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Identification of Novel Genes Critical for CNS Regeneration in L. stagnalis
by
Mila Aleksic
A thesis submitted in conformity with the requirements for the degree of Master of Science
Graduate Department of Physiology University of Toronto
© Copyright by Mila Aleksic 2011
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Identification of Novel Genes Critical for CNS Regeneration in
L. stagnalis
Mila Aleksic
Master of Science
Department of Physiology
University of Toronto
2011
Abstract
Neuronal regeneration in the mammalian central nervous system (CNS) is severely
compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic
regenerative capacity. Understanding the cellular and molecular processes underlying injury and
regeneration in the CNS is necessary for the development of effective therapeutic strategies.
Lymnaea stagnalis, a freshwater pond snail, has proven to be a powerful model for studying the
fundamental mechanisms underlying neurite outgrowth and regeneration. In this study I designed
the first custom L. stagnalis microarray gene chip and carried out microarray analysis to profile
gene expression changes following CNS injury. From a pool of significantly regulated genes, I
provided the first evidence that C/EBP, a transcription factor, plays an integral role in
regeneration by maintaining the viability of the distal neurite. We also proposed a novel
signaling network and demonstrated that BCL 7 regulates neurite regeneration, an effect that
may be mediated through Ca2+
-dependent growth cone formation.
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Table of Contents
Table of Contents ........................................................................................................................... iii
List of Tables ................................................................................................................................. vi
List of Figures ............................................................................................................................... vii
List of Abbreviations ................................................................................................................... viii
1. Introduction ................................................................................................................................ 1
1.1 General Overview ............................................................................................................... 1
1.2 Extrinsic Regulators of Regeneration ................................................................................. 2
1.2.1 Myelin ..................................................................................................................... 2
1.2.2 Guidance Molecules ................................................................................................ 2
1.2.3 The Glial Scar ......................................................................................................... 4
1.3 Intrinsic Regenerative Mechanisms .................................................................................... 4
1.3.1 Gene Expression ..................................................................................................... 5
1.3.2 Protein Synthesis ..................................................................................................... 7
1.3.3 Cytoskeleton and Associated Proteins .................................................................... 8
1.4 Invertebrates in Nerve Regeneration ................................................................................ 11
1.5 Lymnaea stagnalis: a Model for Regeneration ................................................................. 12
1.6 Gene Expression Profiling with Microarrays ................................................................... 14
1.6.1. Microarray Technology ........................................................................................ 14
1.6.2. Applications of Microarrays in Regeneration ....................................................... 15
1.7 RNA interference: A Tool to Identify Molecular Mechanisms ........................................ 17
1.8 Assessment of Real-Time qPCR for Gene Expression Analysis ...................................... 19
1.9 Hypotheses ........................................................................................................................ 20
1.9.1.1 Rational I ................................................................................................ 20
1.9.1.2 Hypothesis I ............................................................................................ 20
1.9.1.3 Specific Aim I ......................................................................................... 20
1.9.2.1 Rational II ............................................................................................... 21
1.9.2.2 Hypothesis II .......................................................................................... 21
1.9.2.3 Specific Aim II ....................................................................................... 21
2. General Materials and Methods ............................................................................................... 23
2.1 Animals ............................................................................................................................. 23
2.2 Surgical procedures and in vivo nerve injury .................................................................... 23
2.3 RNA extraction and cDNA synthesis ............................................................................... 23
2.4 cDNA Microarray ............................................................................................................. 24
2.5 Real-time quantitative PCR (qPCR) ................................................................................. 24
2.6 Primary cell culture ........................................................................................................... 27
2.7 RNAi synthesis and delivery ............................................................................................ 27
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2.8 Axotomy and neurite outgrowth ....................................................................................... 30
2.9 Calcium imaging ............................................................................................................... 30
2.10 Immunocytochemistry and confocal imaging ................................................................... 30
2.11 Bioinformatics ................................................................................................................... 31
2.12 Quantification of locomotion activity ............................................................................... 31
2.13 Statistics ............................................................................................................................ 32
3. Identification of the role of C/EBP in neurite regeneration following microarray analysis
of a L.stagnalis CNS injury model ......................................................................................... 33
3.1 Introduction ....................................................................................................................... 33
3.2 Results ............................................................................................................................... 34
3.2.1 CNS injury model of L. stagnalis ......................................................................... 34
3.2.2 Identification of differentially expressed genes .................................................... 34
3.2.3 Gene expression levels of C/EBP change in a time dependent manner
following injury .................................................................................................... 34
3.2.4 The role of C/EBP in axonal elongation following axotomy ............................... 35
3.2.5 C/EBP siRNA treatment hinders locomotion recovery in vivo following CNS
injury ..................................................................................................................... 36
3.3 Discussion ......................................................................................................................... 50
3.3.1 Regeneration in the CNS of Lymnaea stagnalis ................................................... 50
3.3.2 Microarray Analysis Following CNS Injury ......................................................... 51
3.3.3 Time-dependent Regulation of C/EBP ................................................................. 51
3.3.4 Role of C/EBP in the Distal Neurite ..................................................................... 52
4. Bioinformatic analysis reveals a novel role for BCL 7 in neurite regeneration ....................... 55
4.1 Introduction ....................................................................................................................... 55
4.2 Results ............................................................................................................................... 56
4.2.1 Network mapping using a bioinformatics approach ............................................. 56
4.2.2 Down-regulation of protein phosphatase 1k following CNS injury ..................... 56
4.2.3 A novel role for BCL 7 in CNS regeneration ....................................................... 57
4.2.4 BCL 7 impairs neurite outgrowth following axotomy .......................................... 58
4.3 Discussion ......................................................................................................................... 74
4.3.1 Bioinformatic analysis of gene expression changes following CNS injury ......... 74
4.3.1.1 Calmodulin ............................................................................................. 75
4.3.1.2 Phosphoinostide Kinases ........................................................................ 76
4.3.1.3 B-Cell Lymphoma .................................................................................. 77
4.3.1.4 SPARC-like protein 1 ............................................................................. 77
4.3.1.5 Protein phosphatase 1k ........................................................................... 78
4.3.1.6 Sec24 transport protein and bone sialoprotein ....................................... 78
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4.3.2 Suppression of protein phosphatase 1k does not affect neurite dynamics
following axotomy ................................................................................................ 79
4.3.3 BCL 7 regulates neurite elongation in a regenerative model ................................ 80
4.3.4 Role of BCL 7 in calcium transients following axotomy ..................................... 83
Figure 14 .................................................................................................................................. 86
5. Conclusion ................................................................................................................................ 87
6. Future Directions ...................................................................................................................... 88
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List of Tables
Table 1. Primer sequences, product size for specific genes of interest for qPCR…………26
Table 2. Primer sequences for C/EBP siRNA……………………………………………..29
Table 3. Primer sequences for dsRNA synthesis…………………………………………..29
Table 4. Transcripts up- or down-regulated at least 2-fold after CNS injury……………...40
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List of Figures
Figure 1. Experimental model and changes in gene expression
following injury………………… ……………………………………………………………39
Figure 2. Protein sequence alignment of C/EBP………………………………………………42
Figure 3. Time-dependent changes in C/EBP mRNA expression
following nerve injury…………………………………………………………………………44
Figure 4. Knockdown of C/EBP reduced net growth of the distal
axon following axotomy……………………………………………………………………….46
Figure 5. C/EBP siRNA treatment hinders recovery of locomotion
activity of the snails following CNS injury…………………………………………………....48
Figure 6. Schematic illustration of the potential mechanisms of C/EBP
action in maintaining distal axon integrity following axotomy………………………………..50
Figure 7. Network inferred by PPI spider……………………………………………………...62
Figure 8. Down-regulation of protein phosphatase 1k mRNA
after CNS injury………………………………………………………………………………..64
Figure 9. Knockdown of protein phosphatase 1k had no effect
on neurite regeneration…………………………………………………………………………66
Figure 10. BCL7 mRNA up-regulated 5 hrs after CNS injury………………………………...68
Figure 11. Reduction of Bcl7 mRNA results in impaired neurite
outgrowth following axotomy………………………………………………………………….70
Figure 12. Growth cone development and neurite outgrowth is
hindered in Bcl7 reduced cells following axotomy…………………………………………….72
Figure 13. β-tubulin localization following axotomy in
BCL 7 deficient cells…………………………………………………………………………..74
Figure 14. Intracellular [Ca2+
] gradient after axotomy…………………………………………87
Figure 15. Cytoskeletal profile following axotomy……………………………………………91
Figure 16. Schematic illustrating local and global effects of axotomy………………………...93
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List of Abbreviations
Ago2- Argonaute-2
ANKRD1-Ankyrin repeat domain 1 protein
APC/CCdh1
- Anaphase promoting complex/cyclosome and its activator Cdh1
APC- Adenomatous polyposis coli
APOE- Apolipoprotein E
Arg-1- Arginase-1
ATF3-Activating transcription factor-3
BCL-Burkitts Cell Lymphoma
BCL6-B-cell lymphoma 6
BCL7-B-cell lymphoma 7
BDNF - Brain-derived neurotrophic factor
bHLH- Helix-loop-helix
ΔΔCt-delta delta Ct
cAMP - Cyclic adenosine monophosphate
cDNA -Complimentary deoxyribonucleic acid
CaM –calmodulin
CAMBP- CaM binding proteins
CaMK-Calmodulin kinase
CaMK II - Calcium/calmodulin-dependent protein kinase II
CaMK IV - Calcium/calmodulin-dependent protein kinase IV
CaMKK - Calmodulin-dependent protein kinase kinase
CAP-23- Cytoskeleton-associated protein 23
CD44- Cluster of Differentiation 44
CGC-Cerebral giant cells
ChABC- Chondroitinase ABC
C/EBP - CCAAT/enhancer binding protein
CRMP-2- Collapsin response mediator protein 2
CNS-Central nervous system
CNTF- Ciliary neurotrophic factor
CST-Cortical spinal tract
CSPG- Chondroitin sulfate proteoglycans
CREB - cAMP response element binding protein
DLK- Dual-Leucine zipper Kinase MAPKKK
DRG-Dorsal root ganglion
dsRNA-double stranded RNA
ECM-Extracellular matrix
EGFR-Epidermal growth factor receptor
ERK-Extracellular signal-regulated kinase
EST-Expression sequence tags
GAG- Glycosaminoglycan chains
GAPDH-glyceraldehyde-3-phosphate dehydrogenase
GAP-43-Growth associated protein 43
GAT1-GABA transporter-1
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GC-Growth cone
GCOC- Growth cone organizing center
GFAP- Glial fibrillary acidic protein
Gp130- Glycoprotein 130
GPI- Glycosylphosphatidylinositol
GSK-3β- Glycogen synthase kinase-3 β
GTPase- Guanosinetriphosphatase
Ig-immunoglobulin
IL-6-Interleukin 6
IL-8-Interleukin 8
Id2-Inhibitor of DNA binding 2
INF-Interferon response
LIF- Leukemia inhibitory factor
JAK-Janus kinase
KLF-Kruppel-like factors
MAG- Myelin-associated glycoprotein
MAP-Microtubule-associated protein
MAP1A- Microtubule binding protein 1A
MAP1B-Microtubule binding protein 1B
MAPK-mitogen-activated protein kinase
miRNA-micro RNA
mTOR-mammalian target of rapamycin
MEK-MAPK kinase
N-CAM-Neural cell adhesion molecule
NFIL-3-Nuclear factor IL-3
NGF-Nerve growth factor
NT3-Neurotrophin 3
OEC-Olfactory bulb ensheathing cells
OMgp- Oligodendrocyte glycoprotein
PCR-Polymerase chain reaction
PDE –Phosphodiesterase
PeA-Pedal A
PI-1k- Protein phosphatase 1k
PI3K- Phosphoinositide 3-kinases
PIP2- Phosphatidylinositol (4,5) bisphosphate
PIP3- Phosphatidylinositol (3,4,5) trisphosphate
PKA-Protein kinase A or cAMP-dependent protein kinase
PKC-Protein kinase C
PP2C-Protein phosphatase 2C
PTGS- Posttranscriptional gene silencing
PNS-Peripheral nervous system
PTEN-Phosphate and tension homolog
PtdIns- Phosphatidylinositols
PtdIns4K- PtdIns 4-kinases
PIP5Ks- PtdIns-P (PIP) kinases
RA-Retinoic Acid
x
RCe- Right cerebral
RGC- Retinal ganglion cells
RISC- RNA-induced silencing complex
RISK1-Retrogradely transported kinase 1
RNA-Ribonucleic acid
RNAi- Ribonucleic acid interference
RPa-Right parietal
RTK-Receptor Tyrosine Kinase
RT-PCR-Reverse transcriptase polymerase chain reaction
RTN- reticulon
RXR-Retinoid Receptor
SPARC -Secreted Protein Acidic and Rich in Cysteine
SC1- SPARC-like protein 1
SCI-spinal cord injury
SCG- Superior cervical ganglia
sGC-Soluble guanylate cyclase
siRNA-Small interfering RNA
STAT3- Signal transducer and activator of transcription 3
TCR- T cell receptor
TNF-Tumour necrosis factor
UTR-Untranslated region
Wnt- Drosophila melanogaster wingless
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1. Introduction
1.1 General Overview
Injuries to the central nervous system (CNS), for example from stroke or spinal cord injury,
affect millions of people each year and cause devastating and irreversible losses of function.
Over the past 100 years, scientists have been studying CNS regeneration in order to identify
methods of aiding the process (Blesch and Tuszynski, 2009). Whereas CNS axons in adult
mammals do not spontaneously regenerate after injury, peripheral nervous system (PNS) axons
readily regenerate and allow for functional recovery following nerve injury (Huebner and
Strittmatter, 2009). In the last three decades, this fundamental difference between the two
systems led to the identification of several mechanisms that limit regeneration and functional
recovery in the adult CNS (Baptiste and Fehlings, 2007), involving both extrinsic as well as
intrinsic factors (Curinga and Smith, 2008;Liu et al., 2010).
Although molecular insights into regeneration have been largely gained from vertebrate models,
understanding the mechanisms that are involved in regeneration in invertebrate systems has also
been very advantageous for biomedicine (Sanchez and Tsonis, 2006). In contrast to mammals,
CNS regeneration is permissive in adult invertebrates. Unravelling the mysteries behind why
regeneration and functional recovery occur in some model systems and not in humans can
provide new pathways to therapeutically target.
This study takes advantage of the regenerative capacity of the invertebrate model L. stagnalis in
order to identify genes that are differentially expressed following CNS injury. It identifies
functionally known and novel genes that are up- and down-regulated after injury, and that may
underlie the intrinsic regenerative ability of adult Lymnaea neurons. It also examines the role of
the transcription factor C/EBP on neuron regeneration in vitro. The thesis will conclude by
proposing a novel pathway involved in CNS injury and evaluating the mechanisms behind the
network.
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1.2 Extrinsic Regulators of Regeneration
In the early 1980’s, studies by Aguayo et al., established clear evidence that some populations of
adult CNS neurons possess regenerative capacity when provided with environments such as a
peripheral nerve graft (Aguayo et al., 1981). This study prompted the idea that the PNS milieu is
more conducive for long-distance axon regeneration compared to that of the CNS. Subsequently,
differences in the CNS environment including the expression of myelin inhibitory proteins (an
inhibitory extracellular matrix at the injury site), alterations in axon guidance molecules and a
reduction in growth factor expression were found to contribute to the prevention of regeneration.
1.2.1 Myelin
CNS myelin made by mature oligodendrocytes is a major inhibitor of neurite outgrowth. The
CNS contains several myelin derived inhibitors, including Nogo-A, myelin-associated
glycoprotein (MAG), and oligodendrocyte glycoprotein (OMgp) (Filbin, 2003). Nogo-A is a
member of the reticulon (RTN) family of proteins (Chen et al., 2000), and is comprised of two
inhibitory domains Nogo66 and amino-Nogo (Fournier et al., 2001). Blocking Nogo-A with IN-
antibody or NEP1-40 antagonist peptide, has been reported to enhance corticospinal tract (CST)
regeneration following spinal cord injury in rats (Schnell and Schwab, 1990;Bregman et al.,
1995;Fournier et al., 2002). MAGs are transmembrane proteins that regulate neurite outgrowth in
an age-dependent fashion. The expression of MAG has been shown to promote the growth of
embryonic and neonatal neurons, and inhibit outgrowth from more mature neurons (DeBellard et
al., 1996). The third type of prototypic myelin inhibitors are OMgp’s, a
glycosylphosphatidylinositol (GPI) anchored protein expressed by oligodendrocytes in the
mature CNS (Mikol and Stefansson, 1988). Deletion of the OMgp gene resulted in improved
functional recovery and sprouting of serotonergic fibres following SCI (Curinga et al., 2008).
Taken together, these studies demonstrate that deleting or neutralizing myelin derived inhibitors
enhances neurite outgrowth.
1.2.2 Guidance Molecules
Within the adult CNS myelin, studies have also revealed the presence of several inhibitory
guidance molecules, including members of the semaphorin, ephrin, netrin and Wnt families.
These guidance cues are known to regulate neuronal development, however, many CNS neurons
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continue to express receptors for them into adulthood where they play a different role in
regeneration (Reza et al., 1999).
Semaphorins mainly function as inhibitory guidance cues, such that in vitro application of
Sema3A results in growth cone collapse (Kaneko et al., 2006), and blocking Sema3A from
binding its receptor complex results in enhanced axon regeneration after SCI (Fabes et al., 2006).
Eph/Ephrin signaling has also been correlated with reduced regeneration (Fabes et al., 2006).
Ephrins bind to members of the EphA and EphB receptor tyrosine kinase families. While injury
results in an increase in ephrin B2 expression, accumulation of the EphA4 receptor in injured
neurons and results in retraction of axons, and blocking EphA4 receptors enhances sprouting of
CST axons (Fabes et al., 2006).
In contrast to the other two, Netrin-1 can act as either a chemoattractant or a chemorepellent
depending on the receptor type (Toullec et al., 1992). Following SCI, oligodendrocytes express
Netrin-1 and neurons shift their expression to repulsive receptors, suggesting a role for Netrin 1
in inhibiting neuronal outgrowth (Manitt et al., 2006). Like the netrin family, the Wnt family also
comprises attractive and repulsive cues. Frizzled receptors mediate Wnt-4 attraction and Ryk
receptor tyrosine kinase mediates repulsion of Wnt-5a during development (Lyuksyutova et al.,
2003). After dorsal column injury there is an induction of Wnt-5a and Ryk around the lesion and
as a result blocking Ryk promotes sprouting of CST axons (Liu et al., 2008).
Finally, application of neurotrophins functions to promote regeneration. Successful regeneration
in the PNS is known to be accompanied by a rapid increase in such growth factors as NGF,
BDNF, and CNTF (Giger et al., 2010). In the CNS these neurotrophic factors regulate growth,
guidance and survival of neurons but their expression generally decreases during development.
