark et al. 2006
25
mutants do not interact with Myosin IIA.
Alpha kinase Activity
Annexin I TRPM7 phosphorylation of annexin I may have a role in cell death
TRPM7 phosphorylates annexin I at a conserved serine-5 residue within the amphipathic alpha-helix region.
Dorovkov and Ryazanaov, 2004
N-terminal region
Snapin, synapsin I, synaptotagmin I
TRPM7 vesicular channel activity is essential for neurotransmitter (Ach) release in sympathetic neurons
TRPM7 forms molecular complexes with synaptic vesicle proteins synapsin I, synaptotagmin I and snapin (direct) and mediates acetylcholine release
Krapivinsky et al. 2006
Alpha kinase domain
eEF2 kinase TRPM7 mediates eEF2 phosphorylation through eEF2-K under low Mg2+ availability
Indirect influence via phosphorylation of eEF2-K at Ser77. Phosphorylated eEF2-K in turn phosphorylates eEFT2 at Thr-56
Perraud et al. 2011
1.4 Cofilin – the actin depolymerizing factor
The actin cytoskeleton is a dynamic structure that forms the structural framework of a
cell; it mediates cell attachment, migration, division, and death. In addition, the cellular
cytoskeleton allows cells to respond to their local environment by migrating toward, or away
from chemotactic cues. The cytoskeleton organizes the localization of cellular organelles; it also
contributes to intracellular transport, and cell polarity. Thus, regulation of the actin cytoskeleton
is a fundamental component of cellular physiology. The actin depolymerizing factor
(ADF)/cofilin family of proteins are essential regulators of the actin dynamics. During actin
26
treadmilling ATP bound G-actin monomers are added to the barbed end of actin filaments while
ADP-bound G-actin monomers dissociate from the pointed end thereby enabling the
unidirectional movement of actin filaments. ADF/cofilin family members enhance actin filament
dynamics by contributing to the maintenance of the actin monomer pool through
depolymerisation and dissociation of F-actin (Carlier et al. 1997). However, cofilin can also
promote actin polymerization and nucleation depending on the amount of available active cofilin
(Ichetovkin et al. 2002). Cofilin phosphorylation, by one of its upstream regulators, results in an
inactive cofilin, whereas dephosphorylation results in cofilin activation (Andrianantoandro and
Pollard, 2006). Cofilin activity is essential in development (Gurniak et al. 2005) and has been
implicated in numerous neuronal pathologies including Alzheimer’s disease (Maciver and
Harrington 1995) and stroke (Minamide et al. 2000; Bent 2011).
1.4.1 Cofilin structure and expression
The ADF/cofilin family of proteins are ubiquitously expressed throughout eukaryotes
(Bamburg, 1999). These are relatively small actin binding proteins (19kDa) that play essential
roles in regulating actin dynamics. ADF/cofilin proteins are characterized by the presence of
highly conserved actin binding domains and a single regulatory phosphorylation site on Ser-3
(Bamburg, 1999). In addition, ADF/cofilin proteins contain two PIP2 binding domains and a
nuclear localization sequence (NLS) (Bamburg, 1999). The mammalian ADF/cofilin family can
be divided into three homologous members: cofilin-1, also known as non-muscle cofilin; cofilin-
2, also known as muscle specific cofilin; and ADF (van Troys et al, 2008). The expression
patterns of these proteins vary across development in a tissue specific manner. For instance,
27
cofilin 1 is the predominant isoform during development and it remains ubiquitously expressed
throughout adulthood (Vartiainen et al. 2002, Gurniak et al. 2005). During late embryogenesis
and after birth cofilin 2 replaces cofilin 1 in striated muscle thereby forming the only isoform to
be expressed in skeletal muscle and the predominant isoform in cardiac muscle (Nakashima et al.
2005). Whereas, ADF expression increases post-natally, particularly in epithelial and endothelial
cells (Vartiainen et al. 2002). Despite the fact that multiple cofilin isoforms can be found in
certain cells/tissues, they appear to have distinct functional roles, particularly during
development. For instance, cofilin 1 is critical in development, especially for development of the
nervous system as cofilin 1 knockout mice exhibit defects in neural tube closure, impaired neural
crest cell migration and are embryonically lethal (Gurniak et al. 2005). These Cofilin 1
knockouts had no detectable levels of cofilin2 however they did show a 3 to 4 fold increase in
ADF expression (Gurniak et al. 2005). Given that the enhanced ADF expression did not recover
the nervous system dysfunction nor the embryonic lethality suggests that these actin binding
proteins are not functionally redundant and that they play distinct roles in development (Gurniak
et al. 2005). Further evidence comes from ADF knockout studies, where mice lacking ADF
expression are viable and undergo normal development. However, shortly after birth these ADF
knockouts exhibit hyper-proliferation of the corneal epithelium leading to corneal thickening and
blindness (Ikeda et al. 2003). Despite the presence of 3 ADF/cofilin isoforms in mice and
humans, we will focus on the predominant isoform cofilin 1 which we will simply refer to as
‘cofilin’.
28
1.4.2 Cofilin and the regulation of actin dynamics
Actin is an integral member of the cytoskeleton and it is normally found in two cellular
forms: the unbound globular actin (G-actin) monomer, and the bound or filamentous actin (F-
actin) polymer form. Actin filaments are double helical polymers composed of globular subunits
arranged head-to-tail to give the filament molecular polarity. Under steady state conditions F-
actin undergoes rapid polymerization on the barbed end and depolymerisation on the other end,
called the pointed end (Pollard and Borisy, 2003). ATP bound actin monomers (ATP-G-actin)
are added on the barbed end. Once incorporated into F-actin the monomers undergo hydrolysis
of the bound ATP to ATD and Pi (intermediate state). Dissociation of Pi initiates disassembly
reactions by inducing filament debranching and the binding of cofilin to ADP-loaded actin,
which in turn promotes filament dissociation at the pointed end (Pollard and Borisy, 2003). The
ADP-bound actin monomers can re-enter the polymerization cycle once they are ATP bound
(Pollard and Borisy, 2003). This polarized growth and turnover of actin filaments is an integral
component of cell division, migration and the formation of cellular protrusions including
lamellipodia, filopodia, podosomes, and growth cones (Pollard and Borisy, 2003). In these
dynamic structures actin filaments are organized in polarized arrays that exhibit treadmilling
with the barbed ends facing outward towards the edge of the cell pushing the cell membrane
forward as they undergo polymerization (Pollard and Borisy, 2003). Actin filament turnover can
be enhanced through de novo synthesis, increasing the number of filament ends and/or by
increasing the available monomer pool (Pollard and Borisy, 2003).
Actin filaments can turn over by themselves, but they do so at a much slower rate than
needed for cell movement indicating that other factors must contribute to actin dynamics
(Andrianantoandro and Thomas, 2008).Cofilin, profilin, Arp2/3, tropomyosin, and capping
29
proteins are some of the actin binding proteins (ABPs), which contribute to the regulation of
actin dynamics. Cofilin can bind both filamentous actin and actin monomers. In filamentous
actin, each cofilin molecule binds two actin monomers at the cleft between them (van Troys et
al. 2008). Cofilin preferentially binds to ADP/Pi and ADP bound actin over ATP-bound: cofilin
promotes filament severing and dissociation on the pointed end of F-actin (van Troys et al.
2008). In addition, cofilin binds actin cooperatively; as a result cofilin bound actin subunits are
clustered on actin filaments (Bamburg et al. 1999). In both severing and depolymerisation,
cofilin binding is believed to induce a twist in the actin filament, which through long range
effects destabilizes the actin-actin interactions thereby leading to filament fragmentation
(McGough et al. 1997; Bobkov et al. 2006).
Although the actin severing and depolymerizing function of cofilin are well accepted,
much debate exists regarding the functional role of cofilin in regulating actin dynamics. Initially
it was suggested that cofilin activity, during steady state actin polymerization, contributes to the
generation of the actin monomer pool through depolymerisation of actin filaments on the pointed
end (Carlier et al. 1997). However, follow up studies revealed that cofilin activity contributes to
actin polymerisation by severing actin filaments and exposing new barbed ends that could be
subsequently polymerized (Ichetovkin et al. 2002). Both models are not mutually exclusive,
rather they are thought to operate under different conditions. One factor that determines the
effects of cofilin on actin filaments is the proportion of the available pool of active cofilin
monomers given that a low cofilin pool favours actin filament severing whereas a high cofilin
pool favours actin filament nucleation (Andrianantoandro and Pollard, 2006). When a few
cofilin molecules bind to actin (cofilin:actin ≤ 1:2) the number of torsionally strained interfaces
between twisted filament regions is large thereby leading to filament severing to relieve the
30
torsional stress (Bobkov et al. 2006). However, when F-actin is decorated by cofilin
(cofilin:actin ≈ 1:1 ) the number of torsionally strained interfaces decreases leading to overall
enhanced filament stability (Bobkov et al. 2006; Andrianantoandro and Pollard, 2006). In this
case, filament severing is not observed but disassembly occurs at the pointed end at rates
equivalent to those observed by undecorated F-actin filaments (van Troys et al. 2008). At very
high cofilin concentrations (cofilin: actin > 2:1), G-actin binding and de novo nucleation are
observed leading to filament assembly (Andrianantoandro and Pollard, 2006).
1.4.3 Regulation of cofilin activity
Cofilin activity can be regulated through phosphorylation, and interaction with
intracellular proteins such as PIP2 and other ABPs. Cofilin phosphorylation interferes with its
ability to bind actin (Wriggers et al. 1998). Thus, phosphorylation of the N-terminal Ser-3 leads
to cofilin inactivation, whereas dephosphorylation of Ser-3 residue leads to activation (Bamburg,
1999). This phospho-regulation is finely tuned through a number of signalling pathways that
control the activity state of several key regulatory kinases and phosphatases. These key
regulatory proteins include LIM (Lin-11, Isl1, and Mec-3) kinases (LIMK) and testicular kinases
(TESK), which inhibit cofilin through phosphorylation of Ser-3 ( Edwards et al 1999; Toshima
et al. 2001) and slingshot family phosphatases (SSH) and chronophin phosphatase (CIN) that
reactivate cofilin through dephosphorylation of Ser-3 (Ohta et al. 2003;Mizuno et al. 2013).
Membrane phosphoinositides, particularly PIP2 and PIP3 are well known for their ability to bind
cofilin; this interaction interferes with cofilin’s ability to bind actin due to an overlap between
the G/F-actin and phosphoinositide binding sites (Gorbatyuk et al. 2006). Given that PIP2 binds
31
cofilin with similar affinity irrespective of its phosphorylation state, this represents a regulatory
mechanism that is distinct from that of cofilin phospho-regulation (Gorbatyuk et al. 2006). The
formation of PIP2 rich microdomains in the inner leaflet of the plasma membrane could function
to recruit cofilin to the plasma membrane; stimulus induced PIP2 hydrolysis would contribute to
a high amount of cofilin near the membrane to exert its effects on the actin cytoskeleton
(Gorbatyuk et al. 2006; Van Troys et al. 2008). Cofilin activity is also modulated by other actin
binding proteins. For instance, Arp2/3 complex and cofilin function in a synergistic fashion to
promote F-actin polymerization (DesMarais et al. 2005), whereas the binding of tropomyosin to
actin prevents cofilin mediated actin severing (Ono 2007). Most of the ADF/cofilin proteins
exhibit enhanced actin depolymerisation activity under basic pH than they do at neutral or acidic
pH values (Bamburg1999; Bernstein et al. 2000). However, in vertebrates ADF activity is much
more sensitive to changes in pH in comparison to cofilin. ADF cellular distribution is also
sensitive to intracellular pH, since in the presence of an acidic environment (pH<7) ADF
associates with F-actin in the cytoskeleton whereas cofilin localization remains largely
unaffected (Bernstein et al. 2000).
1.4.4 Cofilin in neurodegeneration
Ischemia, excitotoxicity and oxidative stress are key initiators of neurodegenerative
disease and acute CNS injury (Maloney and Bamburg 2007; Aarts et al, 2003). These initiators
can arise from stroke, trauma, or mitochondrial dysfunction and they can be induced or enhanced
by genetic abnormalities (Maloney and Bamburg 2007). A role for cofilin in neurodegeneration
comes from its identification in Hirano bodies, intracellular rod-shaped eosinophilic inclusions
32
found in numerous neurodegenerative disease including Alzheimer’s, dementia, and kuru
(Maciver and Harrington 1995). Ischemia and mitochondrial dysfunction have both been shown
to result in cofilin and ADF hyper-activation and the subsequent formation of cofilin-actin rods
(Bamburg 1999, Minamide et al. 2000). For instance, blockage of mitochondrial and glycolytic
ATP production in hippocampal and cortical neurons led to a rapid ATP depletion, in a matter of
minutes, and equally rapid cofilin activation through dephosphorylation (Minamide et al. 2000,
Huang et al, 2008). These changes in cofilin activity were accompanied by rapid actin-cofilin rod
formation in the majority of neurons (>80%) within a similar time frame. Expression of a
constitutively active cofilin mutant clearly showed that rod formation was due to cofilin hyper-
activation (Minamide et al. 2000). Interestingly, repletion of neuronal cultures with ATP resulted
in rapid dissolution of cofilin rods (Minamide et al. 2000; Huang et al. 2008). Given that neurons
consume nearly 50% of their energy through actin turnover (Bernstein et al. 2003), the
generation of the actin-cofilin rods could represent a neuroprotective mechanism that responds to
fluctuations in ATP levels during stress. Indeed Bernstein and colleagues (2006) have shown that
the generation of actin-cofilin rods in rat hippocampal neurons can slow ATP decline during
anoxic stress. Prolonged exposure to neuronal stress (i.e. ROS, glutamate excitotoxicity, anoxia)
results in the presence of persistent cofilin-actin rods, which can grow long enough to span the
diameter of neurites (Minamide et al. 2000). Despite their initial protective effects, the prolonged
presence of the actin-cofilin rods is proposed to disrupt cytoskeletal structure, intracellular
transport and contribute to the degeneration of neurites beyond the rods (Minamide et al. 2000).