By mimicking the PNS environment, studies have shown that applying these growth factors
following SCI can promote extensive axonal growth. For example, NGF promoted growth of
nociceptive axons (Tuszynski et al., 1996), NT-3 promoted growth of lesioned dorsal column
sensory axons (Taylor et al., 2006) and BDNF promoted growth of diverse groups of axons (Lu
et al., 2005).
Collectively, all of these studies imply that the inability of axons to regenerate can be in part
attributed to the actions of molecules that are critical for proper CNS development.
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1.2.3 The Glial Scar
In addition to the growth inhibitory nature of the CNS milieu, regenerating axons have a further
obstacle to overcome. The glial scar that forms within days of the injury is composed of reactive
astrocytes, microglia/macrophages, and extracellular matrix molecules (specifically chondroitin
sulfate proteoglycans, CSPGs) which pose a chemical barrier for axonal regeneration (Jones et
al., 2003). CSPG’s are a complex group of glycoproteins that are covalently linked to
glycosaminoglycan chains (GAGs) (Galtrey and Fawcett, 2007). Following CNS injury CSPG’s
are secreted by almost all types of cells, especially astrocytes, and mainly exert an inhibitory
effect on neurite outgrowth (McKeon et al., 1999). By enzymatically degrading the CSPG’s
GAG side chains using chondroitinase ABC (ChABC), neurites showed growth potential which
resulted in functional improvement following CNS injury (McKeon et al., 1995;Garcia-Alias et
al., 2009).
As described above, following injury the CNS milieu is altered, leading to an extensive change
in the expression of numerous molecules, the result of which inhibits axons from regenerating.
Although this inhibitory environment constitutes a major barrier for regeneration, its
neutralization is insufficient in promoting maximal regeneration.
1.3 Intrinsic Regenerative Mechanisms
The intrinsic regenerative ability of neurons governs how they will respond to an injury. The
regenerative capacity of neurons substantially declines as they mature due to the intrinsic cellular
changes that occur following development (Muramatsu et al., 2009). During development axon
growth is promoted by the surrounding environment and intracellular machinery. Once the axon
reaches its target, neuronal function switches from axon growth to synapse formation and
maturation (Liu et al., 2010). Evidence from RGCs demonstrates a dramatic reduction in axon
outgrowth after birth (Chen et al., 1995). The developmental decline in axon growth is likely due
to interactions between extracellular signals and intrinsic mechanisms. Temporal changes in
expression of several molecules including cAMP, mTOR, and KLFs, coincides with the
developmental-dependent decline of axon growth in CNS neurons (Muramatsu et al., 2009;Liu et
al., 2010). Furthermore, PNS axons in adult mammals have greater regenerative ability then
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their CNS counterparts. PNS neurons up-regulate several regeneration associated genes
following injury. These include transcription factors, c-Jun, ATF-3 and growth associated
protein, GAP-43, CAP-23 (Huebner and Strittmatter, 2009). The growth of axons following
injury is sustained primarily by these cellular changes in gene expression, protein synthesis and
cytoskeletal modulation. Therefore, regulation of these molecular mechanisms may be a
powerful strategy for enhancing axonal regeneration and functional recovery.
1.3.1 Gene Expression
In the nervous system, changes in gene expression underlie the morphological alterations in cell
morphology and function. Consequently, successful axon regeneration requires proper activation
of the transcription and translation machinery following injury. Distinct changes in gene
expression are observed after injury to the adult CNS, and this is due to their regulation by
transcription factors (Moore and Goldberg, 2011). Transcription factors are DNA-binding
proteins that activate or repress the transcription of their target gene. A single transcription factor
can bind to multiple locations on the genome and regulate numerous genes and as a result
amplify its effects. After CNS injury the up-regulation of transcription factors results in specific
gene expression changes (Moore and Goldberg, 2011). Several transcription factors have already
been shown to promote outgrowth of injured CNS neurons. One of the most promising
candidates lies in the cAMP-CREB pathway. During neuronal maturation there is an age-
dependent decline in the intracellular concentration of cyclic adenosine monophosphate (cAMP)
(Filbin, 2003). The drop in cAMP levels switches the responsiveness of axons to extracellular
cues and coincides with a reduction in the capacity of neurons to regenerate (Cai et al.,
2001;Shewan et al., 2002). In this cascade, cAMP activates Protein Kinase A (PKA) which
phosphorylates and activates the cAMP responsive element binding protein (CREB) (Yamashita,
2007). Activation of CREB results in the transcription of several growth promoting genes,
including C/EBP, ATF-3, Arg-1, and plays a key role in enhancing axon growth and
regeneration both in the PNS and CNS. For example, in cultured DRG neurons expression of
constitutively active CREB is able to overcome the inhibitory effect of MAG on neurite
outgrowth, and in rat SCI models CREB activation promotes the growth of dorsal column axons
through the lesion site (Cai et al., 2002;Gao et al., 2004;Spencer and Filbin, 2004). Moreover,
pharmacologically increasing cAMP levels by the use of a selective cAMP phosphodiesterase
inhibitor type IV, Rolipram, promotes regeneration and recovery of SCI rats and optic nerve
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axon regeneration across the CNS glia (Nikulina et al., 2004), emphasizing the therapeutic
potential of targeting the cAMP-dependent signaling pathways in CNS regeneration.
The anaphase promoting complex/cyclosome and its activator Cdh1 (APC/CCdh1
) are also
suggested to be involved in the transcriptional regulation of axonal outgrowth. APC/CCdh1
causes
the developmental decline of neurite growth in cerebellar granule cells by targeting inhibitor of
DNA binding 2 (Id2). Id2 is an inhibitory protein that binds other helix-loop-helix (bHLH)
factors to prevent them from activating transcription (Iavarone and Lasorella, 2004;Konishi et
al., 2004). During development, by binding to bHLH factors, Id2 inhibits the expression of
several growth inhibitory molecules and receptors, causing the neuron to be insensitive to
extrinsic inhibitory cues and promoting axon growth (Jackson, 2006;Lasorella et al., 2006).
However, in the adult, the APC/CCdh1
complex induces the proteolysis of Id2, relieving its
inhibitory effect, which triggers the expression of several receptors involved in extrinsic growth-
inhibitory signals (Konishi et al., 2004). As a consequence, the neuron is rendered more sensitive
to negative environmental cues, leading to a reduction in intrinsic growth potential.
Aside from the cAMP-CREB signaling pathway and APC/CCdh1
complex, there are several other
factors involved in the transcriptional regulation of neuronal regeneration. For example, the
transcription factor c-Jun has been implicated in promoting axon regeneration in several models
of nerve injury (Raivich et al., 2004). c-Jun regulates the expression of proteins expressed during
regeneration such as CD44, Galanin, and α7β-1 integrin. Mice which lack c-Jun displayed a
severe defect in the axonal response to injury and target re-innervation (Herdegen et al.,
1997;Raivich et al., 2004). Similar to c-Jun, activating transcription factor-3 (ATF3) is also up-
regulated in regenerating neurons (Campbell et al., 2005). ATF3 and c-Jun actually form a
heterodimer and may act synergistically to induce axon outgrowth (Moore and Goldberg, 2011).
The cytokine leukemia inhibitory factor (LIF) and interleuikin-6 (IL-6) also regulate
transcriptional systems that enhance axonal growth, with IL-6 and LIF-null mice displaying
impaired peripheral and central nerve regeneration and mice constitutively over-expressing IL-6
showing enhanced regeneration (Hirota et al., 1996;Cafferty et al., 2004). IL-6 and LIF bind to
IL-6 receptor alpha and gp130 complex which phosphorylates JAK. JAK in turn phosphorylates
the transcription factor signal transducer and activator of transcription 3 (STAT3), which then
enters the nucleus to activate transcription (Taga, 1996). The phosphorylation of STAT3 acts as a
positive injury signal that promotes transcription and regeneration. Kruppel-like factors (KLFs)
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are another type of potential regulators of neurite growth, whereby KLF6 and KLF7 were shown
to be necessary for RGC re-growth after injury (Veldman et al., 2010). These transcription
factors as well as many that are yet to be identified play an essential role in enhancing CNS
regeneration.
Transcription factors control intrinsic processes of regeneration be regulating the expression of
growth promoting genes. A better understanding of these regulatory networks can lead to better
therapeutic targets for enhancing intrinsic regenerative capacity.
1.3.2 Protein Synthesis
In order to achieve successful regeneration, the injured axon must be able to transform into a
growth cone, a dynamic and motile structure at the tip of the axon. The process of growth cone
re-formation and axon outgrowth requires local and global protein turnover, new proteins need to
be synthesized and old ones degraded (Gumy et al., 2010). Axons contain protein synthesis
machinery, including ribosomal proteins, translation initiation factors and mRNAs. Local axonal
protein synthesis is required for axon outgrowth, and application of protein synthesis inhibitors
significantly impairs the growth cone regenerative ability (Verma et al., 2005). Consequently, the
growth of axons is highly dependent on the synthesis of new proteins and there are several
mechanisms that mediate local and global protein synthesis after injury. For example, the cAMP-
CREB-Arg1 pathway is an important target for promoting gene translation and axon
regeneration. Following cAMP activation, CREB transcriptionally induces the expression of
arginase-1 (Arg1), which results in polyamine synthesis and promotion of nerve regeneration
following an optic nerve crush (Deng et al., 2009). Another pathway in protein synthesis-
dependent axon regeneration involves a PI3K signaling cascade regulated by a dual phosphatase,
phosphatase and tensin homolog (PTEN). Receptor tyrosine kinase (RTK) activation following
injury activates phosphoinositide 3-kinases (PI3K), which phosphorylates and converts the lipid
second messenger phosphatidylinositol (4,5) bisphosphate (PIP2) into phosphatidylinositol
(3,4,5) triphosphate (PIP3), resulting in the activation of AKT (Song et al., 2005). PTEN
antagonizes PI3K and catalyzes the conversion from PIP3 to PIP2, so inactivation of PTEN
results in accumulation of PIP3 and the activation of AKT. There are multiple downstream
effectors of PI3K/AKT. The signaling cascade inactivates glycogen synthase kinase-3β (GSK-
3β) and releases the inhibition on the Mammalian target of rapamycin (mTOR), both of which
8
enhance protein translation to promote regeneration (Zhou et al., 2004;Ma and Blenis, 2009).
Specifically, the deletion of PTEN in retinal ganglion cells (RGC) increased the cells’ survival
and promoted robust axon regeneration after injury (Park et al., 2008). These effects were
potentially mediated by the activation of mTOR. mTOR promotes the synthesis of raw materials
for axon extension, and the enhancement of axonal transport and cytoskeleton assembly in the
axon terminal by signaling molecules such as PI3K and GSK-3β (Park et al., 2010). Other
signaling molecules such as ERK1/2 and PKA were also found to be important for successful
regeneration. It is speculated that they are involved in local protein synthesis of growth related
axonal mRNA which regulates growth cone formation (Chierzi et al., 2005). Alternatively they
can act as signal molecules and retrogradely transported to the cell body to achieve global
translational changes (Sung et al., 2001). This suggests that enhancing protein synthesis is a
valuable approach for enhancing regenerative capacity.
The rapid change in local protein synthesis following injury requires degradation of existing
proteins. Axon outgrowth and regeneration is a dynamic balance between synthesis and
degradation of proteins (Gumy et al., 2010). Degradation of proteins in the axon occurs through
various pathways, including ubiquitin-proteosome system (UPS), calcium-mediated proteolysis
and autophagy-mediated degradation. Inhibition of UPS activity has been shown to correlate
with growth cone formation and successful regeneration (Verma et al., 2005). On the other hand,
calcium-dependent calpain proteolysis is required for growth cone development (Spira et al.,
2001). Autophagy, the degradation of proteins inside lysosomes, may also contribute to axonal
homeostasis. However, no direct evidence exists to associate its activity with regeneration
(Cuervo, 2004).
Axon regeneration and the formation of a growth cone require protein synthesis and degradation
to occur synergistically. This complex network of processes is an important target for promoting
regeneration after injury.
1.3.3 Cytoskeleton and Associated Proteins
Axon elongation is due to the assembly of intracellular cytoskeletal machinery involving
intrinsic signals in response to extracellular signals. In order for successful regeneration to occur,
an injured axon must organize its cytoskeleton and transform into a growth cone. The axonal
cytoskeleton is composed of three main elements: neurofilaments, microtubules and actin
9
microfilaments (Lefcort and Bentley, 1989). The growth cone is a key constituent of the axon
outgrowth machinery and microtubules and actin filaments are its main cytoskeletal components.
The structure of the growth cone, which is fundamental to its function, can be divided into the
peripheral domain, the transition zone and the central domain (Dent and Gertler, 2003). The
peripheral domain consists of long, bundled F-actin filaments that form the finger-like filopodia,
as well as a mesh-like network between the filopodia called the lammelipodia (Schaefer et al.,
2002). The central domain is composed of bundled microtubules which enter the growth cone
through the axon shaft. Lastly, between the peripheral and central domains is the transition zone,
where actinomyosin contractile structures lie perpendicular to the F-actin bundles (Schaefer et
al., 2002). During growth cone advancement, polymerization of F-actin occurs and the filopodia
and lamellipodia move forward, extending the leading edge (Suter and Forscher, 2000;Lee and
Suter, 2008). Microtubules also have a major role in growth cone advancement. Individual
microtubules in the peripheral domain act as guidance sensors and explore the environment,
whereas microtubules in the central domain grow into the newly protruding peripheral zone and
consolidate the growth cone neck to form a new axon shaft, resulting in axon outgrowth
(Gordon-Weeks, 1991;Gordon-Weeks, 2004). The growth of a microtubule is due to the
association of its component α and β tubulin monomers, and the development of its polarized
structure with a “plus’ and a ‘minus’ end. In the axon, the plus end is oriented towards the
growth cone where constant polymerization and depolymerization of the monomers occurs
(Baas, 1997;Buck and Zheng, 2002). This dynamic instability of microtubules enables them to
very rapidly switch from elongation to retraction (Baas, 1997). Microtubule dynamics during
outgrowth are tightly regulated by post-translational modifications as well as interactions with
other growth cone proteins (Gelfand and Bershadsky, 1991;Buck and Zheng, 2002).
Support for a role of cytoskeletal and growth cone associated proteins in promoting axon
regeneration has been demonstrated in several studies. For example, GSK-3β inhibits
microtubule assembly by phosphorylating and inactivating collapsin response mediator protein 2
(CRMP-2), adenomatous polyposis coli (APC) and microtubule binding protein 1B (MAP1B)
(Zhou et al., 2004;Brown et al., 2004;Trivedi et al., 2005). CRMP-2 is a microtubule-binding
protein that promotes microtubule polymerization and stabilization resulting in neurite
elongation. During CNS development, the active form of CRMP-2 is able to counteract the
inhibitory effects of GSK-3β (Yoshimura et al., 2005). However, in the adult there is increased
10
expression of inactive CRMP-2 in neurons, which inhibits regeneration (Akira et al., 1990).
APC, which binds to microtubules to regulate their dynamics in neurons, is also inactivated by
GSK-3β resulting in the inhibition of neurite elongation (Zhou et al., 2004). On the other hand,
MAP1B phosphorylation by GSK-3β results in its activation. MAP1B influences microtubule
dynamics by controlling microtubule stability, such that its phosphorylation results in an increase
in the number of unstable microtubules, leading to a reduction in axon growth (Trivedi et al.,
2005). Consequently, treatment with a GSK-3β inhibitor promotes axon regeneration, and this is
most likely mediated by its effects on CRMP-2, APC and MAP1B (Dill et al., 2008).
cAMP signaling also promotes regeneration by regulating cytoskeletal dynamics. cAMP
activates PKA, which inactivates the Rho family of small GTPases. Through a CRMP-2
dependent mechanism, Rho A kinases become negative regulators of actin and tubulin
polymerization such that their inhibition causes axon regeneration in injured spinal cords (Akira
et al., 1990;Mueller et al., 2005).
The intrinsic regenerative capacity of neurons has also been enhanced by overexpressing
cytoskeletal associated growth cone proteins GAP-43 and CAP-23 in double transgenic mice
(Bomze et al., 2001). Furthermore, axotomized transgenic Purkinje cells from transgenic mice
overexpressing GAP-43, exhibit profuse sprouting (Buffo et al., 1997). GAP-43 or CAP-23
alone can enhance the propensity to initiate axon extension. Both proteins are required, however,
to elicit the extension of very long axons (Bomze et al., 2001).
Adult CNS neurons posses an intrinsic capacity to evoke mechanisms that enhance regeneration
after CNS injury (Muramatsu et al., 2009;Liu et al., 2010). Neonatal CNS neurons and PNS
neurons activate signaling cascades which promote regeneration (Liu et al., 2010). However,
adult CNS neurons need to initiate proper transcriptional regulation, cytoskeletal organization,
and protein synthesis in order to regenerate. Current research has revealed numerous molecules
that regulate intrinsic mechanisms for axonal outgrowth. Understanding the global picture of
these molecular interactions and signaling networks will greatly contribute to the development
and identification of new therapies for CNS injury.
11
1.4 Invertebrates in Nerve Regeneration
In contrast to the adult mammalian CNS, some invertebrates are capable of spontaneous CNS
regeneration following injury. Thus they are an important model to aid our understanding of
factors which may promote regeneration in the mammalian CNS. Invertebrate models have
successfully contributed the advancement of neuroscience. Major scientific discoveries on
synaptic transmission, axonal conductance, and integrative neurobiology can be attributed to
these remarkable animals. The advantage of this model organism for investigating the nervous
system includes their large-sized neurons, simple nervous system, and well-defined behaviors
(Otsuka et al., 1966;Kandel and Schwartz, 1982;Davis, 2005). The first evidence for the
existence of local protein synthesis, an essential component of regeneration, came from studies
in the squid giant axon (Giuditta et al., 1991). Furthermore, the identification of several hundred
mRNAs for ribosomal proteins, cytoskeletal proteins, translation factors, was carried out in
invertebrate axons (Giuditta et al., 1991;Gumy et al., 2010). In 1971, J Nicholls and DA Baylor
investigated the large neurons of the leech and demonstrated regeneration of specific neuronal
connections (Baylor and Nicholls, 1971). Crushing all of the axons in a segment of the CNS
disconnected the anterior and posterior parts of the body compromising the rhythmical
swimming movement. However, after a few weeks regeneration occurred and axons that re-grew
made connections with appropriate targets and the leech regained normal swimming function
(Baylor and Nicholls, 1971;Chiquet and Nicholls, 1987). By examining regeneration of axons in
the leech CNS, it was shown for the first time that microglia play a key part in the regenerative
process and that nitric oxide is crucial for their directed migration, an effect that is mediated by a
soluble guanylate cyclase (Glade-McCulloh et al., 1989;Duan et al., 2003). Insights into CNS
regeneration have also emerged from other invertebrate models. Aplysia have the ability to
transform the cut axonal end into a growth cone (GC), a critical event in the cascade leading to
regeneration (Ashery et al., 1996;Sahly et al., 2006). Using cultured Aplysia neurons as a model
system, it was revealed that the magnitudes of the [Ca2+
]i gradients at the site of injury determine
whether or not the cut axonal end transforms into a competent GC (Kamber et al., 2009).
Imaging in Aplysia neurons also revealed that axotomy resulted in reorientation of microtubule
polarity, a critical step for growth cone formation (Erez et al., 2007). Planarians for example are
capable of profound regenerative ability and can regenerate an entire CNS (Agata et al.,
2007;Agata and Umesono, 2008). This phenomenon is dependent on a population of self-
12
renewing adult stem cells called neoblasts (Salo and Baguna, 1984). Analysis of regeneration in
planarians offers a paradigm for understanding how these stem cells are regulated and integrated
into the pre-existing CNS and have provided valuable insights for the safe use of stem cells to
improve the regenerative capacity in the mammalian nervous system (Cebria, 2007;Aboobaker,
2011). More recently, the nematode C. elegans revealed its potential for discovering new
regulators of axon re-growth with the discovery of the central role of the DLK-kinase pathway in
axon regeneration (Hammarlund et al., 2009;Yan et al., 2009). Time-lapse imaging revealed that
DLK-1 mutants fail to form a growth cone, and the over-expression of DLK-1 increases growth
cone formation and axon regeneration. However DLK-1 is not required for CNS development
and axon growth appears normal in DLK-1 mutants (Hammarlund et al., 2009;Yan et al., 2009).
This demonstrates that different pathways can regulate axon development and regeneration.
The comparative study of invertebrate nerve regeneration has long been recognized for its
potential to provide insights into the cellular and molecular mechanisms of CNS regeneration.
The robust spontaneous regenerative ability in the CNS after injury makes invertebrates excellent
models to study cellular and molecular mechanisms underlying axon regeneration (Liu et al.,
2010). Consequently, invertebrate models of regeneration will continue to play a major role in
neuroscience.
1.5 Lymnaea stagnalis: a Model for Regeneration
Lymnaea stagnalis is an example of an invertebrate model system that exhibits successful
regeneration and restoration of function in the adult CNS (Janse et al., 1979;Hermann et al.,
2000;Hermann et al., 2005). It represents an advantageous preparation in which to analyze in
detail the mechanisms by which damaged neurons grow and reform connections with target cells.
L. stagnalis has a simplified nervous system made up of a small number of neurons with large
identifiable neuronal somata. These characteristics have enabled researchers to determine the
functional elements of the Lymnaea neuronal circuitry. Several neural circuits have been
identified in terms of the connections between sensory cells, interneurons and motor cells (Syed
et al., 1990;Syed et al., 1992a;Magoski et al., 1994). These identified adult Lymnaea neurons can
be isolated individually and maintained in culture, allowing for axonal outgrowth and functional
13
synapse formation between appropriate synaptic partners (Syed et al., 1992b;Feng et al., 1997).
In addition, the neurons of L. stagnalis have very large growth cones that can be transected in
culture and electrophysiological recordings can be conducted from both the soma and the growth
cone allowing for functional analysis of regeneration (van Kesteren et al., 2006;Hui and Feng,
2008). Lastly, L. stagnalis neural genes include homologues of well-known transcription factors,
genes involved in neurotransmission, axon guidance and signaling pathways (Feng et al.,
1997;Hermann et al., 2000;van Kesteren et al., 2006;Feng et al., 2009).
Numerous groups have taken advantage of this model system and tested the roles of
neurotrophins, transmitters and chemoattractants on neurite outgrowth and regeneration
(Ridgway et al., 1991;Spencer et al., 1998;Koert et al., 2001;Wildering et al., 2001). Koert et al.,
studied the regenerative properties of two electrically coupled neurons, the serotonergic cerebral
giant cells (CGCs) of the Lymnaea (Koert et al., 2001). The study suggested that auto-released
serotonin is inhibitory to CGC neurite outgrowth in vitro. During regeneration in vivo serotonin
release might fine-tune axon guidance and branching by inducing local collapse responses in
extending neurites (Koert et al., 2001). Adult Lymnaea neurons provided the first evidence for
the existence of both atRA and 9-cis-RA isomers in the CNS of an invertebrate species.
Additionally the Lymnaea model offered important evidence for a physiological role for 9-cis-
RA in neuronal chemoattraction and regeneration through a novel mechanism requiring de novo
local protein synthesis (Dmetrichuk et al., 2006;Farrar et al., 2009;Carter et al., 2010).
Furthermore, Carter et al., cloned a retinoid receptor from the Lymnaea (LymRXR), and
demonstrated that it is present in the developing embryoand in the cytoplasm of adult CNS
neurons, with a strong localization to the neurite. Using regenerating cultured motor neurons,
they also showed that LymRXR is present in the growth cones and that application of
a RXR pan-agonist produces growth cone turning in isolated neurites, in the absence of the cell
body and nucleus, suggesting a novel, non-genomic role for RXR in growth cone steering (Carter
et al., 2010). These studies directly contribute to our current understanding of the mechanisms
underlying the events of axon retraction, regeneration, identification of appropriate target and
reformation of functional connections after injury. Consequently, L. stagnalis is an important
simple model organism for the study of complex biological processes.
One main limitation of using L. stagnalis in genetic functional studies of neuronal regeneration
has been a lack of large-scale screening tools, such as microarray analysis. To circumvent this
14
limitation, we recently sequenced more than 10, 000 ESTs (expression sequence tags) from the
CNS transcriptome of L. stagnalis (Feng et al., 2009) and established the largest neuronal EST
database in Lymnaea (www.lymnaea.org). In combination with the availability of microarray
technology, these gene sequences provide us with the opportunity to perform a high-throughput
screening for altered gene expression levels following nerve injury in the L. stagnalis. The
identification of genes regulated after CNS injury enables us to identify novel genes involved in
regeneration as well as a new role in regeneration for genes with a known function. This will aid
in our understanding of the molecular mechanisms that promote CNS regeneration and support
the development of novel therapeutic strategies for the treatment of spinal cord injuries.
1.6 Gene Expression Profiling with Microarrays
Microarray based expression profiling is a common and valuable tool for performing large-scale
screening studies (Murphy, 2002). While other techniques (qPCR, Western blotting) are slow,
laborious and allow only a few genes to be investigated at a time, microarrays allow for profiling
of thousands of genes simultaneously and are therefore an effective tool for large scale gene
expression experiments.
1.6.1. Microarray Technology
A microarray consists of thousands of spots of oligonucleotides of a specific sequence (probe),
used to hybridize a cDNA sample. Probes are attached by covalent bonds to a chemical matrix
gene chip. Hybridization of the two strands relies on hydrogen bonding between complementary
nucleic acid sequences.
When performing a microarray experiment, typically either a one-colour or two-colour
microarray approach is used. A one-colour platform involves the hybridization of a single sample
to each microarray after it has been labeled with a single fluorophore. In a two-colour procedure
two samples (experimental, control) are labeled with two different fluorophores and hybridized
onto the same array (Ahmed, 2006a;Ahmed, 2006b). There are advantages and disadvantages to
both approaches. In the two-colour design hybridization of two samples onto the same array is
performed to reduce variability by allowing for a direct comparison. However, dye specific
15
biases can affect the results and many technical replicates need to be performed to enhance
accuracy, which substantially adds to cost. The main advantage of using a one-colour platform is
that it is simple and allows for more flexibility (Ahmed, 2006a;Ahmed, 2006b). The approach
allows both a comparison across microarrays and between groups of samples. Similarly, the
variability between microarrays can be reduced by performing sufficient technical and biological
replicates (Shi et al., 2008). Both approaches have similar shortcomings, including poor
reproducibility and specificity, and studies have shown that data generated from both one-colour
and two-color platforms are approximately equivalent (Patterson et al., 2006). Thus, the decision
to perform a one-colour or two-colour approach is based on the individual experiment, cost and
personal preference.
We designed a custom L. stagnalis 15k gene expression array to study CNS transcriptome
changes following CNS injury. Our gene chip consisted of eight individual arrays each with 15
000 spots. For our experiment we chose a one-color array so that we could have more flexibility
with our analysis and be able to use the data for future studies.
1.6.2. Applications of Microarrays in Regeneration
The CNS is an extremely complex system which makes analysis of transcriptome events
following neural injury difficult. The use of microarrays allows for elucidating global changes in
gene expression after CNS injury and provides insight into this complex biological network
(Munro and Perreau, 2009). Microarray technology has already led to significant advancements
in understanding the molecular cascades following brain and spinal cord injury (Marciano et al.,
2002;Dash et al., 2004). A study by Aimone et al. examined gene expression changes following
rat spinal cord injury at the epicenter as well as both rostral and caudal to the lesion site for up to
45 days after contusion. This elaborate and complex experiment demonstrated the temporal and
spatial complexity of gene expression changes that occur after spinal cord injury. It also
identified affected regulatory pathways in multiple systems, including inflammation,
complement cascade, vasculature and angiogenesis, cholesterol biosynthesis, cell death, calcium
signaling, and synaptic plasticity (Aimone et al., 2004). A subsequent study by De Biase et al.,
examined the gene expression over time (4 hrs, 24 hrs, 7 days) at the impact site, as well as
rostral and caudal regions, following mild, moderate, or severe SCI in rats (De et al., 2005). It
16
showed that gene expression responses following mild injury were relatively rapid (4 hrs and 24
hrs), whereas they were delayed and prolonged after more severe injuries. In addition, the
number and magnitude of gene expression changes were greatest at the injury site after moderate
injury and increased in rostral and caudal regions as a function of injury severity (De et al.,
2005).
The ultimate goal of neuronal regeneration research is to lead to functional recovery. Microarray
technology is currently being applied to various models of nerve regeneration, including in vitro
models of neurite outgrowth and in vivo models of peripheral nerve injury permissive to
regeneration (Bosse et al., 2006;Szpara et al., 2007;Stam et al., 2007). For example, Szpara et al.,
assessed the transcriptional activity in embryonic superior cervical ganglia (SCG) and dorsal root
ganglia (DRG) during a time course of neurite outgrowth in vitro (Szpara et al., 2007). They
found that despite its potent effects as an inhibitory extrinsic cue Sema3A does not appear to
significantly affect the intrinsic transcriptional activities involved in neurite outgrowth. Thus, the
robust effects of semaphorin signaling at sites of injury and regeneration are mediated locally
and not via transcriptional changes in the soma (Szpara et al., 2007). On the other hand Stam et
al., utilized the differential response of DRG neurons following lesion of their peripheral neurite
vs the central neurite in order to gain insight in the early transcriptional events associated with
successful regeneration (Stam et al., 2007). They showed that peripheral and central nerve
crushes elicit very distinct transcriptional activation and identified Ankrd1, a gene known to act
as a transcriptional modulator to be involved in neurite outgrowth (Stam et al., 2007).
Comparison of conserved gene expression changes in regenerating and non-regenerating animals
can also help elucidate genes involved in this response. As such animals that exhibit successful
regeneration such as the immature opossum, salamanders as well as invertebrate models
including the leech, planarians and snails have been used to identify the mechanisms that
underlie CNS regeneration (Baylor and Nicholls, 1971;Lane et al., 2007;Monaghan et al., 2007).
For example, Monaghan et al. designed a custom gene chip to examine gene expression changes
following tail SCI in the salamander. They identified numerous genes that were similarly up-
regulated in the both mammals and salamanders, including hemoxygenase-1, APOE, and genes
involved in the immune response. On the other hand several genes were differentially regulated
between these two models (Monaghan et al., 2007). A microarray study of gene expression
changes in planarian regeneration also revealed genes with similar regulation as mammals, for
17
example N-CAM, and a G-protein subunit, as well as differentially regulated genes (Cebria et al.,
2002), emphasizing the complexity of the regenerative process and the necessity for more gene
expression studies in other regenerative models.
The use of these and other regenerative models for transcriptome analysis has been limited until
now due to the availability of species specific microarrays. However, with the recent
advancements in genome wide sequencing, further studies of regenerative mechanisms can be
undertaken.
1.7 RNA interference: A Tool to Identify Molecular Mechanisms
Only over a decade old, RNA interference (RNAi) is a revolutionary technique for manipulating
gene expression in invertebrates and vertebrates based on a highly-conserved cellular pathway.
RNAi was first described in an invertebrate model by Fire and Mello (Fire et al., 1998). They
found that injecting long dsRNA into C. elegans resulted in degradation of the corresponding
homologous mRNA in both the somatic cells as well as the F1 progeny. RNAi is also related to
other gene-silencing phenomena, including posttranscriptional gene silencing in plants. A form
of PTGS in response to transgene sequences was first documented in petunias, when introduction
of a transgene resulted in plants with white instead of purple flowers (Napoli et al., 1990). This
phenomenon was termed 'co-suppression’ because the expression of both the introduced
transgenes and the homologous endogenous genes were coordinately suppressed. A similar
phenomenon was reported in the fungus N. crassa, a silencing mechanism known as ‘quelling’
(Romano and Macino, 1992). Although these processes at first seemed unrelated, they all use
dsRNA homologous to the silenced gene. In addition, key proteins involved in RNAi are highly
conserved across different organisms (Cogoni and Macino, 2000). However, interest in RNAi
rapidly escalated when Tuschl and colleagues demonstrated that RNAi also occurs in
mammalian cells (Elbashir et al., 2001). Since then, the basic processes involved in RNAi have
been determined in detail. The RNAi response is initiated by the enzyme Dicer, an RNAase III
ribonuclease, which cleaves long dsRNA into double stranded siRNA about 20-25 nt long
(Bernstein et al., 2001). The siRNA is then incorporated into the RNA-induced silencing
complex (RISC) where the sense strand is cleaved by the nuclease Argonaute-2 (Ago2) protein.
This results in separation of the two strands, and the RISC complex with the remaining antisense
strand searches for complementary mRNA that is subsequently cleaved by Ago-2 (Sioud, 2011).
18
RNAi effects mediated by dsRNA have been demonstrated in a number of organisms including
plants, protozoa, nematodes, and insects (Cogoni and Macino, 2000). Unlike invertebrates, long
dsRNA was found to potently activate an immune response in mammals (Judge et al.,
2005;Kleinman et al., 2008). The long dsRNA is interpreted by the cells as a pathogen and
initiates a nonspecific interferon (INF) response similar to a viral infection. Protein kinase R, a
cytoplasmic dsRNA interactive protein binds to the dsRNA and regulates IFN-α and β and
terminates protein synthesis (Clemens and Elia, 1997). In addition, enzymes are induced which
produce 2′-5′-linked oligoadenylates and thereby cause unspecific degradation of single-stranded
RNA (Player and Torrence, 1998). However the INF response is only triggered by dsRNAs
which are longer than 30 nucleotides, chemically synthesized short double stranded RNAs
(siRNAs), can induce gene silencing without activating this immune response (Manche et al.,
1992;Elbashir et al., 2001). The advantage of using an invertebrate model is that the genes that
constitute the IFN response are absent from the known invertebrate genomes. As a result the
invertebrates lack the ability to mount antiviral responses when dsRNA is applied, allowing the
researcher to use various RNAi techniques.
Despite the vast potential of this technique it is not equivalent to modifying a gene by mutation.
RNAi acts to decrease mRNA levels and thus can result in incomplete suppression of gene
expression (Sigoillot and King, 2011). In addition RNAi can potentially induce non-specific
effects. Off-target effects are due to silencing of non-target mRNA molecules that have a high
degree of homology with the target sequence (Jackson et al., 2003). Off-target effects are also
induced through the activation of the immune response. Further undesirable side effects are due
to the ability of siRNA to act as micro RNA (miRNA). In this way siRNAs can interact with the
3′-UTR of mRNAs by partial homology and can inhibit their translation (Saxena et al., 2003).
However, the unspecific effects can be minimized with proper RNAi design. Long dsRNA or
commercial siRNA can be either injected into animals, delivered via bacteria, expressed as
transgenes or delivered into cultured cells by transfection or bathing (Perrimon et al., 2010).
RNAi is an invaluable tool for addressing fundamental questions in the biology of living
organisms. Most organisms possess the cellular machinery for RNAi and, consequently this
approach makes loss-of-function studies possible in organisms where genetic tools do not exist.
RNAi has served as a powerful molecular tool to study gene function and provided novel
strategies to treat a variety of diseases. Neurodegenerative diseases represent one of the preferred
19
targets for the development of therapeutic RNAi (Boudreau and Davidson, 2010). For example
Huntington’s disease, spinocerebellar ataxia, Alzheimer’s are all being heavily studied and there
are promising results from RNAi therapy (Gonzalez-Alegre, 2007). In addition RNAi is also
used in regeneration studies in order to isolate the elements involved in regeneration. In the L.
stagnalis both dsRNA and siRNA have been used to successfully reduce targeted mRNA levels,
either by injecting it into the ganglia or applying it into the culture bath solution (Wagatsuma et
al., 2006;Hui and Feng, 2008).
1.8 Assessment of Real-Time qPCR for Gene Expression Analysis
Quantitative real-time PCR (qPCR) is a valuable tool for quantifying gene expression levels. In
qPCR, the amount of PCR product is determined after each cycle of amplification using a
fluorescent indicator (e.g. SybrGreen) or probe (e.g. TaqMan). The change in fluorescent signal
is directly proportional to the number of PCR products, from which the absolute or relative
amount of mRNA can be quantified (Mackay et al., 2002). Currently there are two predominant
methods for qPCR analysis, the comparative Ct (ΔΔCt) and the efficiency-correction (standard
curve) method (Livak and Schmittgen, 2001;Pfaffl, 2001). Both techniques normalize the target
gene expression to a control gene, whose expression does not change after treatment. The
expression levels are normalized and compared between samples. Both methods require the
determination of the efficiency for each gene in addition to the sample-by-sample quantification.
The ΔΔCt method further requires that the efficiencies be identical between target and control
genes (Livak and Schmittgen, 2001;Pfaffl, 2001).
Recently, there has been a rise in the use of qPCR for gene expression quantification, partly due
to validation of large-scale screening experiments such as microarrays, which have also become
increasing popular (Canales et al., 2006;VanGuilder et al., 2008). Due to demand for more rapid
qPCR analysis techniques our lab has recently developed a new approach using Ct-Ct plots for
analysis of qPCR data (Hui et al., 2009). Ct is the PCR cycle at which the amplification process
crosses a user-defined detection threshold (T) within the early exponential phase of the
amplification process. This value of Ct is an indirect measure of the quantity of mRNA. In order
to minimize variability and cost we construct mini-standard curves using dilution varying
replicates. This way dilution-varying replicates measure both Ct value and efficiency thereby
minimizing extraneous variation. The Ct values for test and control genes can then be plotted
20
against each other and fit with the constraint of identical slopes. The intercepts from these fits are
then estimates of the differences in relative expression of two genes between samples.