In addition to their formation in the presence of energetic stress (ATP depletion), actin-
cofilin rods can form in the presence of ATP. For instance, glutamate or peroxide treatment have
no significant effects on cellular ATP levels, however the formation of actin-cofilin rods is still
33
observed (Minamide et al. 2000). Furthermore, unlike the transient rods that form upon ATP
depletion, peroxide and glutamate treatment lead to the formation of persistent rods. The
persistence of cofilin-actin rods could be due to enhanced cross-linking between cofilin
molecules. Cofilin-actin rods generated in response to excitotoxic stress have higher oxidation
levels than those cofilin-actin rods generated by cofilin overexpression (Bernstein et al 2012).
This enhanced oxidation is thought to promote cofilin’s incorporation into rods through
intermolecular disulfide bridges which in turn enhance rod stability (Bernstein et al. 2012).
Given that glutamate excitotoxicity, oxidative stress, mitochondrial dysfunction and ATP
depletion are all consequences of anoxia (Aarts et al. 2003; Maloney and Bamburg 2007) the
generation of persistent actin-cofilin rods could represent a pathological mechanism observed in
the ischemic brain.
In addition to the formation of actin-cofilin rods, cofilin function is also implicated in cell
death. In response to oxidative stress, cofilin has been shown to translocate to mitochondria
where it mediates the opening of the permeability transition pore resulting in cytochrome C
release and ultimately apoptosis (Klamt et al. 2009). Klamt et al. (2009) suggested that oxidation
of cofilin causes it to lose its affinity for actin and translocate to the mitochondria, moreover,
dephosphorylation of the regulatory Ser-3 is essential for this cofilin mediated translocation
(Klamt et al. 2009). The above finding suggests that sequestration of cofilin into rods in response
to transient stress could serve as a neuroprotective measure to circumvent the induction of
apoptosis. Consistent with this notion, Bernstein et al (2006) revealed that the formation of
cofilin-actin rods hindered mitochondrial membrane potential decline. However, the regulatory
mechanisms that induce actin-cofilin rod formation and disassembly still need to be determined.
34
1.5 Rho associated kinases (ROCKs)
Rho-associated kinases (ROCKs) are the first and best characterized effector proteins of
Rho-GTPases. ROCKs are serine/threonine kinases which consist of an amino-terminal kinase
domain followed by a coiled-coil region, and a pleckstrin homology (PH) domain at the
carboxyl-terminus (Nakagawa et al. 1996). The coiled-coil region contains a Rho-binding
domain (RBD) which is thought to interact with other α-helical proteins, whereas the PH domain
in the carboxyl terminus is thought to be involved in protein localization (Riento and Ridley,
2003). The PH domain and the RBD form the autoinhibitory loop that folds back onto the amino
terminus where it inhibits ROCK activity by interacting directly with the kinase domain (Amano
et al, 1999). Cleavage of the C-terminal end results in a constitutively active ROCK, consistent
with the inhibitory role of the C-terminal end of ROCK (Leung et al. 1996). Their closest
homologue is myotonic dystrophy protein kinase (DMPK) with which they share the above
mentioned structural features.
ROCK proteins exist as two closely related isoforms: ROCK1 and ROCK2 which show
the highest homology in their kinase domains and lowest homology in the coiled-coil domain
(Nakagawa et al, 1996). The diversity within the coiled-coil domain could serve in differential
regulation of ROCKs as well as subcellular localization through interactions with other
regulatory proteins. Both ROCK1 and ROCK2 are ubiquitously expressed in mouse and rat
tissues; ROCK2 is largely expressed in muscle and brain whereas ROCK1 expression is highest
in the liver, spleen, kidney and testis (Nakagawa et al. 1996). These isoforms also exhibit
differential expression within the CNS, with ROCK1 expression observed in astrocytes whereas
ROCK2 expression observed in neurons, especially in presynaptic and postsynaptic elements
35
(Iizuka et al. 2012). The variation in tissue distribution and subcellular localization suggest that
ROCK1 and ROCK2 may play distinct functional roles (Riento and Ridley 2003).
1.5.1 ROCK protein signalling
The Rho subfamily proteins (RhoA, RhoB, RhoC) are the only known GTPases that can
activate ROCK proteins in vivo (Yoneda et al. 2005). These Rho proteins bind to the RBD of
ROCK only in the activated (GTP-bound) form. This interaction between Rho and the RBD is
believed to induce conformational changes that diminish ROCK autoinhibition by exposing the
kinase domain (Riento and Ridley, 2003). In addition to its regulation by upstream Rho-
GTPases, ROCK protein activity is also regulated through Rho independent mechanisms. For
instance, arachidonic acid and phosphoinositides PIP2 and PIP3 have been reported to activate
ROCK2, suggestive that interactions with lipids induce conformational changes that expose the
kinase domain of ROCK2 (Feng et al. 1999; Yoneda et al. 2005). These findings also reveal that
ROCK1 and ROCK2 can be regulated through distinct mechanisms. Further support for this
differential regulation comes from other studies that have shown that Rho E specifically inhibits
ROCK1 (Riento et al. 2003). In addition, during apoptosis caspase-3 mediated cleavage of the
ROCK1 C-terminal end results in a constitutively active kinase (Coleman et al. 2001).
ROCK proteins phosphorylate numerous downstream targets that regulate the cellular
cytoskeleton. Consequently ROCKs are implicated in regulating cell adhesion, motility, division
and cell death (Riento and Ridley 2003). Myosin light chain phosphatase (MLCP), LIMK,
Ezrin/Radaxin/Moesin (ERM) and intermediate filament proteins are well known ROCK
substrates. For instance, site directed mutagenesis studies have shown that ROCK1 and ROCK2
36
phosphorylate LIMK1 and LIMK2 at Thr-508 and Thr-505 residues, respectively, and that these
phosphorylation events are essential for LIMK activity (Ohashi et al. 2000; Sumi et al. 2001).
For many of these substrates only one ROCK isoform has been tested, however given that the
ROCK1 and ROCK2 kinase domains are 92% identical (Nakagawa et al. 1999), both are
believed to phosphorylate the same substrates (Riento and Ridley 2003).
1.5.2 ROCK protein function
ROCK is an important regulator of the actin cytoskeleton through its influence on cell
division, adhesion, motility and death. For instance, ROCK is essential in cell division since
inhibition of ROCK activity prevents cytokinesis in mammalian cells (Yasui et al. 1998). In
fibroblasts, ROCK activity promotes actomyosin contractility given that ROCK activation results
in the formation of stress fibres and focal adhesions through the phosphorylation of MLCP
(Leung et al. 1996; Riento and Ridley 2003). ROCK activity also contributes to stress fiber
formation through phosphorylation of LIMK, which in turn phosphorylates and inactivates
cofilin thereby increasing acting filaments (Amano et al. 2001). In addition to regulating cell-
substratum adhesion, ROCKs influence cell-cell contacts since enhanced ROCK activity
compromises the integrity of tight junctions and adherens junctions (Riento and Ridley, 2003).
ROCKs also regulate numerous apoptotic events. Plasma membrane blebbing, a signature feature
of apoptotic cells, is regulated by myosin light chain phosphorylation and actomyosin
contraction (Mills et al. 1998). Caspase-3 mediated activation of ROCK1 has been shown to be
sufficient and necessary in promoting blebbing through enhancing myosin light chain
phosphorylation and actomyosin contractility (Shi and Wei, 2007). On the other hand, a decrease
37
in ROCK activity appears to mediate phagocytic uptake of apoptotic cells (Tosello-Trampont et
al. 2003).
1.6 P-21 Activated Kinases (PAKs)
1.6.1 The structure and expression of P-21 activated kinases (PAKs)
P21 activated kinases (PAKs) are a family of serine/threonine protein kinases consisting
of 6 different isoforms. These isoforms are subdivided into two groups based on sequence
similarities, as well as structural and biochemical properties; Group I consists of PAK 1-3 and
Group II consists of PAK 4-6 (Eswaran et al. 2008). All PAK isoforms contain an N-terminal
regulatory domain and a C-terminal kinase domain. The regulatory domain of group I PAKs
consists of a highly conserved p21- binding domain (PBD) that overlaps with an auto-inhibitory
domain (AID) (Kreis and Barnier, 2008). PAKs are the main kinase effectors for the Rac1 and
Cdc42 family of GTPases. Thus, Rac1 or Cdc42 binding to the PBD causes a conformational
shift that dissociates the AID from the catalytic domain and allows for its autophosphorylation
which is required for full kinase activity (Benner et al. 1995, Lei et al. 2000). In contrast to
Group I, Group II PAKs do not have an AID domain and thus undergo different regulation.
Indeed, Rac or Cdc42 binding does not substantially enhance kinase activity of Group II PAKs,
instead Rac/Cdc42 interaction is important for the translocation of Group II PAKs to specific
intracellular compartments (Arias-Romero and Chernoff, 2008). These findings in conjunction
with sequence variation between Group I and Group II PAKs indicate that these two groups are
differentially regulated (Arias-Romero and Chernoff 2008). Our focus will be on group I PAKs
with a particular emphasis on PAK3.
38
Expression analysis reveals distinct tissue and cellular distribution patterns for Group I
PAKs. For instance, PAK 1 is highly expressed in the brain and spleen, PAK3 is mainly
expressed in the brain, and PAK 2 shows ubiquitous expression (Kreis and Barnier 2009).
Although all three Group 1 PAKs are expressed in the brain, they show differential expression
with PAK1 expression found in neurons, astrocytes and oligodendrocytes, and PAK3 expression
mainly found in neurons (Kreis et al 2008, Kreis and Barnier 2009). Thus, despite the high
sequence similarity between Group I PAKs, these proteins appear to be involved in different
biological processes. This is especially evident from loss-of function mutants in which PAK1
mutants display immune system impairments while PAK3 mutants display abnormal synaptic
plasticity and learning deficiencies (Hofmann et al. 2004, Meng et al. 2005).
1.6.2 Regulation of PAK activity
Group I PAKs are the main effector proteins of the Rac and Cdc42 family of GTPases.
Although they have been shown to be activated by numerous Rac/Cdc42 members, Group 1
PAKs were recently shown to exhibit differential selectivity for their activators. For instance,
PAK3 was recently shown to have higher affinity for Cdc42 than for Rac1 (Kreis et al. 2007).
Given that the PBD is highly conserved among Group 1 PAKs, the substrate specificity might
arise from regions surrounding PBD that might participate with GTPase interactions (Reeder et
al. 2001), and/or through GTPase interactions with PAK binding partners such as guanine
exchange factor cool/pix (Kreis and Barnier 2009). Group 1 PAKs are fully active once
phosphorylated. The interaction of Rac/Cdc42 with PAK induces a conformational change in the
AID which activates the catalytic domain that performs the autophosphorylation of key residues
39
in the regulatory region important for enzymatic activity (Chong et al. 2001). In this model, the
key event involves phosphorylation of a Threonine residue in the activation loop of Group 1
PAKs which in turn mediates the autophosphorylation (Chong et al. 2001). Chong and
colleagues (2001) identified S-139 and Thr-421 as the phosphorylation sites essential for PAK3
activity. In addition to the Rac/Cdc42 mediated activation of Group 1 PAKs, they can also be
activated through GTPase independent mechanisms. For instance, Group 1 PAKs have been
shown to be activated by the lipid sphingosine (Bokoch 2003; Chong et al. 2001).
1.6.3 PAK protein function
Myosin light chain kinase (MLCK), Mitogen activated kinase (MAPK), Bcl-2/Bcl-XL-
antagonist causing cell death (BAD) and LIM kinase (LIMK) are a few of the well characterized
downstream targets of Group I PAKs (Arias-Romero and Chernoff, 2008). Given the diverse
range of downstream targets, Group 1 PAKs have been implicated in a wide range of biological
processes including cell cycle progression, neuronal cell fate determination, differentiation,
neurite outgrowth, axonal guidance, synaptic plasticity, survival and apoptosis (Kreis and
Barnier, 2009). Despite the high sequence similarity among Group 1 PAKs, numerous studies
have described the involvement of specific Group I PAKs in distinct cellular processes. For
instance, PAK1 activation promotes cell survival whereas PAK3 activity is implicated in cell
death (McPhie et al. 2003). Using an Alzheimer’s disease mutant model, McPhie et al (2003)
reported the specific involvement of PAK3 in amyloid precursor protein (APP) mediated
apoptosis, an effect that was abolished using a dominant negative kinase mutant of PAK3 or by
deletion of the APP binding domain of PAK3. In neurons the establishment of polarity is
40
essential for correct migration and the specification of dendrites and axons. A role for PAK1 in
neuronal polarity has been proposed by Rosso et al. (2004), they suggested PAK1 activation and
subcellular localization promote neurite outgrowth by regulating the actin cytoskeleton via
phosphorylation of LIMK1. On the other hand Cobos et al. (2007) reported a role for PAK3 in
inhibiting neurite growth and promoting the migration of immature GABAergic interneurons as
they migrate tangentially to the cortex.