Specifically, a downward-shift in the plot reflects an increase in relative expression and an
upward-shift reflects a decrease. This new method is simple and has proven to be reliable (Hui et
al., 2009). We took advantage of this rapid and cost-effective technique for qPCR analysis to
validate the data from a microarray experiment and confirm the mRNA expression levels of
several identified genes.
1.9 Hypotheses
1.9.1.1 Rational I
Neuronal regeneration in the adult mammalian central nervous system is severely compromised
due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity.
In contrast, the CNS of adult L. stagnalis is capable of spontaneous regeneration following
neuronal injury. Thus, L. stagnalis has served as an animal model to study the cellular
mechanisms involved in neuronal regeneration.
Transcriptional regulation is a critical component of intrinsic regenerative mechanisms that
promote CNS regeneration in invertebrate and vertebrate organisms. We have recently conducted
microarray analysis following CNS injury in the L. stagnalis and identified the transcription
factor C/EBP to be up-regulated after injury.
1.9.1.2 Hypothesis I
The transcription factor CCAAT enhancer binding protein (C/EBP) is important for proper axon
regeneration following injury in the L. stagnalis.
1.9.1.3 Specific Aim I
1) Perform a high throughput screening for altered gene expression levels following nerve injury
in the L. stagnalis
1A) Develop a custom L.stagnalis 15 k microarray gene chip
1B) Identify differentially regulated genes following CNS injury using the custom microarray
21
2) Characterize the role of C/EBP on neurite regeneration following microarray analysis of the L.
stagnalis CNS injury model
2A) Confirm the gene expression level of C/EBP following CNS injury using qPCR
2B) Determine if C/EBP mRNA expression is regulated in a time-dependent manner
2C) Following axotomy of cultured neurons in vitro use RNAi to knockdown C/EBP mRNA to
test if it plays a role in regeneration
1.9.2.1 Rational II
Through a microarray analysis, we previously identified 67 genes/EST’s that were significantly
regulated following CNS injury in the L. stagnalis. Neuronal regeneration following trauma to
the CNS is a complex process regulated by multiple proteins, thus, molecular pathways that
underlie regeneration are promising targets for the treatment of CNS injury.
We have employed a novel proteomic approach to further analyze the microarray data and
identified a new CaM-signaling pathway in the L. stagnalis. We will apply an RNAi approach to
test the role of two proteins, BCL 7 and protein phosphatase 1k, identified in the novel signaling
network on regeneration in the L. staganlis.
1.9.2.2 Hypothesis II
BCL 7 and protein phosphatase 1k enhance regeneration in the Lymnaea CNS.
1.9.2.3 Specific Aim II
1) Use bioinformatics to identify a novel signaling cascade during CNS injury.
1A) Translate identified L.stagnalis genes and identify their mammalian orthologues
1B) Input the newly derived list into PPI-software in order to determine if a novel signaling
cascade exists.
2) Determine whether BCL 7 and protein phosphatase 1k, genes identified in the PPI interaction
network, are indeed part of the regenerative signaling response in the L.stagnalis.
22
2A) Examine the time dependent regulation of BCL 7 and protein phosphatase 1k after CNS
injury
2B) Following axotomy of cultured neurons in vitro use RNAi to knockdown BCL 7 and protein
phosphatase 1k mRNA levels and test if they play a role in regeneration
3B) Use live-cell imaging to study the rate of distal and proximal neurite elongation after
axotomy in BCL 7 knockdown cells
4B) Examine the effects of BCL 7 knockdown on calcium levels following axotomy.
23
2. General Materials and Methods
2.1 Animals
Fresh water pond snails, L. stagnalis, were obtained from culture at the Free University,
Amsterdam, and maintained in the laboratory under standard conditions, at the University of
Toronto (Hui et al., 2007;Fei et al., 2007). All animals used were kept in water at 20 °C under a
12 h light: dark cycle, and fed green leaf lettuce twice a week. Two-month old animals having a
shell length of ~20mm were used for the experiment
2.2 Surgical procedures and in vivo nerve injury
Animals were anesthetized by injecting 0.5 - 1 ml of 60 mM MgCl2 into the foot of the snail.
Dissections were performed in sterile snail saline containing (in mM): NaCl, 51.3; KCl, 1.7;
CaCl2, 4.1; MgCl2, 1.5 (pH was adjusted to 7.9 with 1 mM HEPES/NaOH) a small incision was
made in the dorsal head region and pinned open, exposing the cerebral and buccal ganglia. The
right parietal (RPa) and right cerebral nerves (RCe) were crushed using forceps (Koert et al.,
2001). Sham animals were operated in the same way as the experimental groups, except that the
RPa and RCe connections were not crushed. Following the operation, both the sham-operated
and crush-operated animals were maintained under standard laboratory conditions as described
above. For studying early gene expression changes (microarray, qPCR), operated animals were
kept for 1 hr, 3 hrs and 5 hrs under standard conditions. For the long term functional study of
recovery, animals were kept for 10 days under standard conditions and fed green leaf lettuce
twice a week.
2.3 RNA extraction and cDNA synthesis
Snails were anesthetized with 10% (v/v) Listerine (Listerine) for 10 min, following which the
central ring ganglia and the buccal ganglia were dissected out for total RNA extraction at 1 hr, 3
hrs or 5 hrs post-injury. TRIzol was used to extract total RNA (Invitrogen, USA). Specifically,
100 μl of Trizol was used per two excised ganglia and the final pellet was resuspended in 10 μl
of RNase free water. RNA quality was checked on an agarose gel prior to cDNA synthesis and
RNA concentration was measured using spectrophotometry. First strand synthesis of cDNA was
24
conducted using SuperScript III reverse transcriptase (Invitrogen, USA) with random hexamer
primer (Fermentas, USA) in a reaction volume of 20 μl for 1 μg of total RNA.
2.4 cDNA Microarray
The L. stagnalis microarray was designed based on the L. stagnalis CNS EST database
(http://www.Lymnaea.org/). The 15K gene chip was made by Agilent Technologies (U.S.A).
Briefly, the array contained a total of 15, 000 spots, 10, 333 probes were from unique
EST/nucleotides and 4, 425 probes included different sequences from the duplicate genes, and 50
technical replicates with 9 replicates per probe. The hybridization was performed by the
University Health Network Microarray Centre (Toronto, Canada). One-Color Microarray-based
Gene Expression Analysis (Agilent technologies) with cyanine 3-labeled targets was used to
measure gene expression in experimental and control samples. For the microarray, 2 ganglia
were collected per group; there were 8 groups in total: 4 sham-operated and 4 crush-operated.
Briefly, 500 μg of total RNA was amplified using a Fluorescent Linear Amplification Kit
(Agilent Technologies), labeled with Cy3-CTP and hybridized to the L. stagnalis cDNA
microarray at 65°C for 17 hours. The images were scanned using a Genepix 4000B microarray
scanner (Axon Instruments, Foster City, CA, USA). Image analysis, spot quality control and
normalization were performed using Feature Extraction software 9.5.3 from Agilent
Technologies. The intensity files were loaded into GeneSpring GX 10.0.2 software (Agilent
Technologies), a 75th percentile signal value was used to normalize Agilent one-color
microarray signals for comparisons between arrays. The signals were log 2 transformed, and the
median of the replicated probes was obtained. An unpaired t-test was performed and p<0.05 was
used as a cutoff; from these results we used a 2.0-fold change in signal intensity as a cutoff line
to consider the differential expression of a gene as significant.
2.5 Real-time quantitative PCR (qPCR)
qPCR was performed using 5 μl of SYBR GreenER Reagent System (Invitrogen), added to 1 μl
of the appropriate primer set (Table 1), and a diluted sample of cDNA that was topped off with
DEPC water to a final volume of 10 μl. For qPCR, 5 new groups of crush-operated and 5 new
groups of sham-operated snails were used, each group contained RNA from two ganglia. For the
qPCR validation and time-course a new set of RNA samples from sham and crush-operated
snails was used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been used as a
25
common control gene for spinal cord injury models (De et al., 2005;Lacroix-Fralish et al., 2006).
The expression of GAPDH was unchanged following CNS injury in the L. stagnalis, therefore, it
was an internal control. The cycling parameters were: 95°C/5 min, 40 cycles of 95°C/0.5 min,
55°C/1 min, followed by a melting curve protocol. Changes in gene expression levels following
CNS injury and drug application were determined using Ct-Ct plots (Hui et al., 2009), this
analysis is a modification of the ΔΔCT and efficiency correction methods (Livak and
Schmittgen, 2001). The efficiency of the amplification is assumed to be independent of the
sample, and the standard curves from all the samples are linear. Assuming that the PCR
efficiencies are independent of the sample, a Ct-Ct plot is created by plotting the Ct (threshold
cycle) values of the test gene against the control gene. This yields a linear correlation between
the gene pair and the slope (m) of the plot that is described as m = log (Ec)/log(Eg). The Y axis of
these plots is described by Ctg = m x Ctc – log(R)/log(Eg); where R is the relative expression
level between the target gene (Qg) and control gene (Qc); Eg is the amplification efficiency of the
target gene; Ctg and Ctc are the threshold cycles of the target gene and control gene, respectively
(Hui et al., 2009). The Y-intercept, a quotient of – log(R)/log (Eg), is used as a measure of R,
because Eg is constant. Thus, a small change in the Y-intercept in the injury group over the
control group indicates a decrease in the relative target-gene expression level.
26
Table 1. Gene specific primer sequences, product size for qPCR
27
2.6 Primary cell culture
Snails were anaesthetized for 10 minutes in 10% (v/v) Listerine, and the ganglia were excised in
snail saline containing (mM): NaCl 51.3, KCl 1.7, CaCl2 4.1, MgCl2 1.5, HEPES 2 (adjusted to
pH 7.9 using 1 mM NaOH). The ganglia were then incubated in 3 mg/ml trypsin (Type III,
Sigma, Ontario, Canada) for 24 min. PeA neurons, which are involved in locomotion (Spencer et
al., 1995), were identified and isolated from pedal ganglion with gentle suction using a
Sigmacoted (Sigma, Ontario, Canada) fire-polished pipette (2 mm, WPI, 1B200F) as previously
described (Syed et al., 1990;Feng et al., 2000). The neurons were then placed onto a poly-L-
lysine coated culture dish and maintained in conditioned medium (CM) at room temperature
(Syed et al., 1990;Spencer et al., 1996;Feng et al., 2000).
2.7 RNAi synthesis and delivery
Similar to previous studies dsRNA and siRNA were used for gene silencing experiments (Hui et
al., 2007;Guo et al., 2010). 27-mer siRNA, based on the L. stagnalis C/EBP sequence was
designed using SciTools RNAi Design online software (IDT DNA). When choosing siRNA we
selected for sequences with moderate to low G/C content, biased towards the 3’- terminus and
purposely avoided sequences encoding the transmembrane domains (Reynolds et al., 2004).
Control siRNA was design such that it had the same composition as the selected siRNA
sequence, but had no obvious homology with the mRNA. The selected sequences were then
purchased from Shanghai GenePharma Co.,Ltd (Table 2).
For the synthesis of the long dsRNA, we designed gene specific primers corresponding to the
mRNA fragments with a T7 phage polymerase promoter sequence added onto the 5’ end (Table
3). Conventional PCR (PTC-100TM Programmable Thermal Controllor) was used to amplify the
gene specific RNA with the T7 promoter sequence. A 10 μl reaction volume was constructed
with 5 μl of 2 X PCR master mix (Fermentas, USA) containing: 0.05 μl Taq DNA Polymerase,
reaction buffer, 4 mM MgCl2 and 0.4 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 1 μl of
gene specific T7 forward and reverse primers (Table 3), 1 μl of cDNA library and 3 μl of RNAse
free water. The sample was amplified using the following temperature profile: 94°C/2 min, 35
cycles of 94°C/0.5 min, and 72°C/0.5 min, and finally 72°C/10 min. Amplified product was then
purified using the PureLinkTM PCR Purification Kit (Invitrogen, USA). Control dsRNA was
28
synthesized from a linearized non-coding gWIZ-pBSK vector with T7 forward and T3 reverse
promoter sequences. RNA was transcribed using MEGAscript High Yield Transcription Kit
(Ambion) following directions provided by the manufacturer. Synthesized RNA nucleotides
were then denatured in 85oC water bath for 10 minutes and allowed to gradually anneal as the
bath cooled.
For whole animal knockdown experiments snails were anaesthetized with 10% (v/v) Listerine,
following which 2 μl of 20 μM control RNAi or gene specific RNAi was injected into the head
above the central ganglion using a microlitre syringe (Hamilton Company, Reno, NV, USA)
(Guo et al., 2010). 48 hrs post-injection the ganglia were removed and RNA extraction was
performed in order to confirm the RNAi knockdown using qPCR.
29
Table 2. Custom siRNA sequences used in the knockdown study for C/EBP
Table 3. Primer sequences for dsRNA synthesis, T-7 promoter sequence (5’-
TAATACGACTCACTATAGGG-3’) has been tagged onto the gene specific sequences in
order to transcribe RNA into dsRNA.
30
2.8 Axotomy and neurite outgrowth
L. stagnalis PeA cells were cultured in CM at room temperature for 48 hours, allowing for
sufficient neurite outgrowth. Transection of the neurite was then performed using a glass
micropipette, controlled by a micromanipulator, and moving it perpendicularly across the neurite
until a complete cut was observed (Erez et al., 2007;Nejatbakhsh et al., 2009). Immediately
following axotomy the cells were treated with either culture media (CM) or a final concentration
of 7 nM C/EBP siRNA/control siRNA or 7 nM gene specific dsRNA/ control dsRNA in a final
volume of 2 mL of culture media. Images of neurites were collected at the time of RNAi
treatment (t = 0), and over a 24 hr period (t = 24), using an inverted microscope (40x objective)
(Olympus CK X41) with a digital camera (Olympus C5050). The lengths of the proximal neurite,
the distal neurite as well as an intact neurite of the axotomized cell were measured at t = 0 and t
= 24 hrs, using ImageTool 3.1 software (UTHSCSA, Texas).
2.9 Calcium imaging
Intracellular Ca2+
concentration ([Ca]i) was measured using a fura-2 ratiometric Ca2+
imaging
system (Feng et al., 2002;Hui et al., 2007). Cultured PeA neurons were washed with snail saline.
Cells were loaded with 2 µM Fura-2 AM (Molecular Probes), and pluronic acid F-127
(Molecular Probes) in the standard external solution for 30 min at 37°C in the dark. The cells are
then washed again three times with the external solution and incubated for another 15 min at
37°C in the dark prior to imaging. All of the experiments were carried out in the dark to prevent
photobleaching of the dye. Fura-2 was alternately excited at 340 and 380 nm by illumination
generated by a 100 W Hg/Xe-arc lamp that had passed through 340 and 380 nm excitation filters;
this process was controlled by Image Pro 5 software (PTI). The fluorescence signal was reflected
via a 430 nm dichroic mirror, passed through a 510 nm emission filter and detected and digitized
by an intensified charged-coupled device (ICCD) camera (Roper Scientific) in Image Pro 5 at 1-
10 Hz. The fluorescence intensity (Poenie-Tsien) ratios of images acquired at 340 and 380 nm
were calculated using Image Pro 5.
2.10 Immunocytochemistry and confocal imaging
Cultured PeA cells were fixed with 1% paraformaldehyde in snail saline solution overnight at
4°C. Cells were then washed three times with snail saline, and incubated with a monoclonal
31
mouse β-tubulin antibody (diluted 1:200, Sigma Aldrich) in snail saline solution containing 0.3%
Trition X-100 for 4 hrs at room temperature. The cells were subsequently washed four times with
snail saline, and incubated with goat anti-mouse 2o antibody (diluted 1:200; Invitrogen) and
rhodamine phalloidin (diluted 1:100; Invitrogen) in snail saline solution containing 0.3% Triton
X-100 for 2 hours at room temperature in the dark. The cells were then washed four times and air
dried for 5 min, and the preparation was mounted with permafluor (Fisher Scientific, Canada).
The fluorescence microscopy was conducted using a TCS SL laser confocal microscope (Leica
Confocal Software, version 2.5, build 1347, Leica Microsystems, Germany). z-series scan
images of labeled cells were viewed under a 40 or 60X oil immersion lens using a Ar and Green
He Ne laser. The goat anti-mouse 2o antibody was excited at 488 nm the phalloidin was excited
at 568 nm. Each plane was averaged three times using a step size between each plane of
approximately 0.3μ m. Background fluorescence was determined using three regions not
encompassing cells throughout the entire z-stack. The resolution of the final image was 1024 by
1024 pixels.
2.11 Bioinformatics
Identified sequences were translated using ExPASy online software
http://ca.expasy.org/tools/dna.html, and the protein sequences were blasted with
http://blast.ncbi.nlm.nih.gov/Blast.cgi and the closest matches recorded. Using Clustal W
program http://www.ebi.ac.uk/clustalw/sequences were aligned and compared with human
database. Orthologues were analyzed using PPI-spider software http://mips.helmholtz-
muenchen.de/proj/ppispider
2.12 Quantification of locomotion activity
Individual sham-operated and crush operated snails were marked and placed in their normal foot
down posture in a large Petri dish (10 cm diameter by 2 cm depth) in 15 ml of water from their
home aquaria. The snails were giver 5 minutes to acclimate to their environment, after this time
they were recorded for 10 minutes with a video camera using Virtual Dub 1.9 software and the
distance each snail travelled was measured and analyzed using ImageTool 3.1 software
(UTHSCSA, Texas) (Fei and Feng, 2008).
32
2.13 Statistics
Data are presented as mean ± S.E.M. Data were imported to Microsoft Excel or OriginPro v8
(Silverdale Scientific Ltd., Bucks, HP, USA) for graphical presentation. Statistical significance
between mean values of experimental group was evaluated using a Student’s t-test for two
groups, for multiple comparisons the data was analyzed using ANOVA with the Holm-Sidak
post hoc test using SigmaStat 3.0 (SPSS, Chicao, IL, USA). Significance was defined by
probability level of lower than P < 0.05.