PAK3 appears to be involved in regulating synapse structure and function. The
phosphorylated (active) form of PAK3 has been observed in dendritic spines (Chen et al. 2007).
Theta burst stimulation markedly increased the numbers of spines containing phosphorylated
PAK3 and phosphorylated cofilin (Chen et al. 2007). More importantly this enhanced
phosphorylation of PAK3 and cofilin corresponded with enlarged synapses thereby implicating
PAK3 activity with synaptic plasticity (Chen et al. 2007). Alterations in PAK3 activity have
also been linked to the pathogenesis of Alzheimer’s disease (AD) and mental retardation. For
instance, a mouse model of Alzheimer’s disease revealed a significant reduction in PAK1 and
PAK3 expression and activity (Zhao et al. 2006). Pharmacological inhibition of PAK activity in
normal mice was sufficient to induce cofilin pathology and memory impairment similar to
Alzheimer’s disease thereby suggesting that PAK signalling is involved in the synaptic
dysfunction and cognitive impairments (Zhao et al. 2006). In addition to AD pathology, PAK3 is
linked to non-syndromic mental retardation with five identified genetic mutants affecting
effector binding and kinase activity (Kreis and Barnier 2009). PAK3 knockout mice reveal
impairments in synaptic plasticity, as well as in learning and memory consistent with the notion
that PAK3 plays an essential role in regulating synapse function and cognition (Meng et al.
2005)
41
1.7 The Slingshot phosphatase (SSH) family
1.7.1 Slingshot phosphatase structure
Slingshot phosphatases (SSHs) are novel family of protein phosphatases that can
specifically dephosphorylate and activate cofilin. In humans and mice the SSH family consists of
three members SSH1, SSH2 and SSH3, each of which has multiple isoforms with the long
isoforms named SSH-1L, SSH-2L and SSH-3L, respectively (Ohta et al. 2003). Unless making
distinctions between the different SSH isoforms I will use the terms SSH1, SSH2, SSH3, to refer
to SSH1L, SSH2L and SSH3L respectively. All 3 SSH members exhibit ubiquitous expression
through many of the tissues examined, with distinct tissue expression patterns between members.
For instance, SSH-1L is highly expressed in the brain, kidney and thymus whereas SSH-2L is
highly expressed in the thymus, testis, heart and brain, and SSH-3L is only expressed in the brain
(Ohta et al. 2000). SSH proteins contain three highly conserved regions starting from the N-
terminal non-catalytic (SSH-N) domain, consisting of subdomain A and B, followed by the
catalytic P subdomain (Kurita et al. 2008). The P-subdomain itself has basal phosphatase activity
but no actin binding ability (Kurita et al. 2008, Yamamoto et al. 2008). However, the B and P
subdomains are essential for phospho-cofilin binding by SSH1 and the cofilin phosphatase
activity of SSH1 (Kurita et al. 2008). The A subdomain, on the other hand is required for F-actin
mediated activation of SSH1 but not for phospho-cofilin binding (Kurita et al. 2008). Unlike the
highly conserved N-terminal region, the C-terminal end of SSH family members is fairly
variable with the exception of the S-domain, a Serine rich region conserved between mammalian
SSH1 and SSH2 but not found in SSH3 (Mizuno et al. 2013). The S-domain of SSH1 and SSH2
contains an actin binding region as well as conserved residues that regulate phosphatase activity
42
(Yamamoto et al. 2006; Soosairahaj et al. 2005). To date SSH1 has 3 defined F-actin binding
regions including an LKR motif within the S-domain, an LHK motif within the B-subdomain,
and Trp-458 flanking the P-subdomain (Yamamoto et al. 2006). Deletion of either the B-
subdomain or S-domain reduces acting binding activity by nearly 50% whereas mutation of Trp-
458 completely abolishes actin binding activity suggesting that the latter residue is essential for
actin binding (Yamamoto et al. 2006).
1.7.2 Regulation of SSH1 activity
Although all three SSH members dephosphorylate phospho-cofilin at the Ser-3 position,
SSH3 is less effective than the other two (Ohta et al. 2003). In addition, SSH3 shows no actin
binding capacity, unlike SSH1 and SSH2 who have been reported to tightly bind to actin (Ohta et
al. 2003). Kurita et al. (2008) previously reported that F-actin binding enhances the cofilin
phosphatase activity of SSH1 by 1200 fold in comparison to a 2.2 fold increase in basal
phosphatase activity towards pNPP, an artificial phosphatase substrate typically used in
phosphatase assays. In addition to the A-subdomain, Trp-458 has also been reported to be
essential for the F-actin mediated SSH1 activation (Nishita et al. 2005). Given the reported actin
mediated activation of SSH1, it is likely that the enhanced cofilin phosphatase activity of SSH2,
in comparison to SSH3, is due to the actin binding activity of the former and not the latter.
In addition to the F-actin binding mediated activation of SSH1, SSH1 activity is also
regulated by phosphorylation and interaction with 14-3-3, a cytosolic scaffolding protein
(Yamamoto et al. 2006; Soosairajah et al. 2005). Incubation of PAK4 with a C-terminally
truncated SSH1 mutant (residues 1-535 out of 1049) has been shown to increase the
43
phosphorylation of this SSH1 mutant (Soosairajah et al. 2005). Although the identity of the
phosphorylated residues is unknown, this phosphorylation of SSH1 decreases its ability to
dephosphorylate LIMK and cofilin, two known substrates of SSH1 (Soosairajah et al. 2005). In
addition, phosphorylation of Ser-937 and Ser-978 within the S-domain of SSH1 promotes 14-3-3
binding (Nagata-Ohashi et al. 2004). This interaction of 14-3-3 with SSH1 is thought to interfere
with the ability of SSH1 to bind to actin. Indeed, it has been shown that pre-incubation of SSH1
with 14-3-3ζ results in a decrease in F-actin binding for SSH1 (Soosairajah et al. 2005) and its
cofilin phosphatase activity (Nagata-Ohashi et al. 2004). Calcineurin (CaN) and protein kinase D
(PKD) have been proposed as potential regulators of SSH1 activity. PKD has been shown to
result in Ser-937 and Ser-978 phosphorylation of SSH1 (Peterbus et al. 2009) whereas CaN has
been reported to dephosphorylate and activate SSH1 although it’s unclear whether Ser-937 and
Ser-978 are among the dephosphorylated residues (Wang et al. 2005). In contrast to 14-3-3
interaction, SSH binding with acting binding proteins, coronin-1B or coronin-2A, is thought to
promote SSH1 activity and regulate lamellipodia dynamics at the leading edge of migrating cells
(Mizuno et al. 2013).
1.7.3 SSH1 function
As previously mentioned cofilin plays an important role in regulating the actin
cytoskeletal dynamics. The functional modulation of cofilin activity is performed by kinases
such as LIMK and TESK which inhibit cofilin via Ser-3 phosphorylation, and by phosphatases
such as SSH and CIN, which active cofilin. Given the various cofilin activators and inhibitors,
the overall effect on cofilin activity is a consequence of the spatiotemporal regulation of the
44
numerous activators and inhibitors. Indeed, the regulation of SSH1 and LIMK has shown to
influence numerous cofilin mediated cellular processes such as cell migration, chemotaxis, and
cell division (Mizuno et al. 2013). Nishita et al. (2005) provide an exquisite model of SSH1 and
LIMK1 spatiotemporal regulation in directional cell migration. Based on data from knockdown
studies of LIMK1 and SSH1 in chemokine stimulated Jurkat T cells, the authors propose that
LIMK1 is initially activated to promote lamellipodia formation followed by SSH1 activation to
prevent membrane extensions in one direction while allowing local cofilin activity to the leading
edge of the migrating cell (Nishita et al. 2005). SSH1 and LIMK1 have also been shown to
function in a complementary fashion during cell division. Early in the cell cycle LIMK1 is
hyper-activated while SSH1 is inhibited, but as cells enter telophase and cytokinesis LIMK1 is
inhibited while SSH1 activity is increased (Amano et al. 2002; Kaji et al. 2003). Both of these
changes in LIMK1 and SSH1 activity correspond well with the enhanced cofilin phosphorylation
observed early in the cell cycle and its progressive decrease in phosphorylation as it enters
telophase and cytokinesis (Amano et al. 2002; Kaji et al. 2003).
45
Chapter 2
Objectives and Hypothesis
TRPM7 channels have been linked to neuronal death both in vitro and in vivo models of
ischemia (Aarts et al. 2003; Sun et al. 2009). The founder study by Aarts and colleagues (2003)
showing that TRPM7 channels play a more important role in ischemic cell death, particularly
during prolonged ischemia (>1.5 hours) led our research group into the identification of potential
signalling relationships operating downstream of TRPM7 activation. This search led to the
identification of cofilin-1, which was shown to be hyperactivated downstream of TRPM7
activity during anoxia; furthermore this enhanced cofilin activity contributed to neuronal death.
Inhibition of TRPM7 activity through Gd3+ or suppression of TRPM7 expression via TRPM7
specific siRNA attenuated cofilin hyperactivation during OGD (Bent 2011). Furthermore,
TRPM7 activity during anoxia corresponded with LIMK inhibition, an effect that was relieved
following TRPM7 inhibition (Bent 2011). Given the observation that LIMK – cofilin signalling
pathway operated downstream of TRPM7 the primary focus of this study is to identify potential
regulators of LIMK and cofilin activity that operate downstream of TRPM7. Rho-kinases are
known regulators of LIMKs (Amano et al. 2001) and recent findings implicate Rho-associated
kinases as contributors of ischemic neuronal cell death (Yamashita et al. 2007; Gisselson et al.
2009; Jeon et al. 2013) Thus, I hypothesize that TRPM7 activity during OGD influences ROCK2
protein activity. However, p-21 activated kinases are also known regulators LIMK function
(Arias-Romero and Chernoff 2008). Given that dysregulation of PAK3 is linked to synaptic
impairments I hypothesize that TRPM7 activity influences PAK3 activity during OGD. In
46
addition, Slingshot-family phosphatase 1 (SSH1) is known to regulate cofilin and LIMK (Kurita
et al. 2008; Soosairajah et al. 2005). Furthermore, oxidative stress and increased intracellular
Ca2+ have been shown to promote the cofilin phosphatase activity of SSH1, suggestive that
SSH1 activity is enhanced by an ischemic microenvironment (Wang et al. 2004; Kim et al.
2009). Based on the previous reports I hypothesize that TRPM7 activity during OGD enhances
SSH1 activity.
The experiments of this study will be split into three aims. First I will establish a neuronal
cell death model that confirms the previous findings of Aarts et al. (2003). This model will then
be used to evaluate the neuroprotective effects of ROCK2 during TRPM7 channel activity in
OGD. Furthermore, this cell death model will also be used to evaluate the cell death signalling
mechanisms that operate during OGD. Secondly, I will evaluate the effects of TRPM7 activity
on ROCK2, PAK3 and SSH1 using phospho-specific antibodies targeting sites that influence
protein activity. Lastly, to determine whether phospho-PAK3 and phospho-SSH1 localization is
influenced by TRPM7 activity during OGD, immunofluorescent images labelling the above two
proteins will be taken during normoxic conditions and compared to those of OGD and TRPM7
inhibition. Co-labeling with LIMK1, should expose any differential localization between LIMK
and its upstream regulators.
47
Chapter 3
3. Materials and Methods
3.1 Antibodies
Table 3: The antibodies used for western blotting and immunofluorescence analysis are listed at
their respective dilutions.
Product description
Specificity Clonality Dilutions used
Species reactivity
Manufacturer
Rabbit Anti-PAK3 (phospho S154) Antibody
Detects endogenous levels of PAK3 when phosphorylated at Ser-154 in humans (Ser139 orthologue in rats)
Polyclonal WB (1:1000) ICC (1:200)
Human Rat
Abcam
Rabbit Anti-PAK3 Antibody
Detects endogenous levels of total PAK3 protein
Monoclonal WB (1:1000) ICC (1:250)
Human Rat Mouse
Millipore
Rabbit Anti-ROCK2 (phospho T249) Antibody
Detects endogenous levels of ROCK2 when phosphorylated at Thr-249
Polyclonal WB(1:1000) ICC(1:200)
Human Rat Mouse
Abcam
Rabbit Anti-ROCK2 Antibody
Detects endogenous levels of ROCK2
Polyclonal WB(1:1000) IF(1:200)
Human Rat Mouse
Abcam
Rabbit Anti-Slingshot-1L (phospho S978)
Detects endogenous levels of Slingshot-1L phosphorylated at Ser-978.