33
3. Identification of the role of C/EBP in neurite regeneration following microarray analysis of a L.stagnalis CNS injury model
i
3.1 Introduction
Injuries of the CNS can lead to devastating and irreversible loss of function because adult
mammalian CNS neurons have a limited regenerative capacity (Aguayo et al., 1981;Blesch and
Tuszynski, 2009). This is partially due to an age dependent reduction in the intrinsic regenerative
potential of CNS neurons (Curinga and Smith, 2008;Liu et al., 2010). In contrast, some adult
invertebrate neurons are capable of spontaneous regeneration following injury (Hasan et al.,
1993;Blackmore and Letourneau, 2006). Lymnaea stagnalis has served as a critical model
system because of its ability to spontaneously regenerate and restore function in the adult central
nervous system (Janse et al., 1979;Hermann et al., 2000;Hermann et al., 2005). Since many
molecular mechanisms are conserved across species, the identification of molecules involved in
neuronal regeneration in the L. stagnalis will aid in our understanding of factors which may
promote regeneration in the mammalian CNS.
One main limitation of using L. stagnalis in genetic functional studies of neuronal regeneration
has been a lack of large-scale screening tools, such as microarray analysis (Marciano et al.,
2002;Dash et al., 2004;Munro and Perreau, 2009). To circumvent this limitation, we have
recently sequenced more than 10, 000 ESTs (expression sequence tags) from the CNS
transcriptome of L. stagnalis (Feng et al., 2009) and established the largest neuronal EST
database in Lymnaea (www.lymnaea.org). In combination with the availability of the microarray
technology, these gene sequences provide us with the opportunity to perform a high throughput
screening for altered gene expression levels following nerve injury in the L. stagnalis. In this
study, we have designed the first microarray chip covering 10, 333 known L. stagnalis genes to
profile the gene expression patterns following CNS injury. We identified 348 genes that were
differentially regulated following CNS crush. Using real-time qPCR analysis we confirmed that
the gene expression level of CCAAT enhancer binding protein (C/EBP), a transcription factor, is
34
up-regulated following nerve injury. Knockdown of C/EBP following axotomy of cultured PeA
cells lead to retraction of the distal axon. This indicates that C/EBP plays a crucial role in
neuronal regeneration of Lymnaea neurons by maintaining the viability of the distal neurite.
3.2 Results
3.2.1 CNS injury model of L. stagnalis
In order to profile the gene expression patterns following injury of the central nervous system,
we modified an established (Koert et al., 2001;Hermann et al., 2005) nerve injury model by
crushing the right parietal (RPa) and right cerebral nerves (RCe) (Figure 1A). In control groups,
sham operations were performed without nerve injury.
3.2.2 Identification of differentially expressed genes
To search for genes that may be involved in neuronal regeneration or degeneration, microarray
analyses were carried out to compare the gene expression levels between the sham-operated and
crush-operated CNS models (Figure 1A). We used an unpaired t-test for comparison of the
15,000 array signals between two groups: sham- and crush-operated groups. This analysis
resulted in the selection of 348 genes or ESTs that were differentially regulated 3 hrs following
CNS injury (p < 0.05). Further screening for genes with a fold-change > 2 resulted in a final
group of 67 differentially expressed genes. Figure 1C shows a heat map representing the
hierarchical clustering of the 67 genes with significant differential expression levels between the
control and injured groups. Overall, more genes were up-regulated (n = 42) than down-regulated
(n = 25), suggesting that diverse regulatory pathways and biological processes are activated
within the first 3 hrs following injury (Figure 1B-1). Among the 67 regulated genes only 16
have been identified with known functions related to development, survival or signal
transduction (Table 4). Interestingly, the functions of the majority of genes (51) have not been
described (Figure 1B-2).
3.2.3 Gene expression levels of C/EBP change in a time dependent manner following injury
The microarray analysis indicated that the mRNA level of CCAAT enhancer binding protein,
C/EBP, was significantly increased 3 hrs following CNS injury, as compared to the sham control
35
group. A protein sequence alignment of C/EBP orthologues from L. stagnalis, Aplysia, rat, and
human revealed that C/EBP has a high homology in the DNA-binding domain, suggesting that
C/EBP may have similar biological functions in these species (Figure 2). To confirm that C/EBP
is indeed up-regulated following nerve injury at 3 hrs, we performed conventional qPCR
analysis. We found that the expression level of C/EBP mRNA significantly increased, compared
to the control gene GAPDH, in the injured CNS group (2.35 ± 0.26 fold; n = 5, P < 0.05) over
the sham groups (Figure 3A).
To study its temporal expression pattern, we further measured the gene level of C/EBP at 1 hr, 3
hrs and 5 hrs post CNS injury using real time qPCR analysis. The standard curves for C/EBP are
shown in Figure 3B with a slope of -3.52, indicating the PCR efficiency for the primers are
sufficient (Figure 3B). To visualize the gene expression level between the injured and sham
groups, the threshold cycles (Ct) of C/EBP (the target gene) and GAPDH (the control gene) were
plotted against each other, as described previously (Hui et al., 2009). Figure 3C shows the
representatives of Ct-Ct correlation plots between the C/EBP and GADPH gene pair at the three
different time points (1 hr, 3 hrs, 5 hrs). The slopes of the plots were consistent in all
experimental samples, indicating the equality in amplification efficiencies between the primers
of the gene pair. The Y-intercepts (– log(R)/log(Eg)) of these plots were smaller in the injury
group (crush) than in the controls (sham) at all the times tested, indicating the relative expression
level of C/EBP mRNA increased following injury. Specifically, C/EBP mRNA was initially
increased by 7.36 ± 0.73, (n = 5) at 1 hr after CNS injury, but appeared to reduce to 2.35 ± 0.26
(n = 5) by 3 hrs and 1.98 ± 0.75, (n = 5) by 5hrs, albeit at levels that are still significantly greater
than in sham-operated (control) animal (Figure 3D). These results suggest that elevation of
C/EBP mRNA expression may be necessary for transcriptional induction of regeneration
associated genes, and thus likely to be a direct target of injury induced signaling cascades that
promote neuronal repair.
3.2.4 The role of C/EBP in axonal elongation following axotomy
The increased expression of C/EBP mRNA following nerve injury in vivo suggests that this
protein may be an upstream gene regulator of either pro-regenerative genes or responsive genes
for cell injury. To elucidate the functional significance of C/EBP mRNA following nerve injury,
we examined the effect of C/EBP on axonal outgrowth and regeneration on PeA neurons in
36
culture using a siRNA gene silencing approach, as described previously (Nejatbakhsh et al.,
2009). Axotomy was performed on cultured PeA neurons, which had undergone adequate neurite
outgrowth (Figure 4A). Immediately following axotomy, cells were treated with either, control
culture media (CM), control siRNA or C/EBP siRNA. The lengths of the proximal and distal
ends of the axotomized neurite, as well as an intact neurite were measured over a 24 hr period, as
described previously (Nejatbakhsh et al., 2009). Compared to control CM, and control siRNA
treated cells, the net increase in length of the proximal end and the intact neurites of C/EBP
siRNA treated cells did not show significant changes over the 24 hours following axotomy.
However, the distal end axons in the C/EBP siRNA treated cells retracted (-12.43 ± 8.95, n = 15)
whereas that in control CM (+17.77± 8.27, n=22) and control siRNA treated cells continued to
elongate (19.68 ± 7.83, n = 24) 24 hours following axotomy (Fig. 4B). The net changes in the
distal axons were significant between the C/EBP siRNA and the control CM and control siRNA
groups (P < 0.05). These findings suggest that sufficient C/EBP levels are required for
maintaining the integrity of the distal axon. Due to the application of siRNAs following
axotomy, the differences found in the distal axons may be associated with local protein synthesis.
3.2.5 C/EBP siRNA treatment hinders locomotion recovery in vivo following CNS injury
In order to determine whether C/EBP siRNA treatment has a role in injured animals, we
compared the locomotion activity of the snails treated with control or C/EBP siRNA followed by
RPa and RCe nerve crush. The distances that the snails crawled in 10 min was measured before
and after nerve crush (1, 5 and 10 days post-crush) and compared between control siRNA and
C/EBP siRNA injected groups. As shown in Figure 5, both groups had severely impaired
movement following the nerve crush-operation, but slowly gained their ability to crawl over the
course of 10 days. However, after 10 days, the distance (per 10 min) measured from the C/EBP
siRNA group (4.99 ± 0.71 cm, n = 3) was significantly shorter than the control siRNA group
(10.17 ± 1.00 cm, n = 5) (P < 0.05). This data suggests that C/EBP siRNA treatment disrupted
the recovery of snails from nerve injury and support the notion that C/EBP knockdown impedes
CNS regeneration in vivo, potentially through its effects on the distal neurites.
37
Figure 1. Experimental model and changes in gene expression following injury
(A) CNS injury protocol: the dorsal surface of the L. stagnalis CNS, both right parietal (RPa)
nerves and right cerebral nerves (RCe) were crushed with fine forceps. Inset: a schematic
diagram of the ganglia and location of injury (black lines). (B) Ratio of the regulated EST
sequences 3 hrs following CNS injury in the L. stagnalis. (B-1) A total of 348 genes or ESTs
were shown to be differentially regulated in signal intensity. Open pie (80.7 %) represents
genes/EST’s changes < 2.0-fold in signal intensity but significant (P <0.05); grey pie (19.3 %):
changes ≥ 2.0-fold (P < 0.05). Dark grey: up-regulated genes (12 %), and light grey: down
regulated genes (7 %). (B-2) Ratio of the 67 differentially regulated genes, 16 (23.8 %) genes
have orthologous with known functions related to development, survival or signal transduction,
whereas 51 (76.2 %) genes have no orthologues. (C) Heat map representing the hierarchical
clustering of 67 genes showing significant differential expression (P < 0.05). The expression
pattern of each gene in the sham and crashed groups is displayed as a horizontal stripe. For each
gene, the expression ratio of the crush-operated to the sham-operated experiments is represented
by the green and red scale at the bottom of the figure. The genes are numbered on the right, and
the experimental groups are labeled on the bottom.
38
Figure 1
39
Table 4. Selected genes with more than 2 fold changes in expression level following CNS injury
in L. stagnalis and their predicted functions, based on their sequence analysis.
40
Figure 2. Protein sequence alignment of C/EBP
Amino acid alignment between CCAAT enhancer binding protein from L. stagnalis [GenBank #:
BAD16556], A. kurodia [GenBank #: AAG61258], R. norvegicus [GenBank#: AAI29072] and
H. sapian [GenBank#: EAW75629] C/EBP. The high degree of sequence similarity at the DNA-
binding domain indicates that C/EBP has a conserved function domain across different species
41
Figure 2
42
Figure 3. Time-dependent changes in C/EBP mRNA expression following nerve injury.
Real-time PCR was performed with specific primers for C/EBP between sham-operated and
crush-operated L. stagnalis CNS at 1 hr, 3 hrs, and 5 hrs post injury.
(A) Relative mRNA levels of C/EBP vs GADPH increased significantly following CNS injury as
compared to the sham-operated controls at 3 hrs (2.35 ± 0.26, n = 5) (t = -3.5, df = 8, P < 0.05).
(B) Standard curves of C/EBP and GAPDH from 6 independent samples. The PCR efficiency of
the primers was estimated by slope (m) = -1/log(efficiency). The expression GAPDH was
unchanged following CNS injury, therefore it was used as an internal control. (C) Representative
Ct-Ct correlation plots between C/EBP and the control gene, GADPH, at three different time
points following injury. C1: 1 hr; C2: 3 hrs; C3: 5 hrs. Relative expression ratio between C/EBP
and GADPH was estimated as Yintercept = -log(ratio)/log(efficiency C/EBP). (D) Relative gene
expression of C/EBP vs. GADPH normalized to corresponding control samples. C/EBP
increased in a time-dependent manner. 1 hr: 7.36 ± 0.73 (n = 5); 3 hrs: 2.35 ± 0.26 (n = 5), and 5
hrs: 1.98 ± 0.75 (n = 5). * indicates significant differences: ANOVA: F(2,12)=17.4, P < 0.05. The
data were presented as means ± S.E.M.
43
Figure 3
44
Figure 4. Knockdown of C/EBP reduced net growth of the distal axon following axotomy.
(A) Representatives of transected axons in culture. Immediately following axotomy (t = 0), either
CM, or control siRNA or C/EBP siRNA was added into culture medium at a final concentration
of 7 nM. The neurons were observed over the next 24 hrs (t = 24 hrs). Arrow indicates the
transection site. (B) The length of the proximal axon, distal axon, and intact neurite of injured
cells were measured after treatment over the next 24 hrs. The distal axon had a significant
reduction in outgrowth following axotomy in the C/EBP siRNA treated cells (-12.43 ± 8.9) (n =
15) as compared to the control CM treated cells (+17.77± 8.27) (n=22) (*P < 0.05) and control
siRNA treated cells (+19.68 ± 7.83) (n = 20) (**P < 0.05). (C) Relative gene expression level of
C/EBP vs GAPDH reduced in the C/EBP siRNA group as compared to control siRNA group (n
= 5) (*P < 0.05). ANOVA: F(2,54) = 4.03, P < 0.05. The data were presented as means ± S.E.M.
45
Figure 4
46
Figure 5. C/EBP siRNA treatment hinders recovery of locomotion activity of the snails
following CNS injury
Distance that the injured snails crawled in 10 min was measured at various time points following
nerve crush procedure. The snails were injected with either control siRNA or C/EBP siRNA. On
the 10th day, the C/EBP siRNA group crawled an average of 4.99 ± 0.71 cm (n = 3) per 10 min
which was significantly less than the control siRNA group did (10.17 ± 1.00 cm, n = 5) (t = 3.6,
df = 6, *P < 0.05). Data are present by mean ± s.e.d.
47
Figure 5
48
Figure 6. Schematic illustration of the potential mechanisms of C/EBP action in
maintaining distal axon integrity following axotomy.
C/EBP mRNA is expressed in both the soma and axon (Nadeau et al., 2005;Yan et al., 2009). In
the soma, C/EBP functions as a transcription factor and results in the transcription of
regenerative-associated genes, such as β-tubulin and GAP-43, via a cAMP/PKA/CREB
dependent signaling pathway (Nadeau et al., 2005). Axon injury also activates an ERK-
dependent pathway that results in the phosphorylation of C/EBP and further activation of pro-
regenerative genes (Sung et al., 2001). In the distal axon the DLK-1 pathway regulates C/EBP
mRNA stability following axotomy (Yan et al., 2009). Local protein synthesis of C/EBP in the
distal axon may be required for regeneration or to prevent degeneration of distal axons following
injury.
49
Figure 6
50
3.3 Discussion
In this study, we designed the first Lymnaea gene chip for microarray analysis and profiled the
gene expression patterns during the early stages of CNS repair following injury. We found that
67 genes were significantly regulated during the first three hours post injury, including the
transcription factor C/EBP. We further studied the temporal gene regulation pattern of C/EBP
following CNS injury using real time qPCR analysis, and found that C/EBP gene levels were
significantly up-regulated 7 fold, 1 hr after CNS injury, and decreased to a 2 fold increase at 3
hrs. Subsequently, C/EBP mRNA levels were maintained at the 2 fold increase for at least 5 hrs.
Reduction of C/EBP mRNA levels by C/EBP siRNA treatment following axotomy caused
retraction of the distal neurite in vitro, suggesting local protein synthesis of C/EBP mRNA in the
distal axon. Our observation that C/EBP siRNA treatment disrupted the recovery of locomotory
function of the snails following nerve injury in vivo further support the notion that C/EBP is
required for nerve regeneration. C/EBP is expressed along the axon, in the cell body, and at the
synapse (Brown et al., 2004), indicating that this gene may act in distal regions of the axon to up-
regulate local mechanisms of regeneration.
3.3.1 Regeneration in the CNS of Lymnaea stagnalis
L. stagnalis has been used as a simple and reliable model to investigate genes involved in
regeneration, due to its ability to functionally recover following CNS injury in vivo. For
example, central ganglia cell (CGC) connections to buccal ganglia were crushed to disrupt
feeding behavior in animals. Two weeks following the procedure the CGC regenerated,
functional synapses formed between proper synaptic partners and the feeding behaviour was
again observed (Koert et al., 2001). In vivo CNS regeneration was also observed following
axotomy of innervating axons to the pneumostome and surrounding areas which prevented the
occurrence of lung respiration in 69% of snails. Several weeks following surgery, axonal
regeneration leading to the reformation of functional synapses was observed and pneumostome
opening returned to normal (Hermann et al., 2000). In our modified CNS injury model, we
crushed both the right parietal (RPa) nerves as well as the right cerebral nerves (RCe) (Figure
1A). We are interested in the gene regulation occurring at the early stages of CNS trauma, thus
most of our in vivo experiments were completed within 5 hours following injury.
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3.3.2 Microarray Analysis Following CNS Injury
The ability to profile the expression of thousands of genes simultaneously makes microarrays an
invaluable biomedical research tool (Murphy, 2002). The technique allows for the quick
identification of genes that may alter regenerative ability in the mammalian CNS. It has
previously been used to assess the genes involved in anti-inflammatory responses in a spinal cord
injury model. The anti-inflammatory response is an important component of secondary tissue
damage following spinal cord injury. The microarray study allowed for identification of a
conserved novel regulator of regeneration, and provided a new mechanism by which to initiate
the regenerative response (Carmel et al., 2001). However, microarrays are not without their
limitations, shortcomings of microarray analysis are associated with poor reliability, and
specificity (Duggan et al., 1999;Murphy, 2002;Asyali and Alci, 2005;Patterson et al., 2006). Due
to the inherent sources of error in microarray technology, it is necessary to confirm the findings
using an independent methodology from separate samples. Real-time quantitative PCR is a
readily used technique to confirm microarray data (Patterson et al., 2006). In our study, we also
conducted qPCR analysis using different samples and verified that the expression of C/EBP was
indeed up-regulated following nerve injury.
3.3.3 Time-dependent Regulation of C/EBP
Recovery from nerve injury and neuronal regeneration are time-dependent events, from the
initial inflammatory response to the development of the glial scar (Hagg and Oudega, 2006).
Inflammation, neurotransmitter dysfunction, ionic imbalance, cytoskeletal alteration and
increased transcription occur as early as 3 hrs following spinal cord injury (Ruda et al.,
2000;Song et al., 2001;Tachibana et al., 2002). Nerve outgrowth and regeneration can be
enhanced by steroids during a critical window within the first 6 hrs following nerve injury
(Tanzer and Jones, 2004). In the C. elegans, axons regenerate following axotomy in vivo and by
24 hrs fully functional synapses are formed (Yanik et al., 2004). We have recently demonstrated
that L. stagnalis regeneration post injury in vitro can be seen as early as 3 hrs, and reformation
of connections between the proximal and distal axon is observed by 24hrs (Nejatbakhsh et al.,
2009). Deducing the gene regulation during the early response following neuronal injury is
important in uncovering the molecular basis of neuronal regeneration. In this study, our
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microarray gene expression analysis identified 67 genes that were significantly regulated
following CNS injury in the L. stagnalis. We examined C/EBP gene expression at three different
time points (1 hr, 3 hrs, 5 hrs) in order to understand how it is regulated temporally in the early
stages of recovery and repair. Our results indicated that the gene is up-regulated in a time-
dependent manner following CNS injury (Figure 3). We further showed the locomotory activity
of the control snails was severely disrupted immediately following nerve injury but recovered
gradually over 10 days. However, C/EBP siRNA treatment prior to nerve crush procedure
disrupted the recovery of locomotory function of the snails (Figure 5), suggesting that C/EBP is
required for nerve regeneration in vivo.
3.3.4 Role of C/EBP in the Distal Neurite
C/EBPs belong to the basic leucine zipper DNA binding protein family and are transcription
factors with diverse cellular roles, including regulating cell growth, differentiation, apoptosis,
and learning and memory (Menard et al., 2002;Marshall et al., 2003;Hatakeyama et al., 2006).
C/EBPs are also responsive to brain injury and ischemia (Kfoury and Kapatos, 2009;Ejarque-
Ortiz et al., 2010). Six isoforms of C/EBP have been found in mammals (Poli, 1998), and
C/EBP is highly homologous to Lymnaea C/EBP (LC/EBP) (Hatakeyama et al., 2004). C/EBP
was first identified as a mediator of the inflammatory response and IL-6 signaling through its
binding to IL-6 response elements in the promoters of acute phase response genes TNF, IL-8,
and G-CSF (Akira et al., 1990). C/EBP expression is enriched in neurons, up-regulated following
brain injury (Cortes-Canteli et al., 2002;Cortes-Canteli et al., 2004;Nadeau et al., 2005) and in
various neuronal regenerative animal models where its expression is regulated by transcription
factors such as CREB and NFIL-3 (Korneev et al., 1997;Sung et al., 2001;MacGillavry et al.,
2011). Aplysia nerve injury activates axoplasmic kinase, RISK-1, which phosphorylates
apC/EBP and initiates the binding of apC/EBP to the ERE enhancer site in vitro. Increases in
RISK-1 and apC/EBP were detected in injured neurons (Sung et al., 2001). In leech, C/EBP
mRNA was increased during neuronal regeneration (Korneev et al., 1997). In this study, we
show that C/EBP mRNA expression is increased following CNS injury in a time-dependent
manner. The 7-fold increase in C/EBP mRNA at 1 hr following injury is likely related to the
inflammatory response. The sustained 2 fold increase over the 5 hr period suggests that it may be
essential for transcriptional induction of regeneration associated genes. Knockdown of C/EBP
mRNA expression following axotomy by target-gene siRNA caused a reduction in neurite
53
outgrowth, as compared to control. Surprisingly this phenomenon was observed only in the distal
axons. Due to the fact that siRNAs were applied following axotomy, our data suggests that local
C/EBP mRNA is likely involved in neuronal regeneration/elongation. It is unexpected to observe
a lack of significant inhibition in the proximal re-growth (Figure 4B) although the net growth of
the proximal length was reduced in C/EBP siRNA group. This could simply reflect that a partial
knockdown (~40%) of C/EBP was not sufficient to cause the disruption of the regenerative
properties of the neurites that are in contact with the cell soma. In contrast to the distal section of
neurites which may rely on local protein synthesis, the cell somata continuously provide
complimentary mechanisms to sustain neurite properties.
Following axotomy, the distal segment undergoes wallerian degeneration and the proximal end
either degenerates or attempts to re-connect with its target (Waller 1850). An alternative
mechanism of regeneration occurs in invertebrates, whereby the proximal axon directly connects
with the distal fragment and prevents it from degenerating (Hoy et al., 1967;Carbonetto and
Muller, 1977;Deriemer et al., 1983). In the C. elegans when mechanosensory neurons are
axotomized the proximal and distal segments will re-grow, if they make contact the distal
fragment survives, otherwise it undergoes wallerian degeneration (Yanik et al., 2004). In this
case the balance between regeneration and degeneration shifts and results in recovery of
neuronal function. A similar process is observed in the Lymnaea, whereby the proximal and
distal ends reconnect after axotomy, which protects the distal end from degenerating. This
illustrates the importance of the distal fragments, and the maintenance of their integrity through
C/EBP, for successful regeneration in this model system.
CEBP-1, a member of the C/EBP family in the C. elegans, is a direct effecter of the DLK-1
cascade (Yan et al., 2009). CEBP-1 mRNA has been found in the axons and presynaptic regions,
and is stabilized via its 3’UTR by activation of the dual-leucine zipper Kinase-1 (DLK-1)
pathway (Yan et al., 2009). The role of C/EBP in axon regeneration is suggested via dual-leucine
zipper kinase MAPKKK (DLK-1)-dependent pathway (Yan et al., 2009). Neuronal regeneration
associated proteins such as -tubulin and GAP-43, are transcriptional targets of C/EBP (Miller et
al., 1989;Menard et al., 2002;Nadeau et al., 2005). Following nerve injury, up-regulation of
C/EBPβ mRNA and C/EBP phosphoprotein coincides with increases in the transcription of the
α-tubulin promoter in the wild-type but not in C/EBPβ −/− mice (Nadeau et al., 2005). DLK-1
acts in a MAPK cascade with MAPMKKK-4 and p38 kinase (Nakata et al., 2005). Both the
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DLK-1 pathway and CEBP-1 were necessary for regenerative growth of adult touch neurons
(Yan et al., 2009). We thus proposed a model by which the DLK-1 pathway regulates C/EBP
mRNA stability, leading to its increased translation at synapses and maintenance of the distal
axon (Figure 6). Local protein synthesis has been shown to increase in injured axons and thus
play an important role in the regeneration-degradation process (Willis and Twiss, 2006;Wang et
al., 2007;Wang et al., 2010). Our study provided the first evidence that local regulation of C/EBP
mRNA in axons is important for outgrowth and regeneration in the L. stagnalis.
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4. Bioinformatic analysis reveals a novel role for BCL 7 in neurite regeneration
4.1 Introduction
Failure of spontaneous regeneration in the adult CNS results in limited recovery following spinal
cord injury. One of the critical steps in successful regeneration is the transformation of the
injured axonal end into a growth cone, a specialized compartment that integrates extracellular
signals into dynamic growth patterns (Erez et al., 2007;Kamber et al., 2009). The transformation
of the injured axon into a retraction bulb rather than a growth cone indicates the presence of
intrinsic or environmental factors which interfere with the regeneration process (Lobato, 2008).
Identification of these factors can lead to novel therapeutic strategies for improving regeneration
in the CNS.
Currently, hundreds of genes and molecules have been implicated in CNS regeneration,
including those involved in both intrinsic and extrinsic processes, and altering the expression of
any one of these may have a limited impact on CNS regeneration (Aguayo et al.,
1981;Muramatsu et al., 2009). Although manipulating molecules such as cAMP, GSK3β and
PTEN have been shown to enhance regeneration, successful functional regeneration has not been
shown to be the result of a single gene (Nikulina et al., 2004;Brown et al., 2004;Park et al.,
2010). Consequently, identifying the molecular pathways that underlie axonal regeneration is a
more promising approach for the treatment of CNS injuries.
Recently, large scale gene-expression profiling and bioinformatics has been applied to
understand the role of known proteins in a biological context and interpret them in the context of
a protein network (Antonov et al., 2009). We have recently taken advantage of the newly
available partial transcriptome for the L. staganlis (Feng et al., 2009), a model for successful
CNS regeneration, to profile the gene expression changes following CNS injury (Figure 1). From
this study, we identified 67 genes whose expressions were significantly altered during injury. In
order to identify a novel signaling network we employed a proteomic analysis approach using
protein-protein interaction (PPI) networks to interpret identified protein lists (Xu et al.,
56
2010;Alberio et al., 2010;Silverman-Gavrila et al., 2011). One such tool is the web-based PPI
spider (http://mips.helmholtz-muenchen.de/proj/ppispider), which develops a PPI network model
based on known protein-protein interactions from the literature. A useful and important feature
of this software is that it takes into account that there may be proteins missing from the list and
automatically includes the most relevant missing proteins. The output is a model of protein-
protein interactions that represent the most probable scenario of how proteins within the list are
functionally connected (Antonov et al., 2009).
Microarray analysis in the L. stagnalis identified various genes potentially involved in CNS
regeneration; we then used PPI software and identified a novel signaling pathway. The proposed
network included BCL 7 and protein phosphatase 1k. In this study an RNAi approach was used
to manipulate the gene expression levels of BCL 7 and protein phosphatase 1k, and examine
their roles following neuronal injury.
4.2 Results
4.2.1 Network mapping using a bioinformatics approach
In order to test if there are potential interactions among the genes that were identified in the
microarray study we used PPI-spider (http://mips.helmholtz-muenchen.de/proj/ppispider)
software. PPI-spider is a newly developed tool for predicting networks based on known protein-
protein interactions (Antonov et al., 2009). Among the 67 genes identified, PPI-spider predicted
a novel signaling network which included 5 proteins: band-4 protein-2, phosphoinositide kinase,
BCL 7, protein phosphatase 1k, and Sec24 (Figure 7). The network also consisted of other
suggested interactor proteins not identified in the microarray: calmodulin-1, SPARC-like protein
1, BCL 6, and bone sialoprotein. The predicted network represents a novel signaling cascade that
may be involved in CNS regeneration in L. stagnalis. Based on this model two genes were tested
for their involvement in neural injury, BCL 7 and protein phosphatase 1k.
4.2.2 Down-regulation of protein phosphatase 1k following CNS injury
Protein phosphorylation and dephosphorylation are major regulatory mechanisms of cellular
functions such as cell signal transduction. Protein phosphatase 1k, which was revealed by PPI-
spider to be part of the proposed signaling network, is a novel PP2C isoform targeted exclusively
to the mitochondria matrix. This family of Serine/threonine phosphatases, share a common
57
catalytic domain which suggests some function is conserved between species (Figure 8A). Our
microarray study revealed that protein phosphatase 1k mRNA expression decreased 3 hrs after
CNS injury in the L. stagnalis model (Table 4). Using qPCR, we further examined the temporal
regulation of protein phosphatase 1k gene expression, compared to control GADPH, and found
that its expression showed a decrease at 1hr (0.43 ± 0.13, n = 5, P < 0.05) and 3 hrs (0.34 ± 0.19,
n = 5, P < 0.05) following CNS injury, but recovered to control levels by 5 hrs (1.2 ± 0.4, n = 5)
(Figure 8B).
The early down regulation of protein phosphatase 1k after CNS crush in vivo suggests it may
play a role in the immediate response to injury. In order to determine if protein phosphatase 1k is
involved in neurite outgrowth or retraction of CNS neurons after injury we used a gene silencing
approach. Axotomy was performed on PeA neurons cultured for 48 hrs to allow for adequate
neurite outgrowth (Figure 9A). Immediately after axotomy, cells were treated with either control
or protein phosphatase 1k dsRNA. Three different neurite lengths were measured over the next
24 hrs, the lengths of the intact neurite as well as the proximal and distal neurites. We found that
a 51.3 ± 9.6% knockdown of protein phosphatase 1k expression, confirmed by whole animal
qPCR, had no significant effect on neurite outgrowth , as no difference was observed between
intact control (29.52 ± 9.2 µm, n = 16) and intact protein phosphatase 1k dsRNA treated (35.4 ±
12 µm, n = 33) cells. Similarly, knockdown of protein phosphatase 1k did not affect regeneration
from the proximal (2.9 ± 2.6 µm, n = 18), and distal (22.34 ± 10.6 µm, n = 18) neurites as
compared to control dsRNA treated cells proximal (13.9 ± 4.1 µm, n = 36) and distal (32 ± 9.2
µm, n = 36) neurites (Figure 9). These results suggest that the down-regulation of protein
phosphatase 1k mRNA is likely not involved in outgrowth and regeneration.
4.2.3 A novel role for BCL 7 in CNS regeneration
The PPI-spider also identified BCL 7 to be involved in the proposed signaling cascade. A protein
sequence alignment of BCL 7 orthologues from L. stagnalis, C.elegans, drosophila, mouse, and
humans reveal a strong sequence similarity at the N-terminus, suggesting that it may be involved
in an evolutionarily conserved function (Figure 10A). However, the function of BCL 7 is
currently unknown, which led us to examine its role in CNS regeneration.
In order to examine the time-dependent regulation of BCL 7 expression during the course of
injury, we performed conventional qPCR analysis on samples 1 hr, 3 hrs, and 5 hrs following
58
CNS injury (Figure 10B). Compared to the control gene GAPDH, we found that the expression
level of BCL 7 mRNA did not increase significantly until 5 hrs after CNS injury (2.5 ± 0.6 fold;
n = 5, P < 0.05). These results suggest that BCL 7 may be important in promoting CNS
regeneration in L.stagnalis.
In order to elucidate the functional significance of BCL 7 up-regulation, we examined the effect
of Bcl7 knockdown on axonal outgrowth and regeneration in cultured PeA neurons using a
dsRNA. One neurite of each PeA neuron was axotomized 48 hours after they were cultured to
allow for adequate neurite outgrowth (Figure 11A). Immediately following axotomy, cells were
treated with either control dsRNA or BCL 7 dsRNA. The knockdown was confirmed by qPCR,
which showed that BCL 7 expression was reduced to 52.1 ± 7.1% of control levels compared to
GADPH (Figure 11C). The lengths of the proximal and distal ends of the axotomized neurite, as
well as an intact neurite from the same cell, were then measured over 24hrs, as described
previously. Compared to the length of control dsRNA treated cells (13.9 ± 4.1 µm, n = 36), there
was a net decrease in length of the proximal neurite (-7.9 ± 6 µm, n = 23) of BCL 7 dsRNA
treated cells. There was also a reduction in the distal neurite length in BCL 7 dsRNA treated cells
(2.2 ± 9.3 µm, n = 23) compared to control dsRNA (32 ± 9.2 µm, n = 36). Likewise, there was a
reduction in the lengths of the intact neurite in BCL 7 dsRNA treated cells (-7.7 ± 10.7 µm, n =
19) compared to control dsRNA treated cells (35.4 ± 12 µm, n = 33) (Figure 11B). These
findings suggest that BCL 7 prevents neurite degeneration and promotes neurite outgrowth
following axotomy.
4.2.4 BCL 7 impairs neurite outgrowth following axotomy
The transformation of the cut neurite end into a growth cone is a critical step in the regenerative
process after injury. Following axotomy, in BCL 7 knockdown cells, there was a retraction of
both the distal and proximal ends after 24 hrs (Figure 12). In order to determine whether BCL 7
has an effect on growth cone formation, we used live cell-imaging to examine the transformation
of the axon into a growth cone following axotomy in control dsRNA and BCL 7 dsRNA treated
cells over the course of 1.5 hrs (Figure 12). Five minutes after axotomy, the control dsRNA
treated cells showed an initial retraction of the proximal (-79 ± 16.2, n = 5) and distal neurites
(18.5 ± 9.9, n = 5). Approximately 50 minutes later, there was swelling of the proximal end (-
24.3 ± 16.3, n = 5), and lamellipodium could be seen extending laterally from the swollen
59
compartment. After about 1.5 hours, the cut end of the proximal segment extended out (+12.7 ±
9.8, n = 5) and its lamellipodia branches made contact with the growing distal axon (+21.4 ±
15.6, n = 5), which itself generated small lamellipodia (Figure 12A). However, preliminary data
from BCL 7 dsRNA treated cells, demonstrates a different restructuring of the neurite following
injury. The BCL 7 dsRNA treated cells also showed a retraction of the proximal (-95.9 ± 33.1, n
= 3) and distal (-157.3 ± 34.5, n = 3) cut ends 5 min after axotomy (Figure 12B). In contrast to
the control cells these neurons did not appear to form growth cones and as a result the proximal
(-86.01 ± 28, n = 3) and distal (-167.4 ± 69.9, n = 3) ends were unable to successfully re-grow
and regenerate their neurites after 1.5 hrs.
Following axotomy, cultured Aplysia neurons form a specialized microtubule-based vesicle trap
at the growth cone organizing center (GCOC) just proximal to the site of transection (Erez et al.,
2007;Erez and Spira, 2008;Kamber et al., 2009). Microtubules depolymerize and repolymerize to
form two microtubule based vesicle traps which sort and concentrate Golgi-derived vesicles that
subsequently fuse with the plasma membrane and begin the formation of the growth cone (Erez
et al., 2007). In this study, axotomy of Lymnaea neurites results in growth cone formation and re-
connection of the proximal and distal ends (Figure 12A). Following knockdown of BCL 7,
however, our preliminary data shows that transected neurites did not successfully form growth
cones (Figure 12B). This suggests that BCL 7 may regulate microtubule dynamics in the injured
neurite and impact proper growth cone formation, resulting in unsuccessful regeneration.
Microtubules are assembled from tubulin heterodimers, which contain different α- tubulin and β-
tubulin isoforms (Lopata and Cleveland, 1987). We used immunohistochemistry to examine the
structure of β-tubulin in the control dsRNA and BCL 7 dsRNA treated cells. In control dsRNA
treated cells, β-tubulin bundles were observed in the cut proximal and distal neurite as well as in
the intact neurite. However, in BCL 7 knockdown neurons, the distribution of tubulin appeared
to be segmented, and fragmentation of tubulin was observed (Figure 13). These results suggest
that a decrease in BCL 7 expression may result in greater microtubule instability and more
depolymerization, leading to the lack of growth cone formation and degeneration of the axon.
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Figure 7. Network inferred by PPI spider
Suggested network mapping of proteins involved in CNS regeneration. Sequences identified
from the Lymnaea microarray study were blasted (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and the
closest matches were aligned using Clustal W program (http://www.ebi.ac.uk/clustalw/) and
compared with human database. The proteins indentified in the Lymnaea study are represented
by the filled squares, open circles highlight suggested interactors predicted to be part of the
network. (In collaboration with Dr. Silverman-Gavrila)
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Figure 7
62
Figure 8. Down-regulation of protein phosphatase 1k mRNA after CNS injury
(A) Amino acid alignment between protein phosphatase 1k protein from L. stagnalis [GenBank
#: ES574299], X. laevis [GenBank #: NP_001085111], M. musculus [GenBank #: AAH92238],
R. norvegicus [GenBank #: NP_001101333] and H. sapian [GenBank #: NP_689755] sequence.
(B) Relative gene expression of PP1k vs. GADPH after 1 hr 3 hrs and 5 hrs following CNS
injury was normalized to corresponding control sham-operated samples. PP1k decreased in a
time-dependent manner. 1 hr: (0.43 ± 0.13) (n = 5); 3 hrs: (0.34 ± 0.19) (n = 5).* indicates the
difference was statistically significant as compared to the sham-operated control (P < 0.05). The
data were presented as means ± S.E.M.