Polyclonal WB (1:1000) IF (1:200)
Human Rat Mouse Chicken
ECM Biosciences
Rabbit Anti-Slingshot-1L (C-terminal region)
Detects endogenous levels of total Slingshot 1L
Polyclonal WB(1:1000) IF(1:200)
Human Rat Mouse
ECM Biosciences
Mouse Anti-LIMK1 Antibody
Detects endogenous levels of total LIMK1
Monoclonal IF (1:200) Human Rat Mouse
Abcam
WB = western blot; IF = Immunofluorescene
48
3.2 Rat Primary Neuronal Culture
Cortical neurons were isolated from embryonic day 18 (E18) Spargue-Dawley rats as
described by Choi et al. (1987), but with minor modifications. In short neurons were removed
from the uterus and placed in chilled DMEM (Invitrogen). Embryos were then removed from
their amniotic sac and placed in a petri dish containing chilled DMEM (Invitrogen). Using iris
microscissors, a horizontal incision was made along the dorsal-midline at the brain stem – spinal
cord border. The same incision was then used to make a vertical cut upwards the nose along the
midline skull suture. The iris microscissors were used to apply pressure along the line between
the eye and the ear to push the brain out of the skull. The olfactory bulbs were removed and the
two hemispheres were spread apart along the midline. An incision was made to cut each cortical
hemisphere from the striatum. The meninges were then removed and cortices collected in a six-
well plate containing chilled DMEM. Following collection of all the cortices, the DMEM was
removed, and cortices were incubated at 37˚C for 7 minutes with pre-heated (37˚C) Trypsin-
EDTA (0.05%, Invitrogen). After incubation, the Trypsin-EDTA solution was aspirated and the
cortices were washed twice in plating media (MEM supplemented with 10% fetal bovine serum,
10% heat-inactivated horse serum, and 2M dextrose). This was followed by tituration with a
flamed glass Pasteur pipette to dissociate the cells. The resultant homogeneous mixture of cells
was then filtered through a 0.75µm cell strainer, into fresh media. Tryphan blue staining and a
haemocytometer were used to determine the concentration of live cells. The cells were diluted in
media consisting of 50% plating media and 50% growth media (Neurobasal supplemented with
B-27 supplement, 1% fetal bovine serum, and 1% glutamine) and were then plated in Poly-D-
Lysine coated Corning CellBind plates (VWR) at a density of 6 x 105cells/cm2 or on Poly-D-
Lysine coated, acid-washed, coverslips at a density of 3.75 x 104cells/cm2. The cells were left
49
overnight in a humidified, 5% CO2 incubator at 37˚C. The following day (day in vitro 1 or
DIV1) a half media change was done with growth media, and on DIV4 a half media change was
done with growth media containing 10µM 5-fluoro-deoxyuridine (Sigma) to inhibit glial growth.
Half-media changes were then performed every 3 days until DIV12 when they were used for
experiments.
3.3 In vitro stroke model – Oxygen Glucose Deprivation
Oxygen Glucose Deprivation (OGD) was used as the in vitro model of ischemia. DIV12
neurons were washed three times in degassed, glucose-free Hanks Balanced Salt Solution
(HBSS) and maintained at 37˚C in an anoxic chamber (Thermo EC) with a 5% CO2, and 95% N2
(<0.01% O2) atmosphere. HBSS containing MCN was used to isolate for the ‘TRPM7 like’
currents. MCN consists of MK-801 (10µM, Sigma-Aldrich), CNQX (25µM, Sigma-Aldrich),
and Nimodipine (2µM, Sigma-Aldrich) was used to block glutamate and voltage gated calcium
channels and isolate the ‘TRPM7-like’ currents. Gd3+(10µM) treatment was used to inhibit
TRPM currents and Y-27632(10 µM) was used to block ROCK2 proteins. In control
experiments neurons treated with HBSS alone or with HBSS in combination with MCN, MCN
Gd3+ or MCN Y-27632, were incubated for two hours in a humidified 5% CO2 incubator.
Following the 2 hour treatment (OGD or HBSS) the cells were either placed in fresh growth
media (for cytotoxicity, viability, and apoptosis assay), or fixed (for immunofluorescent
analysis), or proteins were extracted (for western blot analysis). HBSS was comprised of
5.33mM KCl, 0.441mM KH2PO4, 4.17 mM NaHCO3, 137.93 mM NaCl, 0.338mM Na2HPO4,
1.7mM CaCl2, 2.3mM MgCl2 and 5.56mM D-Glucose (all Sigma-Aldrich), with a pH 7.4.
50
Glucose and MgCl2 were completely removed from the glucose-free solution which was
subsequently degassed overnight to form the OGD media.
3.4 Protein Extraction
Following the experimental treatment, cells were washed twice in PBS and collected in
pre-chilled microtubes with a cell scraper. The cells were centrifuged at 1000 rpm for 5 minutes.
The supernatant was then removed and the pellet was resuspended in an equal volume of ice-
cold Cell Lytic Mammalian Cell Lysis Reagent (Sigma-Aldrich) containing phosphatase and
protease inhibitors (phosphatase inhibitor cocktail II [Sigma-Aldrich] and protease inhibitor
cocktail [Roche]). The cells were incubated for 15 minutes on ice with mixing at the halfway
point and then centrifuged for 10 minutes at 10,000xg to pellet the unlysed cells and debris. The
supernatant was transferred to a fresh microtube which was then stored at -80˚C for later use.
3.5 DC-Lowry Protein Assay
Cell extracts were quantified in 96 well plates using the DC protein assay (Bio-Rad). Cell
extracts of proteins samples were made in 1/10th dilution. Protein standards ranging from 0.0625
to 1mg/ml were made using bovine serum albumin (Bio-Rad). Five µL of each standard and
sample was loaded into a separate well; followed by the addition of 25µL of reagent A’ and
200µL of reagent B. After 15 minutes incubation at room temperature, absorbance was measured
at 750nm using Synergy-HT (Bio-Tek) microplate reader. If protein concentrations were outside
51
the standard curve, the assay was repeated with new protein dilutions to ensure correct
determination of protein concentration.
3.6 SDS – Polyacrylamide Gel Electrophoresis (SDS – PAGE)
Standardized protein extracts of the DIV12 experiments were separated by size using the
Bio-Rad Mini-Protein SDS-PAGE system. The proteins were run on discontinuous gels with a
4% stacking layer and a 7% or 10% separating layer depending on the size of the proteins. The
stacking gel consists of 0.5M Tris-HCl, pH 6.8 and 4% SDS; the separating gel consists of 1.5M
Tris-HCl, pH8.8, and 0.4% SDS. Gels were run at 90V for 20 minutes, until the proteins reached
the separating layer, and then were run at 100V for 90 – 150 minutes. Once running was
complete the proteins were transferred onto 0.4 µm pore-sized nitrocellulose membranes (Bio-
Rad) in 1X Transfer Buffer (292mM Tris, 192mM Glycine, 20% methanol) at 100V for 70
minutes. To verify proper transfer, the nitrocellulose membranes were temporarily stained with
Ponceau S (0.1% w/v Ponceau in 3% v/v acetic acid). The stain was then removed by washing 3
times in TBST for 5 minutes each.
3.7 Western Blotting
Nitrocellulose membranes containing proteins were blocked for 1 hour at room
temperature in TBST containing 5% BSA with gentle agitation. Membranes were then incubated
with primary antibody solution overnight, at 4˚C with gentle agitation. The primary antibody
solutions were made in TBST with 2% BSA (see 3.1 for specific antibody dilutions). The
52
following day blots were washed 3 times in TBST (5 minutes each). After washing, the blots
were incubated for 1 hour at room temperature under gentle agitation with secondary antibody
solutions consisting of TBST with 1% BSA and HRP-conjugated secondary antibody goat anti-
mouse (Sigma-Aldrich) or mouse anti-rabbit (ECM biosciences). Blots were washed 5 times in
TBST (5 minutes each), developed with chemiluminescent substrate (Thermo Scientific) and
immediately visualized in ChemiDoc XRS imager (Bio-Rad) using Image Lab software (Bio-
Rad). Densiometric analysis of the bands of interest was performed using NIH ImageJ analysis
software.
3.8 Immunofluorescent Analysis and Imaging
Primary cortical neurons were plated on poly-D-Lysine coated glass coverslips (1µg/µl)
and then subjected to the experimental treatment on DIV12 as outlined in section 3.3. Following
treatment the cells were washed twice in PBS 1X, (Sigma-Aldrich) and then fixed with ice cold
1X PBS containing 4% paraformaldehyde for 15 minutes at room temperature. After fixing, cells
were washed 3 times (5 minutes each) with PBS and permeabilized using PBS containing 0.04%
Tween-20 (Sigma-Aldrich) for 15 minutes at room temperature. Cells were then washed 3 times
(5minutes each) with PBS 1X and blocked in blocking solution (consisting of PBS with 5%
BSA) for 1 hour at room temperature. After blocking, cells were incubated overnight at 4˚C with
primary antibody solution (see 3.1 for specific antibody dilutions) containing the antibody of
interest in PBS with 2% BSA. The following day the cells were washed 3 times (5 minutes each)
with PBS and incubated, for 1 hour at room temperature, with secondary antibody solutions
consisting of PBS with 2% BSA and secondary antibodies (Invitrogen): Alexa Fluor 488 donkey
53
anti- rabbit (1:1000) and Alexa Fluor 555 donkey anti-mouse (1:1000). The cells were then
washed 3 times with PBS and coverslips mounted on glass slides using ProLong® Gold anti-fade
reagent with DAPI (Life technologies). Epifluorescent images were captured with an Olympus
IX81 microscope imaging system with a Q-imaging Fast 1394 Rolera-MGi Plus camera using
Metamorph software.
3.9 Cell death, viability, and apoptosis assay
Primary rat cortical neurons were plated at 3 x 105cells/cm2 in 96 well plates and
subjected to 2 hours of HBSS (HBSS, HBSS MCN, HBSS MCN Gd3+) or OGD (OGD, OGD
MCN, OGD MCN Gd3+) treatment on DIV12. Then the treatment solution (HBSS or OGD) was
aspirated and fresh growth media was added to the cells. Cytotoxicity, viability and apoptosis
were measured at 24 hours following treatment, using the ApoTox-Glo Triplex Assay
(Promega). In short, at the 24 hour time point, viability/cytotoxicity reagent was added to each
well followed by 40-50 minute incubation in humidified 5% CO2 incubator at 37˚C. After
incubation cell viability fluorescence was measured at 360Ex/485Em and cytotoxicity fluorescence
was measured at 485Ex/520Em using Synergy-HT (Bio-Tek) microplate reader. Caspase-Glo 3/7
reagent was then added to each well and the cells were again incubated for 40-50 minutes in a
humidified 5% CO2 incubator at 37˚C. Apoptosis was then quantified by measuring
luminescence with Synergy-HT (Bio-Tek) microplate reader.
Cytotoxicity was also measured using Hoechst and Propridium iodide staining in primary
rat cortical neurons plated in 12 well plates. After the experimental procedure on DIV12,
treatment solution (HBSS or OGD) was aspirated and fresh growth media was added to cells,
54
which were then placed in humidified, 5% CO2 incubator. After 24 hours, Hoechst and
propidium iodide (PI) was added to the cells incubated for 30 minutes in a humidified, 5%CO2
incubator. Following incubation epifluorescent images (20X) were taken with an Olympus IX81
microscope imaging system with a Q-imaging Fast 1394 Rolera MGi Plus camera using
Metamorph software. Cytotoxicity was quantified by counting the number of dead (PI stained)
cells over the total number of cells (Hoechst stained) in each captured field.
3.10 Statistical Analysis
The data are presented as the mean ± SEM. Statistical comparisons were performed by
either Student’s T-test or one way analysis of variance (ANOVA). Dunnett’s multiple
comparisons test was performed for multiple comparisons in situations where significant
statistical differences were observed. All experiments were repeated at least three times (N=3)
unless otherwise noted.
55
Chapter 4
4 Results
4.1 Neuronal survival following OGD: cytotoxicity and viability
Rodent cortical neurons exposed to oxygen glucose deprivation (OGD) for periods up to
1 hour undergo neuronal cell death that is predominantly mediated by excitotoxicity (Goldberg
and Choi, 1993). Excitotoxicity is also the predominant mechanism responsible for ischemic
neuronal death in vivo (Goldberg and Choi 1993). More recently non-excitotoxic mechanisms of
neuronal death, mediated by cell surface and intracellular cation channels have been implicated
in mediating ischemic and anoxic neurotoxicity. Aarts and colleagues (2003) first showed that
although excitotoxicity is an early contributor of ischemic neuronal death, TRPM7 channels are
likely to be more important in neuronal death during prolonged exposure (>1.5 hours) to anoxia.
TRPM7 knockdown or Gd3+ treatment can prevent cell death in primary neurons exposed to up
to 3 hours of anoxia (Aarts et al. 2003). In addition, it has recently been proposed that ROCK
acts as a mediator of excitotoxic cell death (Jeon et al. 2013; Wang and Liao 20103). ROCK
protein inhibition protects against ischemic damage both in vitro and in vivo (Yamashita et al.
2007; Gisselsson et al. 2010). Recent data from our research group shows that TRPM7 activation
impairs neuronal survival through hyper-activation of the actin depolymerizing protein, cofilin-1
(Bent 2011). Since ROCK proteins can regulate cofilin activity through LIM kinase, it is possible
that ROCK signalling is responsible for the cofilin mediated neuronal pathology observed during
TRPM7 activation.
56
In order to study the potential downstream mediators of TRPM7 in anoxia I first needed
to become proficient in primary neuronal culture techniques and reproduce cell death
experiments using prolonged OGD. I exposed primary cortical neurons to 2 hours of OGD in the
presence of MCN (to block excitotoxicity and isolate for TRPM7 currents), MCN Gd3+ (to
inhibit TRPM7-channels) and MCN Y-27632 (to inhibit ROCK2). Control experiments were
performed by incubating neurons for 2 hours in HBSS, HBSS MCN, HBSS MCN Gd3+ and
HBSS MCN Y-27632 (see Figure 2B, 2D, and 2B). Cytotoxicity, viability and apoptosis
(discussed in 3.9) were assessed 24 hours following treatment by using ApoTox-Glo Triplex
assay (see Figure 2A - D), a new assay to our lab, which allows for the evaluation of
cytotoxicity, viability and apoptosis in the same cell-based assay well.