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Figure 8
64
Figure 9. Knockdown of protein phosphatase 1k had no effect on neurite regeneration
Neurite outgrowth following axotomy of cultured PeA neurons after treatment with control
dsRNA and protein phosphatase 1k dsRNA. (A) After axotomy (t=0) control dsRNA or PP1k
dsRNA was added to culture. The neurite outgrowth was measured over the next 24 hrs. Arrows
indicate site of transection (B) The length of the proximal axon, distal axon, and intact neurite of
injured cells were measured after dsRNA treatment over the 24 hrs. The proximal (2.9 ± 2.6 µm,
n = 18) distal (22.34 ± 10.6 µm, n = 18) and intact (29.52 ± 9.2 µm, n = 16) neurite lengths did
not significantly change following axotomy in the protein phosphatase 1k dsRNA treated cells as
compared to the control dsRNA treated cells proximal (13.9 ± 4.1 µm, n = 36), distal (32 ± 9.2
µm, n = 36) and intact (35.4 ± 12 µm, n = 33) neurites. (C) Relative gene expression level of
protein phosphatase 1k vs GAPDH reduced in the protein phosphatase 1k dsRNA group (n = 5)
as compared to control dsRNA group (n = 5). * indicates the difference between the protein
phosphatase 1k dsRNA group and the control dsRNA group was statistically significant (P <
0.05).
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Figure 9
66
Figure 10. BCL7 mRNA up-regulated 5 hrs after CNS injury
(A)Amino acid alignment between BCL7 protein from L. stagnalis [GenBank #: ES576803], C.
elegans [GenBank #: Q09242], D. melanogaster [GenBank #: O76857], M. musculus [GenBank
#: NP_033875] and H. sapian [GenBank #: CAA62012] sequences. This demonstrates the
conserved BCL 7 N-terminal sequence. (B) Relative gene expression of BCL7 vs. GADPH after
1 hr, 3 hrs and 5 hrs following CNS injury, normalized to corresponding sham-operated control
samples. BCL7 mRNA expression is significantly increased 5 hrs after injury: 5 hrs (2.5 ± 0.6)
(n = 5). * indicates the difference was statistically significant as compared to the sham-operated
control (P < 0.05). The data were presented as means ± S.E.M.
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Figure 10
68
Figure 11. Reduction of Bcl7 mRNA results in impaired neurite outgrowth following
axotomy.
(A) Representatives of transected axons in culture. Immediately following axotomy (t = 0),
control dsRNA or BCL 7 dsRNA was added into culture medium. The neurons were observed
over the next 24 hrs (t = 24 hrs). Arrow indicates the transection site. (B) The length of the
proximal axon, distal axon, and intact neurite of injured cells were measured after dsRNA
treatment over 24 hrs. The proximal (-7.9 ± 6 µm) (n = 23) distal (2.2 ± 9.3 µm) (n = 23) and
intact (-7.7 ± 10.7 µm) (n = 19) neurite lengths were significantly reduced following axotomy in
the BCL7 dsRNA treated cells as compared to the control dsRNA treated cells proximal (13.9 ±
4.1 µm) (n = 36) (t = 3.7, df = 57, P < 0.05), distal (32 ± 9.2 µm) (n = 36) (t = 2.17, df = 60, P <
0.05)and intact (35.4 ± 12 µm) (n = 33) (t = 2.4, df = 50, P< 0.05) neurite lengths. (C) Relative
gene expression level of Bcl7 vs GAPDH reduced in the BCL7 dsRNA group (n = 5) as
compared to control dsRNA group (n = 5) (t = 3.98, df = 8, P< 0.05). * indicates the difference
between the BCL7 dsRNA group and the control dsRNA group was statistically significant (P <
0.05).
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Figure 11
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Figure 12. Growth cone development and neurite outgrowth is hindered in Bcl7 reduced
cells following axotomy
Neurite outgrowth following axotomy in control dsRNA treated cells over 100 minutes. (A) Live
cell imaging of PeA neurons transected in culture containing control dsRNA. 5 minutes after
axotomy the proximal and distal neurites retract. Following retraction the proximal neurite
begins to develop a growth cone and the distal neurite extends lamellipodia (t = 50 min). After
approximately 100 minutes the proximal growth cone lamellipodia touches the distal neurite
lammellipodia and they begin to bridge the gap and regenerate the injured neurite. (B) Live cell
imaging of PeA neurons transected in culture containing BCL 7 dsRNA. Neurites retracted after
transection but did not appear to form a growth cone and elongate over the 100 minutes. (C) Rate
of proximal and distal neurite outgrowth in control dsRNA and BCL 7 dsRNA treated cells. 5
minutes after axotomy the proximal neurites retracted in both control dsRNA treated cells (-79 ±
16.2) (n = 5) and BCL 7 dsRNA treated cells (-95.9 ± 33.1) (n = 3). After 100 minutes control
dsRNA treated cells had significantly greater elongation (+12.7 ± 9.8) (n = 5) as compared to
BCL 7 dsRNA treated cells (-86.01 ± 28) (n = 3) (t = 5.2, df = 6, P < 0.05). The distal neurites
also retracted in control (18.5 ± 9.9) (n = 5) and BCL 7 (-157.3 ± 34.5) (n = 3) dsRNA treated
cells 5 min after axotomy. BCL 7 dsRNA treated cells had significant reduction in distal neurite
outgrowth (+21.4 ± 15.6) (n = 5) after 100 minutes as compared to control dsRNA treated cells (-
167.4 ± 69.9) (n = 3) (t = 3.4, df = 6), P<0.05). * indicates the difference between the BCL7
dsRNA group and the control dsRNA group was statistically significant (P < 0.05).
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Figure 12
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Figure 13. β-tubulin localization following axotomy in BCL 7 deficient cells
Cultured PeA neurons were fixed with 1% paraformaldehyde 100 minutes after axotomy and
immunostained with anti-β-tubulin in (A) Control dsRNA and (B) BCL 7 dsRNA treated cells.
Magnification of the cut site demonstrates β-tubulin integrity at the growth cone. (In
collaboration with Nasrin Nejatbakhsh and Andrew Barszczyk)
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Figure 13
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4.3 Discussion
Microarray analysis revealed 67 significantly regulated genes following CNS injury in the
L.stagnalis. PPI-spider software was used to analyze their mammalian protein orthologues and
identified 5 proteins potentially interacting and belonging to a common signaling pathway. These
included band-4 protein-2, phosphoinositide kinase, BCL 7, protein phosphatase 1k, and Sec24.
In addition the software identified missing interactors which include calmodulin-1, SPARC-like
protein 1, BCL 6, and bone sialoprotein. However, few of these genes have been previously
implicated in CNS regeneration. In order to examine the specific role of these identified genes in
regeneration we further analyzed the effects of BCL 7 and protein phosphatase 1k dsRNA
knockdown on regeneration in vitro. Knockdown of protein phosphatase 1k in vitro, and
mimicking the effects of CNS injury on its mRNA levels, resulted in no affects on neurite
outgrowth and regeneration following axotomy. On the other hand knockdown of BCL 7 caused
a reduction in the lengths of the proximal and distal neurites 24 hrs after axotomy. Furthermore
live cell imaging of control dsRNA and BCL 7 dsRNA during the first hour following axotomy
revealed that the BCL 7 dsRNA treated cells did not form proper growth cones as compared to
control. BCL 7 may affect cytoskeletal dynamics and growth cone development and this may be
mediated by CaM and the PPI spider proposed network.
4.3.1 Bioinformatic analysis of gene expression changes following CNS injury
Protein-protein interaction (PPI) networks can be used for the interpretation of bioinformatics
studies. PPI spider (http://mips.gsf.de/proj/ppispider) is a freely available web-based tool
for inferring a network model from an experimentally identified protein list. Several studies have
used this approach to interpret protein lists (Martin et al., 2008;Yue et al., 2008), and
demonstrated that in most cases PPI spider can provide statistically significant hypotheses that
are helpful for understanding protein lists (Antonov et al., 2009). PPI spider uses a Global
Network statistical framework to analyze protein list using a global protein-protein interaction
network from IntAct, an open data molecular interaction database for 9 organisms, as reference
knowledge (Aranda et al., 2010). For the human genome, the reference network covers about
7960 genes involved in approximately 40,000 unique pairwise interactions. An experimentally
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derived protein list is translated into a network model. PPI software takes into account that there
may be missing proteins in the list, and automatically includes in the network model the most
relevant proteins that are missing. The software provides a model of protein interactions that
represent the most probable scenario of how proteins within the list are connected (Antonov et
al., 2009). Due to the limited transcriptome information available for the L. stagnalis, the PPI-
software does not have a Lymnaea database. In order to bypass this limitation we generated a list
for the mammalian protein orthologues of the 67 genes produced from our microarray study.
The newly derived protein list was used as the input for the PPI-software; the proposed network
included 5 proteins identified from the microarray study, band-4 protein-2, phosphoinositide
kinase, BCL 7, protein phosphatase 1k, and Sec24. As well as missing interactor proteins which
include calmodulin-1, SPARC-like protein 1, BCL 6, and bone sialoprotein.
4.3.1.1 Calmodulin
The majority of the proteins in the proposed PPI spider network have not directly been
implicated in CNS regeneration. However, some have been proposed to have an effect on neurite
outgrowth. For example, CaM was identified as a missing interactor protein in the proposed PPI
network and it has been shown to affect growth cone formation and neuronal elongation by a
variety of pathways involving microtubules, kinases, phosphatases, or GTPases. CaM is an
ubiquitously expressed calcium-binding protein that upon binding of calcium undergoes a large
conformational change which allows it to bind to various CaM binding proteins (CAMBP`s)
(Klee et al., 1980). Upon binding to CAMBP’s, CaM regulates neuronal outgrowth and growth
cone turning by modulating microtubule dynamics. Specifically, CaM binds CRMP-2 in a Ca2+
-
dependent manner resulting in an increase in the number and length of F-actin in the growth cone
and promotion of neurite outgrowth (Tateishi et al., 1997;Stenmark et al., 2007;Zhang et al.,
2009). Contrastingly, CaM regulates GAP-43 binding to F-actin by blocking actin
polymerization in the CaM bound form, resulting in repulsion of the growth cone (He et al.,
1997;Zakharov and Mosevitsky, 2007). CaM also regulates cytoskeletal dynamics by regulating
microtubule stability through interactions with MAPs (Lee and Wolff, 1984). Aside from
cytoskeletal regulation, CaM may exert its effects on neurite outgrowth by affecting non-receptor
tyrosine kinases. CaM binding to neural cell adhesion molecule (NCAM), activates non-receptor
tyrosine kinases fyn and fak to facilitate neurite outgrowth (Kolkova et al., 2000;Nakata et al.,
2005). Upon activation of fyn and fak, there is nuclear translocation of a N-terminal fak
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fragment, a C-terminal NCAM fragment and CaM, where the complex most likely associates
with nuclear proteins to regulate gene expression and promote neurite outgrowth (Kleene et al.,
2010). CaM also activates a variety of calmodulin kinases (CaMKs) in order to regulate neurite
elongation. Activation of CaMKIα leads to axonal growth, whereas CaMKIγ promotes dendritic
outgrowth (Neal et al., 2010). CaMKII and CaMIV have also been shown to be important in
axon outgrowth (Takemura et al., 2009;Li et al., 2009). In addition to activating various kinases,
CaM also binds and activates calcineurin, a major Ca2+
- calmodulin-dependent protein
phosphatase in the brain (Klee et al., 1998). Activated calcineurin has been reported to either
stimulate or inhibit neurite outgrowth depending on the cell type (Ferreira et al., 1993;Chang et
al., 1995). CaM regulation of axonal elongation and growth cone turning has been reported in a
variety of pathways, as a result it is a critical component of the proposed PPI spider, which
suggests a novel signaling cascade for CaM.
4.3.1.2 Phosphoinostide Kinases
In addition to CaM, the proposed network identified a phosphoinositide kinase as part of the
signaling cascade. Phosphoinositide kinases play a crucial role in signal transduction by
transferring a phosphate to the inositol ring of phosphatidylinositols (PtdIns), which then act as
the precursors of several second-messenger molecules (Fruman et al., 1998). Phosphoinositide
kinases are categorized into three general families: phosphoinositide 3-kinases (PI3Ks), PtdIns 4-
kinases (PtdIns4Ks), and PtdIns-P (PIP) kinases (PIP5Ks). PI 3-kinases have been implicated in
numerous cellular activities including, neuronal survival, differentiation and motility (Fruman et
al., 1998). PI 3-kinase is additionally required for neurite outgrowth and regeneration, and
inhibition of PI 3-kinase was shown to block the outgrowth of processes in PC12 cells, sensory,
motor, and retinal neurons (Kimura et al., 1994;Lavie et al., 1997;Kita et al., 1998;Eickholt et al.,
2007). Moreover, PI 3-kinase inhibitors cause the retraction of previously
extended neurites in PC12 cells and sensory neurons, suggesting that PI 3-kinase is required not
only for the initiation, but also the maintenance of neurites (Sanchez et al., 2001). PtdIns 4-
kinases on the other hand play an important role in vesicle trafficking and lipid transport (Balla
and Balla, 2006). At the plasma membrane PtdIns 4-kinase regulate the synthesis
of PtdIns(4,5)P2 which recruit several signaling proteins, such as adaptors for endocytosis and
proteins that are involved in actin polymerization (Balla and Balla, 2006). Lastly, PIP5Ks
produce PtdIns(4,5)P2 which play a role in cell adhesion, secretion, endocytosis, and neurite
77
remodelling through actin cytoskeletal reorganization (Pike, 1992;Honda et al., 1999).
Phosphoinositide kinases are important components of signal transduction and generate vital
second-messenger molecules (PtdIns), consequently they are critical elements in the PPI spider
pathway.
4.3.1.3 B-Cell Lymphoma
The third group of proteins identified in the proposed signaling cascade are the B-Cell
Lymphoma genes. B-Cell Lymphoma 6 (BCL 6) is a transcription factor whose abnormal
expression has been implicated in the tumorigenesis of non-Hodgkin lymphoma. It functions as a
transcription repressor important for regulating lymphoid development. It is highly expressed in
B- and T-cells, and is essential for germinal center formation and antibody responses by
regulating TH2-type inflammation (Halstead et al., 2010). BCL 6
–/– mice have
a high rate of TH2-
mediated hyperimmune disease and die at an early age (Ye et al., 1997). In macrophages and B-
cells, BCL 6 represses chemokine gene transcription and attenuates
the inflammatory response
(Toney et al., 2000;Shaffer et al., 2000). However, its expression is not exclusive to immune
cells; it has been detected in most tissues and cell types where it exerts mostly anti-
inflammatory effects (Takata et al., 2008). BCL 6 expression has been associated with
terminal
differentiation, and it has been proposed as anti-apoptotic protein in mouse myocytes (Kumagai
et al., 1999). In other non-immune cells, however, BCL 6 over-expression induced apoptosis via
down-regulation of Bcl-2 and Bcl-XL(Yamochi et al., 1999) and delayed S phase
progression(Albagli et al., 1999), suggesting that its effect on cell survival or death is cell type
specific. In contrast to BCL 6, the function of BCL 7 is unknown. The gene for BCL 7 is deleted
in the neurodevelopmental disorder Williams Syndrome, suggesting that it may be involved in
early development (Zani et al., 1996;Jadayel et al., 1998). The B-cell lymphoma genes have not
been implicated in neuronal outgrowth previously; consequently the PPI spider suggests a novel
role for these genes in regeneration.
4.3.1.4 SPARC-like protein 1
SPARC-like protein 1 (SC1) was also suggested as a missing interactor protein in the PPI spider
network. SC1 is a member of the SPARC (Secreted Protein Acidic and Rich in Cysteine) family
of extracellular matrix-associated proteins with a highly acidic domain I, a follistatin-like
domain, and an extracellular calcium binding domain (Brekken and Sage, 2000). SC1 is
78
expressed in the developing as well as the adult CNS. In the adult rodent brain SC1, co-localizes
with the astrocyte marker glial fibrillary acidic protein (GFAP) (McKinnon and Margolskee,
1996). Additionally, SC1 was found to localize to synapses, particularly the postsynaptic
terminal as well as the perisynaptic glial processes that surround synapses (Lively et al., 2007).
Following brain injury, SC1 is rapidly up-regulated in reactive astrocytes around the site of
injury (McKinnon and Margolskee, 1996;Lively et al., 2007). This suggests that SC1 could be an
important modulator of CNS injury and regeneration, possibly through the novel PPI spider
pathway.
4.3.1.5 Protein phosphatase 1k
Protein phosphorylation/dephosphorylation is a major regulatory mechanism of cellular function,
such as cell signal transduction. Protein phosphatase 2C (PP2C) is one of four major protein
serine/ threonine phosphatases (PP1, PP2A, PP2B and PP2C) (Barford, 1996;Barford et al.,
1998). It is distinguished from other groups of phosphatases by its requirement for divalent
cations (Mg2+ or Mn2+) and insensitivity to the okadaic acid (Barford, 1996;Barford et al.,
1998). Protein phosphatase 1k is novel PP2C isoform, and it was suggested to be part of the
proposed PPI signaling network. Protein phosphatase 1k is targeted exclusively to the
mitochondria matrix via a mitochondrial targeting sequence at its N-terminus (Joshi et al., 2007).
Protein phosphatase 1k is an essential protein for cellular survival and mitochondria
permeability transition pore (MPTP) regulation as demonstrated by both in vivo and in vitro
studies. Loss of protein phosphatase 1k expression leads to cell death and MPTP opening in
response to calcium overload as well as developmental defects in the CNS of zebrafish (Lu et al.,
2007). This evidence suggests protein phosphatase 1k plays a critical role regulating cell death
and development, and PPI spider proposes a novel role for this gene in CNS injury.