Live cell protease activity is measured using GF-AFC, a membrane permeable peptide
substrate, which enters cells and is cleaved by the live-cell protease activity to generate a
fluorescent signal proportional to the number of living cells. These live-cell proteases become
inactive in cells that have lost their membrane integrity thereby allowing GF-AFC to label live
cells exclusively. Dead cell protease activity is measured using bis-AAF-R110 (membrane
impermeable substrate) which is cleaved by dead cell proteases released from cells that have lost
their membrane integrity. The fluorogenic products AFC and R110 have different excitation and
emission spectra (AFC = 400Ex/505Em; R110= 485Ex/520Em) thereby allowing viability and
cytotoxicity to be measured simultaneously. The plate reader used in this assays did not have a
filter to match the recommended excitation and emission values for the viability assay thus the
next closest filter was used for viability (360Ex/485Em); cytotoxicity was measured at the
recommended values (485Ex/520Em). The 2 hour OGD treatment resulted in a 79 ± 6 % increase
in cytotoxicity over HBSS control (see Figure 2A), and this difference between HBSS and
57
OGD was significant (p<0.05). This is consistent with previous reports showing enhanced
neuronal cell death following exposure to OGD (Goldberg and Choi, 1993, Aarts et al. 2003).
However, in the MCN treated neurons exposed to OGD, cell death values significantly fell back
to control levels (p<0.01). Equivalent significant decreases in cell death were also observed for
the MCN Gd3+ and the MCN Y-27632 treated neurons exposed to OGD (p<0.01 for both). The
cytotoxicity assay results were complemented by the viability assay (see Figure 2C), which
revealed a 30 ± 3 % decrease (p<0.01) in viability for the OGD treated groups over HBSS
control, whereas the OGD MCN and OGD MCN Gd3+ treated cultures exhibit viability values
equivalent to the HBSS controls (P<0.01 for both). Control experiments reveal that the 2 hour
incubation with HBSS, HBSS MCN, HBSS MCN Gd3+ and HBSS MCN Y-27632 had no
observable cytotoxic effects (see Figure 2B, 2D).
Surprisingly, the OGD MCN, OGD MCN Gd3+, and OGD MCN Y-27632 treated groups
exhibited equivalent levels of neuroprotection; no significant differences in cytotoxicity or
viability were observed between OGD MCN, OGD MCN Gd3+and OGD MCN Y-27632 treated
cultures suggesting that MCN treatment alone was sufficient at rescuing the OGD phenotype
(see Figure 2A, 2B). To confirm the validity of the ApoTox Triplex assay data, another
cytotoxicity assay was performed using Hoechst (which labels the nuclei of all cells) and
propidium iodide (PI; labels only dead cells) double staining. Hoechst and PI positive cells were
counted in HBSS Ctrl, OGD, OGD MCN, OGD MCN Gd3+, and OGD MCN Y-27632 treated
neuronal cultures. Exposure to OGD resulted in a 246 ± 86 % increase in cell death relative to
the HBSS control (p<0.001) as indicated by the increased number of PI positive neurons (see
Figure 3). OGD MCN, OGD MCN Gd3+, and OGD MCN Y-27632 treatments cause equivalent
reductions in cell death that were highly significantly different (p<0.001) in comparison to OGD
58
exposure alone. Similar to the ApoTox Triplex assay data, MCN alone appeared sufficient to
prevent OGD-mediated cell death. The cell death studies show that I was successful at growing
neurons that are susceptible to OGD and can be protected by MCN. However, these findings are
inconsistent with the Aarts et al (2003) which showed significant neuronal cell death during
prolonged anoxia (>1.5 hours) despite MCN treatment. Variability in the cell culture models (rat
vs mouse) as well as contamination by glia might account for the increased effectiveness of the
MCN treatment.
Figure 2. Cytotoxicity following oxygen glucose deprivation.
Embryonic cortical neurons were exposed to 2 hours of oxygen glucose deprivation (OGD) in
the presence of MCN, MCN Gd3+ and MCN Y-27632. Control treatments were exposed for 2
hours with HBSS, HBSS MCN, HBSS MCN Gd3+, and HBSS MCN Y-27632. Cytotoxicity and
viability were quantified according to the emission spectra of the cytotoxic product (R110) and
viability product (AFC) (n=3). (A) OGD treatment alone displays a significant 79 ± 6 %
increase in cytotoxicity relative to control, while OGD treatment in combination with MCN,
MCN Gd3+ or MCN Y-27632 displayed significantly lower cytotoxicity values in comparison to
OGD alone. (B) The HBSS MCN, HBSS MCN Gd3+, and HBSS MCN Y-7632 control
treatments had no apparent effect on neuronal cytotoxicity. (C) Viability significantly decreased
by 30 ± 3 % for the OGD treatment in comparison to HBSS control, whereas OGD MCN and
OGD MCN Gd3+ treatments showed significantly enhanced viability in comparison to OGD
alone. (D) The HBSS MCN, HBSS MCN Gd3+ and HBSS MCN Y-7632 control treatments had
no apparent effect on neuronal viability (n=3). All values are normalized to HBSS control, each
59
column represents mean ± SD. Results were analyzed via one-way ANOVA followed by
Dunnett’s multiple comparison test; * and ** indicate P<0.05 and P<0.01, respectively, in
comparison to control (HBSS Ctrl); # and # # indicate P<0.05 and P< 0.01, respectively, in
comparison to OGD treatment. MCN = MK801 (10µM), CNQX (25 µM) and Nimodipine (2
µM) (used to isolate for TRPM7 currents); Gd3+ = Gadolinium (10 µM); Y-27632 = ROCK
inhibitor (10µM).
HBSS Ctrl
OG
D
OG
D MCN
OG
D MCN G
d3+
OGD MCN Y
-276
320
50
100
150
200
250
% C
yto
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A
*
# ## #
# #
60
HBSS Ctrl
HBSS MCN C
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HBSS MCN G
d3+ C
trl
HBSS MCN Y
-276
32 C
trl0
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B
HBSS Ctrl
OG
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OGD M
CN
OG
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d3+
OGD MCN Y
-276
320
50
100
150
% V
iab
ility
**
# # # #
C
61
Figure 3: Evaluation of OGD mediated cytotoxicity using Hoechst and
propidium iodide staining.
Embryonic cortical neurons were exposed to 2 hours of oxygen glucose deprivation (OGD) in
the presence of MCN, MCN Gd3+ and MCN Y-27632. Control treatments were exposed for 2
hours with HBSS. Cytotoxicity was quantified by comparing the number of dead cells (PI
positive) to total cells (Hoechst positive) (A) Hoechst and PI double staining reveal a 246 ± 86 %
increase in cell death (p<0.001) for the OGD treated samples relative to the HBSS control (n=4).
(B) Epifluorescent images (20X) of the Hoechst and PI double labelling assay; PI = labels dead
HBSS Ctrl
HBSS MCN C
trl
HBSS MCN G
d3+ C
trl
HBSS MCN Y
-276
32 C
trl0
50
100
150
% V
iab
ility
D
62
cells only; Hoechst = labels all cell nuclei. All values are normalized to HBSS control, each
column represents mean ± SD. Results were analyzed via one-way ANOVA followed by
Dunnett’s multiple comparison test, *** indicate P<0.001 in comparison to control (HBSS Ctrl);
# # # indicates P<0.001, in comparison to OGD treatment. MCN = MK801 (10µM), CNQX (25
µM) and Nimodipine (2 µM) (used to isolate for TRPM7 currents); Gd3+ = Gadolinium (10 µM);
Y-27632 = ROCK inhibitor (10µM).
HBSS Ctrl
OG
D
OGD M
CN
OG
D MCN G
d3+
OGD MCN Y
-276
320
100
200
300
400
500
% C
yto
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icity
(PI/H
oe
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st)
Re
lativ
e to
HB
SS
Ctr
l ***
# # # # # # # # #
A
63
64
4.2 Necrosis is the dominant form of cell death following OGD exposure while
apoptosis is more prevalent following NMDAR and TRPM7 inhibition
To gain some insight into the post ischemic cell death mechanisms, 24 hours after
exposure to OGD neuronal cultures were probed for caspase activity using the Caspase-Glo3/7
reagent (of ApoTox Triplex Assay). The caspase-Glo® 3/7 reagent contains a luminogenic
DEVD-peptide substrate for caspase3/7 and a thermostable luciferase. Caspase3/7 cleavage of
DEVD-peptide substrate releases luciferin, a substrate for luciferase that generates light. The
light generated is quantified using a luminometer. The 2 hour exposure to OGD resulted in a 31
± 4 % decrease (p<0.05) in apoptosis in comparison to HBSS control group suggesting that non-
apoptotic mechanisms contribute to the OGD mediated neuronal cell death (see Figure 4A). The
OGD MCN, OGD MCN Gd3+ and OGD MCN Y-27632 treatment groups displayed a 28±8%,
35±8% and 32±9% increase in apoptosis in comparison to HBSS Ctrl, respectively. In
comparison to OGD treatment, OGD MCN, OGD MCN Gd3+ and OGD MCN Y-27632
displayed a 59 ± 24%, 66 ± 22% and 63 ± 25% increase respectively and the latter two increases
were significant in comparison to OGD treatment (P<0.05). Some neuronal apoptosis was
observed among all control groups, likely due to stress associated with the experimental
procedure (i.e. removal of cultures out of incubator, adding and removing treatment solution).
However, apoptosis values did not differ significantly between HBSS, HBSS MCN, HBSS MCN
Gd3+, and HBSS MCN Y-27632 control groups suggesting that MCN, Gd3+, and Y-27632
treatments do not affect cell death fate under control conditions (see Figure 4B). The decreased
apoptosis during OGD indicates that the majority of neuronal cell death reported by the
cytotoxicity assays is necrotic, consistent with previous models of ischemic cell death (Martin et
al. 1998). Interestingly, inhibition of both excitotoxicity (MCN) and TRPM7 (Gd3+) was
65
sufficient at changing neuronal cell death fate towards apoptosis. Similarly inhibition of
excitotoxicity and ROCK activity was equally effective at changing neuronal cell death fate
towards apoptosis suggesting that TRPM7 mediated and ROCK mediated signalling influences
neuronal cell death fate in OGD.
Figure 4. Apoptosis is not the predominant cell death mechanism observed in
neurons following OGD exposure.
Embryonic cortical neurons were exposed to OGD for 2 hours. Apoptosis was assayed 24 hours
post-OGD treatment using a caspase 3/7 dependent luciferase assay. (A) OGD treated cultures
display a 31 ± 4 % reduction in apoptosis relative to HBSS controls. On the other hand, OGD
MCN, OGD MCN Gd3+ and OGD MCN Y-27632 treated neurons display a 28±8%, 35±8% and
32±9% increase in apoptosis, respectively, in comparison to HBSS Ctrl, and 59 ± 24%, 66 ±
22% and 63 ± 25% increase in apoptosis respectively, relative to OGD. (B) HBSS, HBSS MCN,
HBSS MCN Gd3+, and HBSS MCN Y-27632 treated controls cultures display equivalent levels
of apoptosis. All values are normalized to HBSS control, each column represents mean ± SD.
HBSS control and OGD values were analyzed via paired T-test; * indicates P<0.05 relative to
HBSS Ctrl; whereas all other comparisons with OGD were performed via unpaired T-test; #
indicates P<0.05 relative to OGD. MCN = MK801 (10µM), CNQX (25 µM) and Nimodipine (2
µM) (used to isolate for TRPM7 currents); Gd3+ = Gadolinium (10 µM); Y-27632 = ROCK2
inhibitor (10µM).
66
HBSS Ctrl
OG
D
OGD M
CN
OG
D MCN G
d3+
OGD MCN Y
-276
320
50
100
150
200
% A
po
pto
sis
*
A
# #
HBSS Ctrl
HBSS MCN C
trl
HBSS MCN G
d3+ C
trl
HBSS MCN Y
-276
32 C
trl0
50
100
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% A
po
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B
67
4.3 The effects of oxygen glucose deprivation on ROCK 2 activity
Recent findings from our research group reveal a decrease in LIMK and cofilin
phosphorylation following a two hour exposure to OGD (Bent 2011). In addition, TRPM7
inhibition during OGD significantly enhanced LIMK and cofilin phosphorylation relative to
OGD alone (Bent 2011). Given that LIMKs are downstream targets of ROCKs, ROCK2 activity
was measured to investigate a potential involvement in the OGD and TRPM7 mediated effects of
LIMK previously reported by our research group. It has previously been suggested that trans-
autophosphorylation of the ROCK activation loop is essential for protein activity (Chen et al.
2002). Thus, to determine whether ROCK2 activation is involved in OGD or TRPM7 mediated
cell death, phosphorylation of ROCK2 levels within its activation loop was measured using an
anti-ROCK2 (phospho – T249) antibody. DIV12 primary cortical neurons were exposed to 2
hours of OGD with or without MCN, MCN Gd3+, and MCN Y-2732. Results reveal that ROCK2
phosphorylation was only statistically different from HBSS control in the presence of the ROCK
specific inhibitor Y-27632 (see Figure 5). Y-27632 treatment resulted in a 34 ± 3% decrease
ROCK2 phosphorylation (p<0.05) in comparison to HBSS control. Neither OGD MCN nor Gd3+
treatment had any observable effect on ROCK2 phosphorylation. Although previous studies
suggest ROCK family proteins play a role in ischemic pathology (Yamashita et al. 2007;
Gisselsson et al. 2010) the current data suggests that the activating phosphorylation at Threonine
249 is not involved in ROCK2 activity following in vitro exposure to anoxia.
68
Figure 5. OGD, MCN, and Gd3+ do not affect phosphorylation of ROCK within the
activation loop.
Proteins were extracted from DIV12 neurons following a 2 hour treatment with HBSS, HBSS
MCN, HBSS MCN Gd3+, OGD, OGD MCN, OGD MCN Gd3+, and OGD MCN Y27632. To
determine whether phosphorylation within the activation loop of ROCK2 changes following the
various treatments, cell extracts were separated via SDS-PAGE and probed for anti-ROCK2
(phospho-T249) antibody and mouse anti-rabbit-HRP secondary. Total ROCK2 was measured
using anti-ROCK2 antibody and mouse anti-rabbit-HRP secondary. Results show a significant
decrease (34± 3%) in ROCK2 phosphorylation only for the OGD MCN Y-27632 treated
cultures. All values are normalized to HBSS control, each column represents mean ± SD. Results
were analyzed via one-way ANOVA followed by Dunnett’s multiple comparison test, as well as
via an unpaired T-test, ** signifies P<0.01 relative to HBSS control. HBSS= Hanks Buffered
Salt Solution; MCN = MK801 (10µM), CNQX (25 µM) and Nimodipine (2 µM) (used to isolate
for TRPM7 currents); Gd3+ = Gadolinium (10 µM); Y-27632 = ROCK inhibitor (10µM).
69
4.4 Oxygen Glucose Deprivation attenuates P-21 activated kinase 3 activity
Recent findings from our research group report a decrease in LIMK1 activity
(dephosphorylation) following OGD and this decrease correlates with enhanced cofilin activity
(dephosphorylation) (Bent 2011). Furthermore, inhibition of TRPM7 currents with Gd3+ or
suppression of TRPM7 expression, using siRNA specific for TRPM7, impaired the cofilin
hyperactivation observed in the OGD treatment (Bent 2011). Group I PAKs are known to
activate LIMK proteins via phosphorylation of critical threonine residues (Arias-Romero and
Chernoff 2008). In addition, Group I PAK protein activity is regulated by phosphorylation of
70
critical residues within and outside the kinase domain (Edwards and Gill, 1999). Thus, PAK3
phosphorylation at Ser-144, a site known to contribute to PAK3 kinase activation (Chong et al.
2001), was measured to determine whether PAK3 activation is altered following anoxia and
whether TRPM7 lies upstream of PAK3 regulation. TRPM7 activity was isolated during OGD
using MCN and subsequently blocked with Gd3+; changes in PAK3 activity were then
determined as a measure of PAK3 phosphorylation (see Figure 6). The HBSS MCN control
group displayed similar levels of PAK3 phosphorylation as HBSS control, indicating NMDAR,
AMPA/kainite receptor, and voltage-gated Ca2+ channels have no baseline effect on PAK3
activity. Despite an apparent 18 ± 2% increase in PAK3 phosphorylation for the HBSS MCN
Gd3+ control group this difference was not significant relative to the other two controls further
suggesting that inhibition of TRPM7 channels has no baseline effect on PAK3 activity. A
significant reduction in PAK3 phosphorylation was observed for the OGD (38±5 %;p<0.001),
OGD MCN (26±3 %;p<0.05), and OGD MCN Gd3+ (25± 0.5% ;p<0.05) cultures compared to
control. Given that PAK3 activity is regulated by phosphorylation; these results indicate that
PAK3 activity is reduced following oxygen glucose deprivation and may therefore lie upstream
of reduced LIMK activity in anoxia. Furthermore, this reduction in PAK3 activity during OGD,
in comparison to HBSS control, appears to be independent of glutamate excitotoxicity and
TRPM7 inhibition as neither MCN nor MCN Gd3+ treatment were able to rescue the decrease in
PAK3 activity during OGD.
71
Figure 6. OGD attenuates PAK3 phosphorylation
Proteins were extracted from DIV12 neurons following a 2 hour treatment with HBSS, HBSS
MCN, HBSS MCN Gd3+, OGD, OGD MCN and OGD MCN Gd3+. To determine the effects of
OGD and TRPM7 activity on PAK3 phosphorylation cell extracts were separated via SDS-
PAGE and probed for anti-phospho PAK3 antibody and mouse anti-rabbit-HRP secondary. Total
PAK3 was measured using anti-PAK3 antibody and mouse anti-rabbit-HRP secondary. All
values are normalized to HBSS control, each column represents mean ± SD. Results were
analyzed via one-way ANOVA followed by Dunnett’s multiple comparison test, * and ***
represent P < 0.05 and P< 0.001,respectively, in comparison to HBSS control. HBSS= Hanks
Buffered Salt Solution; MCN = MK801 (10µM), CNQX (25 µM) and Nimodipine (2 µM) (used
to isolate for TRPM7 currents); Gd3+ = Gadolinium (10 µM).
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4.5 Modulation of SSH1 activity by TRPM7 following oxygen glucose
deprivation.
As previously mentioned LIMK activation is mediated via phosphorylation by both Rho-
activated kinases and p-21 activated kinases; whereas dephosphorylation of LIMK via
phosphatases results in inhibition of kinase activity. Slingshot phosphatase 1 is known to
dephosphorylate both LIMK and cofilin (Ohta et al. 2003, Soosairajah et al. 2005).
Phosphorylation of C-terminal residues Ser-937 and Ser-978 of SSH1 promotes 14-3-3 binding
which inhibits SSH1 phosphatase activity (Nagata-Ohashi et al. 2004). To investigate whether
SSH1 plays a role in the LIMK and cofilin dephosphorylation following OGD, cortical neurons
were exposed to 2 hours of OGD. The role of TRPM7 in regulating SSH1 activity was also
investigated by isolating for TRPM7 currents through MCN, and then inhibiting those currents
with Gd3+. The HBSS, HBSS MCN, and HBSS MCN Gd3+ controls did not show statistically
significant differences in SSH1 phosphorylation (see Figure 7). A small but statistically
significant decrease in SSH1 phosphorylation (13± .7%), was observed for the OGD treatment
group in comparison to HBSS control (paired T-test; p= 0.02). OGD MCN treatment caused a
25± 9% increase in SSH1 phosphorylation relative to OGD; however, this difference was not
statistically significant. A 76 ± 20% increase in SSH1 phosphorylation was observed for the
OGD MCN Gd3+ cultures, relative to OGD alone; this result was statistically significant (p <
0.01). Overall these results suggest that TRPM7 may be involved in SSH1 regulation during
oxygen glucose deprivation.
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Figure 7. TRPM7 modulates SSH1 activity
Proteins were extracted from DIV12 neurons following a 2 hour treatment with HBSS, HBSS
MCN, HBSS MCN Gd3+, OGD, OGD MCN and OGD MCN Gd3+. To determine whether SSH1
activity is modulated by OGD and TRPM7, cell extracts were separated via SDS-PAGE and
probed for anti-phospho S978 SSH1 antibody and mouse anti-rabbit-HRP secondary. Total
SSH1 was measured using anti-SSH1 antibody and mouse anti-rabbit-HRP secondary. All values
are normalized to HBSS control, each column represents mean ± SD. Results were analyzed via
one-way ANOVA followed by Dunnett’s multiple comparison test, as well as paired T-test. *
represents P < 0.05 relative to HBSS control, and ## represents P<0.01 relative to OGD. HBSS=
Hanks Buffered Salt Solution; MCN = MK801 (10µM), CNQX (25 µM) and Nimodipine (2 µM)
(used to isolate for TRPM7 currents); Gd3+ = Gadolinium (10 µM).
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4.6 Immunofluorescent analysis of intracellular protein localization
Work from our research group has focused on identifying the proteins which interact with
TRPM7, and proteins that form a signalling relationship with TRPM7 in mediating neuronal cell
death. This work led to the identification of LIMK1 and cofilin-1 signalling transduction partners
downstream of TRPM7 in OGD. Although a direct interaction between TRPM7 and LIMK1 or
cofilin has not been shown yet, biochemical analysis of the signalling pathways behind TRPM7
activation during OGD and its consequent inhibition following Gd3+ treatment has implicated
TRPM7 in mediating cofilin hyperactivation (dephosphorylation) during OGD (Bent, 2011). To
determine whether the observed changes in PAK3 and SSH1 phosphorylation influenced protein
localization, immunofluorescent analysis of cortical neurons exposed to 2 hours of HBSS, OGD
and OGD MCN Gd3+ was performed. Successful immunostaining was achieved for LIMK1, P-
SSH1 and P-PAK3 (see Figures 8 and 9). LIMK1 labelling was largely concentrated in the
cytoplasm of the cell with strong staining along the perinuclear area, and no staining in the
nucleus and along the cell processes (see Figure 8 and Figure 9). P-PAK3 shows strong
fluorescence in the cell soma particularly around the perinuclear area; weak staining is observed
along cellular processes with no staining in the nucleus (see Figure 8). P-SSH1 labelling reveals
similar results as P-PAK3, with strong staining in the cell body and along the perinuclear area,
weak labelling in the cellular process, and no labelling in the cell nucleus (see Figure 9). Merged
images reveal colocalization of P-PAK3 and LIMK1 labelling (see Figure 8), similar
colocalization is observed for P-SSH1 and LIMK1 (see Figure 9). When comparing the
immunofluorescent staining patterns of LIMK1 no observable differences in cellular localization
can be seen between the three different treatment groups (HBSS, OGD, and OGD MCN Gd3+).
The same is true for P-PAK3 and P-SSH1 cellular localization. The data suggests that the
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intracellular localization of LIMK1 and its upstream regulators P-PAK3 and P-SSH1 is not
affected by OGD treatment or OGD MCN Gd3+ treatment.
Figure 8. LIMK1 and P-PAK3 co-immunostaining in cortical neurons
Following a two hour exposure of DIV12 cortical neuronal cultures to HBSS, OGD, or OGD
MCN Gd3+, co-immunostaining was performed with mouse-anti-LIMK1 antibody (abcam) and
rabbit-anti-PAK3 (phospho S154) antibody (abcam) followed by goat-anti-mouse Alexa Fluor
555 secondary and goat-anti-rabbit Alexa Fluor 488 secondary, respectively. Epifluorescent
images of cells were taken with a conventional microscope with a 60X objective. Nuclei were
counterstained with DAPI (present in the Prolong Gold® Antifade mounting reagent).
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Figure 9. LIMK1 and P-SSH1 co-immunostaining in cortical neurons
Following a two hour exposure of DIV12 cortical neuronal cultures to HBSS, OGD, or OGD
MCN Gd3+, co-immunostaining was performed with mouse-anti-LIMK1 antibody (abcam) and
rabbit-anti-SSH1 (phospho S978) antibody (ECM biosciences) followed by goat-anti-mouse
Alexa Fluor 555 secondary and goat-anti-rabbit Alexa Fluor 488 secondary, respectively.
Epifluorescent images of cells were taken with a conventional microscope with a 60X objective.
Nuclei were counterstained with DAPI (present in the Prolong Gold® Antifade mounting
reagent).
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Chapter 5
5 Discussion
In this study primary neuronal cultures, isolated from E18 Sprague Dawley rats, were
used to investigate neuronal survival and apoptosis following exposure to oxygen glucose
deprivation. Inhibition of the excitotoxic cascade via MCN (which also isolates for TRPM7
currents); and TRPM7 currents with Gd3+ were used to evaluate the neuroprotective effects of
TRPM7 channels. Consistent with previous findings from our research group, a two hour
exposure to oxygen glucose deprivation significantly enhanced neuronal death. Surprisingly,
MCN treatment alone was sufficient at restoring neuronal survival to control levels. As a
consequence of the complete recovery of neuronal cell death by the OGD MCN treatment
conclusions regarding TRPM7’s contribution to neuronal pathology during OGD could not be
made potentially due to the MCN treatment masking any neuroprotective effects from TRPM7
inhibition in the OGD MCN Gd3+ treatment. Similarly, due to the complete rescue of neuronal
cell death by MCN during 2 hours of OGD, it is still unclear whether inhibition of ROCK
activity contributes to any additive neuroprotection through non-excitotoxic mechanisms.
Recent findings from our research group linking TRPM7 activation to LIMK and cofilin
dephosphorylation led to the investigation of three upstream regulators of LIMK kinase activity,
ROCK2, PAK3 and SSH1, the latter of which is also a direct regulator of cofilin activity (Amano
et al. 2001,Ohta et al. 2003). Western blot analysis suggests that activating phosphorylation of
Thr-249 is not involved in ROCK2 activity following in vitro ischemia (OGD). Given numerous
studies linking ROCK2 to ischemic neuropathology (Yamashita et al. 2007;Jeon et al. 2013;
Wang and Liao 2012;Gisselson et al. 2009) it is tempting to speculate that ROCK2
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autophosphorylation is not essential for regulating ROCK2 activity during in vitro ischemia,
however, more work needs to be done before any conclusions can be made. PAK3
phosphorylation decreased following OGD and did not recover with either MCN or MCN Gd3+
treatment suggesting that glutamate and TRPM7 independent signalling mechanisms are
responsible for the suppression of PAK3 activity during OGD. A small decrease in SSH1
phosphorylation was observed during OGD treatment, and TRPM7 inhibition dramatically
enhanced SSH1 phosphorylation suggestive of a potential TRPM7 mediated regulation of SSH1
during ischemia.
To investigate whether the observed changes in SSH1 and PAK3 phosphorylation would
affect their subcellular localization and that of their downstream target LIMK1, E18 cortical
neurons exposed to two hours of HBSS, OGD or OBD MCN Gd3+ were co-stained with
antibodies against phospho-SSH1 and LIMK1 , and phospho-PAK3 and LIMK1.
Immunofluorescent analysis reveals strong labelling in the cell soma particularly in the
pericellular region for all three proteins LIMK1, P-PAK3 and P-SSH1. Surprisingly, differential
subcellular localization was not observed among the HBSS, OGD, and OGD MCN Gd3+ treated
groups for all three proteins.
5.1 Anti-excitotoxic treatment protects neuronal cultures against 2 hours of
oxygen glucose deprivation
The findings by Aarts and colleagues (2003) showing that TRPM7 channels may play a
more important role in ischemic cell death particularly during prolonged ischemia (>1.5 hours)
led to the evaluation of this model in the present study. OGD exposure led to significant
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increases in neuronal cell death (see Figure 2, 3) consistent with previous reports (Goldberg and
Choi 1993; Aarts et al. (2003). In contrast to the findings by Aarts et al. (2003), MCN treatment
alone was sufficient at restoring neuronal survival to control levels based on both cytotoxicity
and viability values of the ApoTox Glo Triplex assay (see Figure 2A and 2C). PI and Hoechst
co-staining has been previously used to measure excitotoxic cell death and was used here (see
Figure 3) to validate the results of the newer ApoTox Glo Triplex assay. The results reveal that
TRPM7 inhibition with Gd3+ did not yield any additive neuroprotective effect over MCN alone;
however, in my experiments MCN restored cell survival to control levels with no additional
room for the Gd3+ effects. It is likely that TRPM7 channels were activated as both OGD
treatment and the absence of Mg2+in the extracellular solution would contribute to enhanced
TRPM7 channel activity (Kozak and Cahalan, 2003; Aarts et al. 2003). Consistent with previous
research TRPM7 inhibition was achieved with 10µM Gd3+, a concentration shown to inhibit the
TRPM7 current as well as promote neuronal survival (Aarts et al. 2003; Bent 2011). Evaluation
of neuronal viability with longer OGD exposure (i.e. 3 hours) may yield differences between
MCN and Gd3+ treatments however, this would not fit with our previous research evaluating
TRPM7 signal transduction at 2 hours of OGD injury. There is an inherent variability in primary
neuronal cultures which need to differentiate in vitro, consist of mixed population of cells, and
come from different animals with each set of experiments and thus, my cultures may have been
more readily rescued by MCN than previous studies.
In addition to cytotoxicity, viability was also measured with the ApoTox-Glo Triplex
assay (promega). Interestingly, the viability measurements did not show the same magnitude of
changes across as observed for cytotoxicity. The cytotoxicity assay values revealed a 79 ± 6 %
increase in neuronal cell death (see Figure 2A); however, the viability assay reported a 30 ± 3 %
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decrease in neuronal survival (see Figure 2C) when an a difference of equivalent magnitude
between the cytotoxicity and viability assay values was expected. Due to the limitations of the
filters present in the Synergy-HT (Bio-Tek) microplate reader, viability was measured using an
excitation emission spectrum (360Ex/485Em) that was slightly different from the manufacturers’
(promega) recommended values (400Ex505Em). This use of a suboptimal excitation and emission
spectrum could explain the difference in magnitude between the cytotoxicity and viability values
of the ApoTox-Glo Triplex assay (promega). Despite the variations in cytotoxicity and viability
assay results, the overall data trends reveal that two hours of OGD severely impair neuronal
survival and that inhibition of the excitotoxic cascade (via MCN) is sufficient at rescuing
neuronal viability.
5.2 Different cell death mechanisms appear to dominate during OGD versus
TRPM7 inhibition.
The cell death mechanisms operating during ischemia are largely dependent on the
severity and duration the ischemic insult; consequently both necrotic and apoptotic phenotypes
have been observed in vitro as well as in vivo models (Martin et al. 1998, Lipton 1999; Unal-
Cevik et al. 2004). Apoptotic mechanisms are initiated following exposure to an adverse
stimulus such as ischemia (Lipton 1999); however, whether these mechanisms results in
apoptotic cell death is largely determined on the amount of available intracellular ATP (Leist et
al. 1997; Nicotera et al. 1999). Apoptosis is an energy dependent process (Leist et al. 1997;
Single et al. 2001), thus the dramatic depletion of ATP observed during a severe ischemic insult
would favor necrotic cell death. Cell death fate has profound implications for stroke
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pathophysiology because unlike the removal of cells with minimal disruption of tissue
architecture that occurs in apoptosis, necrotic cell death is pro-inflammatory causing damage to
surrounding regions that result in scarring (Ren and Savill 1998), which can profoundly
influence brain structure and ultimately impair critical brain functions and cognitive processes.
To gain some insight into the cell death mechanisms operating in our in vitro model of
ischemia (OGD) caspase activity was measured to quantify apoptosis. The apoptosis assay data
reveals a significant decrease in apoptosis for OGD treated cultures (Figure 4 A, B) in
comparison to HBSS control. Given the enhanced neuronal cell death observed for OGD (Figure
2, 3), the apoptosis assay data are suggestive that the majority of ischemic neuronal cell death is
non-apoptotic. These results are consistent with findings from Almeida et al. (2002) showing
that 1 hour of OGD results in necrotic neuronal cell death. Surprisingly, of the enhanced
apoptosis observed for the OGD MCN and OGD MCN Gd3+ cultures, increases for only the
latter group were significant indicating that inhibition of TRPM7 activity may influence neuronal
cell death fate. If inhibition of TRPM7 activity promotes apoptotic rather than necrotic cell death
in vivo, it can help explain the decreased tissue damage and preservation of memory tasks
observed in TRPM7 suppressed rodent hemispheres following global ischemia (Sun et al. 2009).
Furthermore, it allows for the potential recovery of neurons through anti-apoptotic interventions.
However, conclusions regarding the effects of TRPM7 activity on neuronal fate cannot
be made based on the apoptosis assay results. Since the OGD MCN and OGD MCN Gd3+ treated
groups show similar increases in neuronal apoptosis (see Figure 4), we cannot distinguish the
AET mediated effects on cell death from those of TRPM7 inhibition. Variation within samples is
a possible contributor to the non-significant enhancement of apoptosis for the OGD MCN
cultures; inhibition of NMDA and AMPA/kainite channels has been previously reported to
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expose apoptotic cell death in OGD treated neurons (Gwag et al. 1995). Furthermore, only
caspase 3 and 7 activity was recorded in the present study, providing a limited view of neuronal
apoptosis. Investigation of other pro-apoptotic factors (i.e. Bak, Bax) and nuclear markers (i.e.
DNA ladder pattern, TUNEL staining) would allow for a more comprehensive study of the AET
and TRPM7 mediated effects on apoptosis. In addition, the use of RNAi to knockdown TRPM7
expression would provide more direct evidence of the TRPM7 mediated effects in comparison to
the Gd3+ treatment.
5.3 Investigation of ROCK signalling in OGD mediated neuronal cell death
Recent reports from our research group show that during OGD, TRPM7 mediated
inhibition of LIMK and subsequent hyperactivation of cofilin result in the formation of cofilin-
actin rods (Bent 2011), aberrant structures associated with neurodegenerative disease (Maciver
and Harrington). Suppression of cofilin expression via siRNA led to a 20% reduction in neuronal
cell death in comparison to OGD, suggesting that the TRPM7 mediated cofilin hyper-activation
contributes neuronal cell death during OGD. Given that Rho associated kinases are known
regulators of LIMK activity (Amano et al. 2001; Ohashi et al. 2000) I decided to investigate
whether the TRPM7 mediated effects during OGD extend to the regulation of ROCK2. Based on
previous reports suggesting that trans-autophosphorylation within the activation loop of ROCK2
is essential for protein activity (Chen et al. 2002), phosphorylation of Thr-249 (located within
activation loop of ROCK2) was used to measure ROCK2 activity (Figure 5). Surprisingly, no
significant difference in ROCK2 phosphorylation was observed following OGD in comparison
to control and no differences in phosphorylation were observed across treatment groups with the
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exception of the cultures treated with ROCK2 inhibitor (OGD MCN Y-27632 treatment), which
showed a significant decrease in phosphorylation. Although this data suggests that ROCK2
activity is not affected during in vitro ischemia (OGD), numerous studies linking ROCK to
ischemia pathology suggest otherwise (Yamashita et al. 2007;Jeon et al. 2013; Wang and Liao
2012;Gisselson et al. 2009). It is possible that ROCK2 signalling may operate during the initial
stages of OGD, which would cause us to miss this signalling event by the 2 hour time point.
Indeed, ROCK activity has been shown to significantly increase in the striatum at 5 min
following in vivo ischemia and then progressively fall to control levels at 2 hours of ischemia
(Yamashita et al. 2007). This enhanced ROCK activity during initial stages of ischemia is
supported by studies linking ROCK protein signalling to glutamate mediated excitotoxicity
which operates within the same time course (Jeon et al. 2002; Gisselson et al. 2009). For
instance, induction of glutamate excitotoxicity in H19-7/IGF-IR (immortalized hippocampal cell
line) results in enhanced ROCK activity as early as 2 minutes and was sustained to 30 minutes
consistent with ROCK signalling events operating during early stages of the excitotoxic cascade
(Jeon et al. 2002). Thus, measuring ROCK2 activity at different time points during OGD
exposure would provide a more complete picture of its temporal regulation.
An alternative possibility is that ROCK2 activity is not regulated by Thr-249
phosphorylation, or that phosphorylation within the activation loop of ROCK2 is not necessary
for enzyme activity. Indeed analysis of the X-ray crystal structure of ROCK suggests the protein
kinase domain has a catalytically competent conformation in the absence of phosphorylation
(Jacobs et al. 2006). Thus, it still remains to be determined whether TRPM7 regulates ROCK
function during OGD, and whether the potential changes in ROCK activity contribute to the
LIMK and cofilin mediated effects observed by our research group.
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Given that upregulation of ROCK2 activity during ischemia onset contributes to neuronal
cell death (Yamashita et al. 2007), I wanted to determine whether disruption of ROCK signalling
in the presence of TRPM7 activity (OGC MCN Y-27632 treatment) would contribute to
enhanced neuroprotection. This way, the presence of additive neuroprotective effects over OGD
MCN treatment would link TRPM7 activity to ROCK. Previous in vitro and in vivo studies have
reported that inhibition of ROCK activity via fasudil or Y-277632 confers similar
neuroprotective effects as does NMDA channel inhibition (Yamashita et al. 2007; Gisselson et
al. 2009); however, the neuroprotective effects of ROCK inhibition in conjunction with anti-
excitotoxic therapy (MCN treatment) have not yet been investigated.
The cytotoxicity and viability assay results reveal no differences in neuroprotection
between OGD MCN, OGD MCN Gd3+ and OGD MCN Y-27632 treated groups (Figure 2,3). As
previously stated the observation that MCN alone completely recovers neuronal viability,
precludes any potential additive neuroprotective effects from being observed. Exposure of
neuronal cultures to 3 hours of OGD might expose differences in neuronal viability between
OGD MCN, OGD MCN Gd3+ and OGD MCN Y-27632 treatment groups. However, the
possibility that ROCK inhibition in the presence of MCN treatment does not result in additional
neuroprotection cannot be excluded as the cytotoxic effects of ROCK2 have been described to
operate downstream of NMDA channel activation (Jeon et al. 2002; Gisselson et al. 2009). Thus,
whether TRPM7 activity contributes to ROCK mediated cytotoxicity during OGD still remains
to be determined.
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5.4 Ischemic depression of PAK3 activity
Like ROCKs, P-21 activated kinases are another family of Ser/Thr protein kinases known
to phosphorylate LIMK (Edwards et al. 1999) and this LIMK phosphorylation is essential for its
kinase activity (Edwards and Gill 1999). Rac and Cdc42 are two members of the Rho family of
GTPases known to regulate PAK activity (Arias-Romero and Chernoff 2008) and recent studies
have implicated both Rac and Cdc42 in ischemic neuronal cell death (Zhao et al. 2007;
Lazarovici et al. 2012; Gutierrez-Vargas et al. 2010). For instance, a decrease in Rac activity has
been observed both in vivo and in vitro following a 2 hour exposure to ischemia (Lazarovici et
al. 2012). This depression in Rac activity has been shown to persist for up to 1 month post
ischemia (Gutierrez-Vargas et al. 2010). In contrast, Cdc42 activity is enhanced following
ischemia/reperfusion, and this Cdc42 upregulation is shown to contribute to delayed neuronal
cell death (Zhao et al. 2007).
Despite the recent interest in the role of Rho GTPases in ischemic pathology, there are
virtually no studies investigating the involvement of PAK proteins in ischemia. Given the
decrease in LIMK and cofilin phosphorylation following OGD observed by our research group,
an investigation into the role of PAK proteins was necessary. Evaluation of P-PAK3 cellular
organization during OGD and TRPM7 inhibition, reveal no differences in P- PAK3 localization
between treatments (Figure 8), suggestive that OGD treatment does not influence PAK3
localization. It is possible that P-PAK3 may exert its signalling effects in the cell soma. PAK3
phosphorylation levels were also measured to evaluate PAK3 activity in response OGD, AET
and TRPM7 inhibition. Results (Figure 6) reveal a significant reduction in PAK3 activity during
in vitro ischemia (OGD). However, these levels did not show significant recovery with either
anti-excitotoxic therapy (AET) or TRPM7 inhibition suggesting that neither the excitotoxic
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cascade nor TRPM7 activity contribute to the reduction in PAK3 activity during in vitro
ischemia. It is possible that the previously reported depression in Rac activity following ischemia
(Gutierrez-Vargas et al. 2010; Lazarovici et al. 2012) contributes to the observed decrease in
PAK3 activity. However, Rac activity levels following OGD exposure need to be evaluated to
confirm a potential involvement in PAK3 regulation. Never the less, the observed decrease in
PAK3 activity during OGD is suggestive of a potential contribution to the reduced
phosphorylation of LIMK and cofilin previously reported by our research group, albeit through
Glutamate independent and TRPM7 independent mechanisms.
PAK1, PAK2 and PAK3 share ~ 90% sequence homology in their regulatory PBD-AID
domain and over 93% homology in their kinase domain (Arias-Romero and Chernoff, 2008),
hence it is of interest to investigate whether PAK1 and PAK2 exhibit similar regulation during
OGD as does PAK3. Although some functional overlap between Group I PAKs exists, they also
exhibit distinct roles, as in regulating cell death. For instance, PAK1 activity is associated with
neuronal survival whereas PAK3 activation is linked to neuronal cell death (Kreis et al. 2009);
thus suppression of PAK3 during OGD could represent an endogenous neuroprotective
mechanism.
5.5 A role for SSH1 downstream of TRPM7 mediated activation in oxygen
glucose deprivation
In addition to evaluating the effects of known up-regulators of LIMK activity (i.e. ROCK
and PAK) during OGD and TRPM7 inhibition, attention was diverted to potential down-
regulators of LIMK activity. Slingshot phosphatase 1 (SSH1) has been described as a cofilin
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phosphatase, which removes the inhibitory phosphate group on Ser-3 and activates cofilin
(Kurita et al. 2008 ). In addition to cofilin dephosphorylation, SSH1 inhibits LIMK via
dephosphorylation of Thr-508, a critical phosphorylation site essential for LIMK activity
(Soosairajah et al 2005). Given that the phosphatase activity of SSH1 is restricted to LIMK and
cofilin, it is an ideal candidate for mediating the changes in LIMK and cofilin activity reported
by our research group. Thus, SSH1 activity was determined by the level of Ser-978
phosphorylation which suppresses the phosphatase activity of SSH1 (Nagata-Ohashi et al. 2004).
14-3-3 binding to phosphorylated C-terminal serine residues (Ser-937, Ser-978) performs this
SSH1 inhibition by interfering with the ability of SSH1 to bind to F-actin, which in turn prevents
the F-actin mediated activation of SSH1 (Nagata-Ohashi et al. 2004).
The enhanced SSH1 activity observed during OGD exposure (see Figure 7) is consistent
with previously reported reductions in phosphorylation of LIMK1 and cofilin during OGD (Bent
2011). In addition, inhibition of TRPM7 activity during OGD resulted in dramatic decrease in
SSH1 activity, which is in support with previous reports from our research group showing
enhanced LIMK and cofilin phosphorylation in the TRPM7 inhibited cultures exposed to OGD.
To determine whether these changes in SSH1 activity influenced its cellular organization
immunofluorescent images SSH1 were taken during OGD and TRPM7 inhibition. The
immunofluorescent analysis (Figure 9) revealed that the inactive pool of SSH1 (P-SSH1)
remained perinuclear and did not change between treatments suggesting that the ischemia does
not affect the localization of inactive SSH1. To date no studies have been performed to evaluate
the effects of anoxia/ischemia on SSH1 activity. The results of this study are the first to describe
changes in SSH1 activity in response to in vitro ischemia (OGD). Furthermore, these results are
the first to implicate TRPM7 channel signalling in the regulation of SSH1 activity. Thus, the
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western analysis data support a potential TRPM7 mediated signalling relationship with SSH1
which can be added to our working model of cofilin regulation during OGD.
Numerous studies provide support for a potential SSH1 mediated effect on cofilin activity
during anoxia. For instance, exposure of HeLa cells to H2O2 results in cofilin activation through
SSH1 phosphatase; oxidation of 14-3-3ζ promotes its dissociation from SSH1 allowing the
dissociated SSH1 to become activated by F-actin binding and consequently activate
(dephosphorylate) cofilin (Kim et al. 2009). In addition, SSH1 has been shown to mediate Ca2+
induced cofilin dephosphorylation via calcineurin in 293T and HeLa cells (Wang et al. 2004).
The authors describe a pathway whereby Ca2+ influx results in calcineurin activation; the active
calcineurin then dephosphorylates SSH1, increasing its cofilin-phosphatase activity (Wang et al.
2004). Given that TRPM7 activation during OGD has been shown to contribute to calcium influx
and oxidative stress (Aarts et al. 2003) it is possible that a calcineurin-SSH1-cofilin signalling
pathway could operate downstream of TRPM7 activity. In addition, TRPM7 may also regulate
SSH1 activity directly through 14-3-3 since co-immunoprecipitation experiments from our
research group show that 14-3-3 pulls down along with TRPM7. Additional studies are
necessary to confirm the western analysis data of SSH1 including in vitro phosphatase assays of
SSH1 to evaluate its cofilin-phosphatase activity; SSH1 knockdown and expression studies to
confirm that changes in SSH1 activity are sufficient at influencing cofilin phosphorylation; and
Ca2+ chelation and anti-oxidant treatments to evaluate the mechanisms behind SSH1 modulation.
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5.6 Model of TRPM7 mediated cell death.
The present study provides a new potential link into the TRPM7 mediated cofilin
hyperactivation recently described by our research group. The model outlined in Figure 10
reflects what may occur when a cell is under stress through OGD. Initially TRPM7 channels are
activated by the ensuing ATP depletion (Katsura et al. 1994), oxidative stress and Ca2+influx
from upstream channel activity (i.e. NMDA, AMPA) (Aarts et al. 2003; Arundine and
Tymianski 2003). TRPM7 channel activation results in further Ca2+ influx which contributes to
the generation of nitric oxide mediated ROS production (Aarts et al. 2003). The ensuing Ca2+
influx promotes further TRPM7 channel activity and ROS production through positive feedback
(Aarts et al. 2003). The Ca2+ overload can activate calcineurin which in turn activates SSH1
(Wang et al. 2005). The oxidative stress may further enhance SSH1 activity by promoting its
dissociation from 14-3-3ζ (Kim et al. 2009). The cofilin-phosphatase activity of active SSH1
would result in cofilin hyperactivation (dephosphorylation) and LIMK1 inhibition
(dephosphorylation). Decreased LIMK activity could also result from suppression of PAK3
activity, albeit through TRPM7 and NMDAR independent signalling events. Ultimately, cofilin
hyperactivation results in the formation of cofilin-actin rods (Bamrug 1999, Minamide et al.
2000, Bent 2011).
Given that neurons consume nearly 50% of their energy through actin turnover, the
generation of actin-cofilin rods is believed to be neuroprotective at first by turning off actin-
treadmilling (Bernstein et al. 2003).However, during prolonged exposure to neuronal stress, as is
the case with OGD, the actin-cofilin rods can grow long enough to span the diameter of neurites
which in turn disrupts cytoskeletal structure and intracellular transport ultimately leading to
degeneration of neurites beyond the rods (Minamide et al. 2000). By disrupting intracellular
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transport and neuronal communication, the actin-cofilin rods ultimately result in neuronal death
(Maloney and Bamburg). Indeed, suppression of cofilin activity during OGD via siRNA
contributed to a 20% reduction in neuronal cell death, affirming a cofilin mediated effect in
ischemia pathology (Bent 2011).
Figure 10. Proposed model of TRPM7 mediated cofilin regulation during OGD
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5.7 Future studies
Evaluation of ROCK2 activity downstream of TRPM7 and its contribution to stroke
pathology was inconclusive. Additional studies are necessary to evaluate ROCK2 activity
downstream of NMDA signalling and TRPM7 signalling. Purification of ROCK2 from cell
lysates and its subsequent use in vitro kinase assays would provide a more direct evaluation of
protein activity. Measuring phosphorylation levels of known ROCK2 substrates (i.e. LIMK,
myosin light chain phosphatase) as well as the activity of ROCK2 upstream regulators (i.e.
RhoA) would provide additional insight into ROCK2 activity levels during the various treatment
employed in the present study. Furthermore, stimulation as well as inhibition of ROCK2 activity
could be useful at evaluating the ROCK2 mediated signalling events and its contribution to
stroke pathology.
Although the present results are in agreement with previous reports linking SSH1 activity
to ischemic pathology, the western analysis data is only correlative and additional studies are
necessary to confirm that SSH1 activity is indeed influenced by TRPM7. Initially, SSH1 activity
should be investigated more thoroughly via in vitro phosphatase assays, as well as through
measuring the phosphorylation levels of LIMK and cofilin. The use of a constitutively active
form of SSH1 and phosphatase dead mutants in the various treatments used in this study would
further reaffirm the capacity of SSH1 to regulate LIMK and cofilin activity. In addition,
knockdown of SSH1 expression would unveil other potential regulators of cofilin activity such
as chronophin phosphatase. Chronophin is known to enhance cofilin activity via
dephosphorylation (Mizuno et al. 2013). The potential effects of chronophin on cofilin activity
during OGD cannot be excluded from the present study. Thus, future studies should elucidate
96
the effects of TRPM7 channels on chronophin and evaluate choronophin’s ability to influence
cofilin during OGD.
In addition, the recent observation from our research group showing 14-3-3 to pull down
along with TRPM7 are suggestive of a potential link between TRPM7 and SSH1 through 14-3-3.
Future studies should be aimed at identifying the TRPM7 regions responsible for 14-3-3 binding
(i.e. via TRPM7 point mutations and Co-IP), and then use this knowledge to determine the
functional outcome of disrupting the 14-3-3 interaction with TRPM7. In addition, the affinity of
TRPM7 for 14-3-3 should be evaluated when the channel is active as well as when suppressed.
It is possible that, the recruitment of 14-3-3 along with its binding partners such as SSH1, to
TRPM7 channels could present a potential mechanism of SSH1 activation during OGD by
exposing 14-3-3 bound SSH1 to a microenvironment that favours its activation. The TRPM7
interaction with 14-3-3 extends beyond SSH1 signalling; 14-3-3 proteins have been described to
have anti-apoptotic functions (Steinacker et al. 2011). Thus, examination of 14-3-3 activity along
with TRPM7 could expose additional mechanisms of TRPM7 mediated cell death.
The TRPM7 mediated regulation of cofilin is only part of the story, since knockdown of
cofilin expression during OGD suppressed neuronal cell death by only 20%. Future studies
should focus on the identification of potential TRPM7 binding partners through the use of co-
immunoprecipitation. In addition to identifying potential downstream mechanisms through
which TRPM7 channels are involved, co-immunoprecipitation can also identify potential
upstream regulators of TRPM7 activity. Such knowledge could help the development of TRPM7
specific inhibitors as to date none have been developed. Conservation of the TRPM7 alpha-
kinase for millennia suggests that its functional role is important. Co-immunoprecipitation
experiments could be coupled with in vitro kinase assays to identify endogenous TRPM7
97
substrates. Indeed myosin IIA, annexin I, and eEF2 kinase have been shown to be
phosphorylated by TRPM7 alpha kinase activity (Clark et al. 2006; Dorvkov and Ryazanov,
2004; Perraud et al. 2011) however the functional significance of such events is still unclear.
Future studies should evaluate TRPM7 substrate relationships both under homeostatic conditions
as well as during pathological states such as ischemia in order to identify direct contributors of
ischemic pathology. Furthermore, the bi-modal nature of TRPM7 makes it difficult to distinguish
the channel mediated effects from those of the alpha kinase. The expression of kinase dead and
channel dead TRPM7 proteins could help isolate the channel mediated signalling events from
those of the alpha kinase.
98
Chapter 6
6. Summary
The primary aim of this project was to identify new molecular interactors that operate
downstream of TRPM7 during OGD. Through western analysis, TRPM7 activity was shown to
influence SSH1 activity during OGD. These changes in SSH1 activity correspond with
previously reported TRPM7 mediated changes in LIMK and cofilin activity. These results
suggest that SSH1 is regulated downstream of TRPM7 and could serve as a potential contributor
to the cofilin mediated neurodegeneration previously reported by our research group. However,
additional studies are necessary to evaluate SSH1 activity during OGD and whether it’s
sufficient at regulating LIMK and cofilin. TRPM7 and NMDA-AMPPA/Kainate signalling did
not appear to contribute to the observed reductions in PAK3 activity. Other studies suggest that
Rac may contribute to PAK3 depression; whether this is indeed the case for OGD still needs to
be determined. Unfortunately, cell death assays and western analyses were inconclusive at
linking ROCK2 mediated neurodegeneration downstream of TRPM7. The use of methods that
directly measure ROCK2 activity would provide more conclusive data. In an attempt to visualize
P-SSH1 and P-PAK3 localization during OGD or TRPM7 inhibition revealed that P-SSH1 and
P-PAK3 localization does not change between treatments. Lastly I was able to develop a cell
death model that resulted in neuronal cell death during OGD, and was completely rescued by
MCN treatment. The use of a 3 hour exposure period (instead of 2 hours) might have caused
differences between OGD MCN and OGD MCN Gd3+ and OGD MCN Y-37632 to emerge. It
will be interesting to see whether ROCK2 mediated neurodegeneration is implicated with
TRPM7 signalling and whether the reported changes in SSH1 influence cofilin activity during
99
OGD. Although, it has been a decade since TRPM7 channels have been implicated in neuronal
pathology, much is unknown about the mechanisms that operate downstream of TRPM7
channels during ischemia. The identification of direct TRPM7 interacting partners and the
development of TRPM7 specific pharmacological inhibitors are essential to enhancing the
progress of TRPM7 research.
100
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