4.3.1.6 Sec24 transport protein and bone sialoprotein
Lastly, PPI spider identified Sec24 transport protein and bone sialoprotein as components of the
novel signaling cascade. Bone sialoprotein is a secreted bone matrix protein involved in bone
metabolism, known to be expressed by macrophages and regulate calcification of necrotic tissue
(Hirota et al., 1995;Wu et al., 1995). Recently, a role for this protein in protecting cells from
hypoxia, by reducing the intracellular Ca2+
was demonstrated in vitro (Denhardt et al., 1995).The
deposition of bone sialoprotein was observed in swollen and calcified axons at the outer edges of
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a cerebral ischemic lesion, particularly in the process of axonal calcification (Gupta et al.,
2010).The role of bone sialoprotein deposition in swollen and calcified axons remains unknown,
however it is speculated that its ability to modulate Ca2+
secretion may be important for its
function during axon calcification. Sec 24 on the other hand, is part of COP-II dependent
intracellular vesicle transport. COPII vesicle assembly is initiated by a transmembrane protein
within the ER membrane, Sec12, which catalyzes guanine nucleotide exchange on the small
GTPase Sar1(Nakano and Muramatsu, 1989). Activated Sar1p-GTP recruits the Sec23/24p
complex by binding to Sec23p, and the cytoplasmically exposed signal of the transmembrane
cargo is captured by direct contact with Sec24p to form a prebudding complex (Kuehn et al.,
1998;Bi et al., 2002). The prebudding complex then recruits Sec13/31p onto Sec23/24p, which
then polymerizes the prebudding complexes to form COPII vesicles (Antonny and Schekman,
2001). The growth of dendrites and axons involves coordinated cytoskeletal reorganization and
membrane trafficking through this intracellular secretory pathway (Lecuit and Pilot, 2003;Ye et
al., 2007). Consequently Sec 24 has already been demonstrated to play an important role in
neurite outgrowth and development by targeting GABA transporter-1(GAT1) to the axon
terminal (Reiterer et al., 2008). Bone sialoprotein and Sec24 have been implicated in regulating
axonal integrit, PPI-spider suggests a novel role for these genes following CNS injury in a
common signaling pathway.
We used PPI spider to interpret our microarray data and generated a novel signaling cascade.
From the proposed signaling pathway we identified several genes with known functions in
neurite outgrowth and regeneration as well as genes with no identified roles in axonal elongation.
The PPI spider software provided us with a novel approach to analyze our gene list in terms of a
global interaction network. We used the suggested pathway to select two genes, protein
phosphatase 1k and BCL7, and examine their roles in regeneration in more detail.
4.3.2 Suppression of protein phosphatase 1k does not affect neurite dynamics following axotomy
Protein phosphatase 1k is new member of the Ser/Thr protein phosphatase from the PP2C
family, specifically targeted to the mitochondria (Joshi et al., 2007). The expression of protein
phosphatase 1k is highest in the brain and heart. However, both the mRNA and protein levels are
significantly reduced in hypertrophied and failing heart (Lu et al., 2007). Additionally, reduced
expression in the zebrafish leads to induced apoptosis and defects in CNS development. The
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induction of cell death in these models was associated with dissipated mitochondrial membrane
potential (MPTP), suggesting that protein phosphatase 1k is essential for cell survival by
maintaining MPTP (Lu et al., 2007). Protein phosphatase 1k mRNA expression was also
reduced in the L. stagnalis 3 hrs after CNS injury. Time-dependent analysis of gene expression
revealed that this early down-regulation of protein phosphatase 1k mRNA quickly recovers to
control levels by 5 hrs. These data suggest that protein phosphatase 1k expression is highly
associated with metabolic status, and its loss of expression correlates with CNS injury and the
progression of cardiac dysfunction. The L. stagnalis CNS is able to undergo successful
regeneration after injury and the recovery of protein phosphatase 1k mRNA levels reflects this
capability. Furthermore, phosphorylation is an important mechanism for regulating
mitochondrial processes (Pagliarini and Dixon, 2006). There is evidence for the
potential phosphorylation of many proteins in the mitochondria including BCL 2, therefore
phosphatases must exist in the mitochondrial membrane for their dephosphorylation (Bassik et
al., 2004). BCL 2 levels have been correlated with enhanced axonal outgrowth (Holm and
Isacson, 1999), and its function may be regulated by dephosphorylation by protein phosphatase
1k. Successful regeneration is a balance between neurite retraction and outgrowth and protein
phosphatase 1k may be part of either of these two processes. However, reduction of protein
phosphatase 1k mRNA levels in vitro, following axotomy of cultured PeA neurons, resulted in
no change in neuronal elongation over 24 hrs. Even though the data suggests that protein
phosphatase 1k does not have a role in regeneration there are caveats that should be considered.
We used RNA interference to knockdown the expression of protein phosphatase 1k and test the
role of protein phosphatase 1k on neurite outgrowth and regeneration. However, one limitation
of this experiment is that dsRNA only reduces mRNA levels, and consequently if there is
adequate protein already present and the turnover rate is slow we may not be able to see an
effect. Furthermore, the ~40 % knockdown of protein phosphatase 1k mRNA may not be a
sufficient reduction, and as a result no effect was observed. Currently our observations suggest
that protein phosphatase 1k is not involved in neuronal retraction or outgrowth following
axotomy, however further confirmation of this hypothesis is necessary.
4.3.3 BCL 7 regulates neurite elongation in a regenerative model
Chromosomal translocations involving the immunoglobulin (Ig) and T cell receptor (TCR) loci
represent common cytogenetic abnormalities in lymphoid malignancies. These translocations
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result in loss of regulation of proto-oncogenes adjacent to the breakpoints, which play important
roles in lymphomagenesis (Korsmeyer, 1992). Molecular cloning of these breakpoints has led to
the identification of several Burkitts Cell Lymphoma (BCL) gene families involved in the
control of normal cell differentiation, proliferation and death. Among the affected genes is BCL
1 at 11q13 and encoding cyclin D1 protein (Rosenberg et al., 1991). BCL 1 is transcriptionally
silent in normal tissues, and expression of the protein promotes neoplastic cell proliferation by
perpetuating the cell cycle transition from G1 to S phase (Baldin et al., 1993). BCL 2 is the most
common translocation in human lymphoid malignancies (Pezzella et al., 1990). The BCL 2
protein is associated with the mitochondria and has a well document role in preventing apoptosis
in many cell types (Yang and Korsmeyer, 1996). The BCL 3 oncogene was first identified in
patients with chronic lymphocytic leukemia (Crossen, 1989;Ohno et al., 1990). BCL 3 is an
atypical member of the inhibitor of NF-kappa B family proteins, because it functions as a co-
activator of transcription (Bours et al., 1993). The over-expression of BCL 3 in tumors results in
increased cell proliferation by activation of the cyclin D1 promoter and inhibition of p53 activity
(Zamora et al., 2010). BCL 6 chromosomal translocation was identified in about 50% of
lymphomas (Butler et al., 2002). BCL 6 is required for germinal center formation and T-helper-
2–mediated responses (Ye et al., 1997). However, it also induces apoptosis by regulating the
expression of BCL-2 and BCL-XL, apoptosis repressor genes (Yamochi et al., 1999). Lastly, the
BCL7 gene family was identified in a complex translocation seen in a Burkitt lymphoma cell line
(Zani et al., 1996). The roles of the BCL 7 family are unknown and the proteins lack any
domains associated with known functions. Alignment of three mammalian BCL 7 proteins shows
homology within the N-terminal 51 amino acids. Secondary structure prediction suggested an α-
helix region followed by a β-strand structure. The conserved N-terminus region also contains
four potential phosphorylation sites (Jadayel et al., 1998). We demonstrated that the L.stagnalis
EST clone also shares some homology with amino-terminal of the BCL 7 gene families from
H.sapien, M. musculus, M. musculus, D. melanogaster, and C. elegans. The BCL 7 gene is
expressed abundantly in most tissues, including the brain (Jadayel et al., 1998). We confirmed
this, and identified BCL 7 in our microarray screen following CNS injury in the L. stagnalis; its
expression was increased 5 hrs after injury. This suggested that BCL 7 may play a role in the
regenerative process following CNS injury, and indeed knockdown of BCL 7 in vitro after
axotomy resulted in a reduction of proximal and distal neurite lengths. Knockdown of BCL 7 did
not appear to impair the retraction response following axotomy as seen in the control. However,
82
the proximal and distal neurites did not form proper growth cones and elongation was severely
compromised within the first hour following transection.
In vivo imaging following nerve injury has allowed researches to observe the dynamic events
that take place at the injury site, revealing key details of axonal degeneration and regeneration
(Bareyre et al., 2005;Kerschensteiner et al., 2005). Kerschensteiner et al., labeled DRG neurons
and monitored them over the course of several hours after injury. In the first half hour the
proximal and distal segments underwent symmetrical degeneration. The distal fragment began
proper Wallerian degeneration approximately 1 to 2 days after the injury. Whereas the proximal
segment showed active re-growth as early as 6 hrs after injury (Kerschensteiner et al., 2005). In
this model, degeneration of the distal stump is required for regeneration of the proximal segment.
However, in the simpler invertebrate models such as the C. elegans, the proximal and distal
segments of the severed axon re-grow and re-connect. This prevents degeneration of the distal
fragment and leads to recovery of neuronal function (Yanik et al., 2004). A similar process was
observed in Lymnaea during in vivo imaging of control dsRNA treated cells, whereby the
proximal and distal fragment re-grew and connected (Figure 12A). On the other hand, the BCL 7
dsRNA treated cells failed to regenerate their proximal and distal segments after axotomy
(Figure 12B), suggesting a role for this protein following neural injury.
The distal neurite exhibited the most dramatic response, and minimal outgrowth was detected.
Whereas, in the proximal neurite even though outgrowth was stunted in the knockdown cells,
more elongation was observed. The difference in proximal and distal axon outgrowth has also
been observed in the earthworm lateral giant axon (Lyckman et al., 1992). This may be due to
the fact that distal neurites are not connected to the soma and consequently the only source of
proteins is via de novo protein synthesis. Evidence from isolated neuritic segments suggests that
neurons are capable of protein synthesis for up to 5 days after axotomy (Spira et al., 1996).
Additionally, isolated axons of Lymnaea are able to synthesize proteins when foreign mRNA is
injected into axons lacking their somatas (van et al., 1997). Furthermore, application of
anisomycin, a protein synthesis inhibitor, disrupts axonal rejoining in Aplysia sensory neurons
following injury (Bedi and Glanzman, 2001). These facts suggest that in our study the survival
and outgrowth from the distal segment was indeed mediated by local protein synthesis.
Alternatively, the proximal neurite is still connected to the soma, and axotomy results in
retrograde signaling to the nucleus and gene transcription (Funakoshi et al., 1993;Gumy et al.,
83
2010). Consequently, BCL 7 is continuously being produced in the proximal segment and a less
dramatic change is seen on neurite outgrowth compared to the distal segment, which has finite
mRNA available. Nevertheless this is the first evidence to suggest that BCL 7 has a role in
neurite outgrowth after axotomy.
The only proposed role for BCL 7 is in development. Currently there are only two lines of
evidence that suggest the BCL7 gene family may be involved in early development. First,
both human and the D. melanogaster BCL 7 genes have been detected in
early embryo and blastocyst cDNA libraries. Second, BCL 7 is commonly deleted in Williams
Syndrome (Jadayel et al., 1998).Williams syndrome is a rare neurodevelopmental disorder
characterized by cardiovascular problems, hypertension, dental abnormalities, hernias and mental
retardation (Morris et al., 1988). Due to the limited functional information on BCL 7 we wish to
further study the mechanisms underlying its role in neurite outgrowth. Cytoskeletal dynamics are
a central component of neurite outgrowth and it is important to determine if BCL 7 is involved in
modulating actin and β-tubulin integrity. We employed immunocytochemistry and stained for β-
tubulin (Figure 13). The similar methodology will be used in the future to compare β-tubulin and
actin expression patterns and localization in control and BCL 7 knockdown cells to determine
whether BCL7 regulates β-tubulin and/or actin dynamics.
4.3.4 Role of BCL 7 in calcium transients following axotomy
After an axon is transected there is a sudden increase in intracellular calcium concentration due
to the influx of calcium ions through the damaged membrane, the opening of voltage-gated
calcium channels, inversion of the Na+/Ca
2+ exchanger , or intracellular stores {Ziv, 1997 285
/id;Mandolesi, 2004 286 /id}. This increase in calcium has a major role in growth cone re-
formation and axon regeneration after injury {Spira, 2001 287 /id}. Studies suggest that the key
elements in the cellular cascades that lead to the growth cone formation are specifically activated
by a transient increase in calcium at the injury site. In addition, the structural changes at the cut
tip only occur after the intracellular calcium has returned to its original level {Ziv, 1997 285
/id;Spira, 1993 288 /id}. Preliminary data in BCL 7 knockdown cells suggests that calcium
influx at the injury site is not appropriately buffered after axotomy. The calcium signal in the
axon fluctuates over the course of the ten minutes and the axon is unable to properly regulate the
intracellular calcium levels. In the growth cone calcium signals can differ in amplitude, spatial
84
spread and kinetics and as a result have varied behavioral effect (Gomez and Zheng, 2006).
Consequently, a minor change in spatial and temporal axonal intracellular calcium may activate a
completely different signaling cascade, possibly one involved in degeneration. Furthermore,
somal calcium concentration transients are also key indicators of transected cell fate and a large
increase in somal calcium triggers apoptosis (Nguyen et al., 2005). Our preliminary data shows
that BCL 7 knockdown cells exhibited a larger increase in somal calcium concentration as
compared to control, which may have initiated a degenerative response and resulted in the BCL 7
knockdown phenotype that we observed (Figure 14). It is crucial in future studies to validate the
effects of BCL 7 on intracellular calcium and/or Ca2+
/CaM dependent signaling pathway.
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Figure 14. Intracellular [Ca2+
] gradient after axotomy
Fura-2 ratio imaging of the [Ca2+
]i after axotomy of a cultured Lymnaea neurons. (A-1) Control
dsRNA treated cell phase contrast image (A-2) Fura-2 ratio image at t = 0 before axotomy and t
= 50 seconds, immediately following axotomy in control cells (B-1) BCL 7 dsRNA treated
neurons phase contrast image (B-2) Fura-2 ratio image at t = 0 before axotomy and t = 50
seconds immediately following axotomy in BCL 7 knockdown cells. Following the resealing of
the ruptured membrane (100 s), the [Ca2+
]i recovers to near control levels, however the BCL 7
treated neuron displayed fluctuating [Ca2+
]i, and the soma had a greater influx of calcium as
compared to control. (In collaboration with Dr. Alex J. Smith)
86
Figure 14
87
5. Conclusion
This is the first microarray study in L. stagnalis, allowing us to identify genes that are
differentially regulated following CNS injury. These genes may be involved in regeneration and
provide a new database for the study of specific molecules in response to CNS trauma. I selected
several potential genes and analyzed their time-dependent gene expression changes following
CNS injury and employed RNAi knockdown to elucidate the role of these genes on regeneration.
Through this method I identified the role of the transcription factor C/EBP in neuronal
regeneration following axotomy of adult neurons in vitro. We also took advantage of the gene
expression study and applied PPI-spider software to analyze the 67 candidate genes identified
from the microarray. Five gene products that were suggested to be potentially interacting in a
novel signaling network, including band-4 protein-2, phosphoinositide kinase, BCL7, protein
phosphatase 1k, and Sec24. I also provided the first evidence that BCL 7 plays a role in neurite
regeneration. BCL 7 may exert this function by regulating growth cone dynamics through a
Ca2+
/CaM dependent pathway.
Enhancing the intrinsic potential of neurons has been shown to favour a pro-regenerative
response (Curinga and Smith, 2008;Liu et al., 2010). Improving proximal and distal neurite
outgrowth is important for the fate of axonal regeneration and re-establishment of proper
functional connections. Following axonal injury in the proximal neurite retrograde signals trigger
a local response at the growth cone, promoting growth cone retraction and extension, and a long
distance response at the nucleus, regulating gene transcription. This study suggests a novel role
for BCL 7 in regulating local growth cone dynamics as well the transcriptional regulation of
growth promoting factors by C/EBP (Figure 16). In the distal neurite local protein synthesis
increases after axonal injury and plays an important role in the regenerative process (Willis and
Twiss, 2006;Wang et al., 2010). Our study provides the first evidence that local regulation of
C/EBP mRNA and BCL 7 mRNA in axons is important for outgrowth and regeneration in the L.
Stagnalis (Figure 16).
The CNS is a highly integrated and complex biological system. In order for successful
regeneration to occur after injury, numerous molecular pathways need to work together in a
coordinated manner. The damaged axons must be able to form and extend their growth cone,
88
navigate to appropriate targets and form functional synapses. Individual genes and proteins play
roles in several of these mechanisms; however, only the synchronized actions of these pathways
can induce effective regeneration. Understanding the basic mechanisms that underlie this
complex response to injury remains a challenge for researches nonetheless it is a pre-requisite for
developing appropriate and effective therapeutic strategies.
6. Future Directions
The present study examines early gene expression following CNS injury in the L. stagnalis. The
study provides evidence that siRNA silencing of C/EBP inhibits regrowth of the distal axon. In
order to strengthen this conclusion additional experiments should be conducted. In the study only
one type of C/EBP siRNA was employed. In order to confirm the observed effect multiple
C/EBP siRNA’s should be applied following axotomy and the outgrowth of the distal and
proximal neurite measured. This would further validate the effects of C/EBP on the distal
neurite. The use of one siRNA and the partial (60%) knockdown of C/EBP could also reflect the
lack of an effect on the proximal neurite after injury. In order to determine if C/EBP actually
plays a role in the proximal neurite a more effective knockdown needs to be established,
likewise, neurite regeneration can be measure in a C/EBP knockout animal. It is also critical to
demonstrate whether the failure of growth from the distal stump after silencing of C/EBP has a
consequence for regeneration in a more complete nerve regeneration model. For example, using
organ-cultured CNS as a regenerative model in the L. stagnalis and backfilling the regenerating
axons with a retrograde tracer. This will demonstrate if C/EBP is indeed a critical factor for
successful regeneration. The study also reveals a novel role for BCL 7 in L. stagnalis CNS
regeneration. Knockdown of BCL 7 using dsRNA resulted in reduced neurite outgrowth from
both the proximal and distal neurites. In order to confirm that these effects are valid multiple
dsRNA’s and siRNA’s should be applied after injury. A similar approach should be applied to
the protein phosphatase 1k experiments. Protein phosphatase 1k knockdown did not appear to
affect neurite outgrowth following axotomy, however this may be due to the partial knockdown
of protein phosphatase 1k and the results would be further validated by applying different
dsRNA and siRNA’s. Furthermore, BCL 7 and protein phosphatase 1k are only partial
89
sequences, cloning the full length gene would confirm that these EST’s are indeed the identified
Lymnaea proteins. BCL 7 silencing appeared to inhibit growth cone formation potentially by
regulating the cytoskeleton. In order to determine if silencing BCL 7 causes deregulation of
cytoskeletal dynamics immunocytchemistry can be performed. Immunolabelling with antibodies
to β-tubulin and actin can be used to compare the cytoskeletal profile in regenerating control and
BCL 7 knockdown neurons. Furthermore, co-immunopercipitation can be carried out to
determine if BCL 7 interacts directly with actin or tubulin.
The process of axonal regeneration is a dynamic event involving degeneration and regeneration
of the injured axon. The mechanisms of degeneration involve activation of NAD-consuming
resulting in NAD decrease and NAD effector inactivation (Wang et al., 2005). This leads to
mitochondrial dysfunction and ATP depletion, followed by Ca2+
influx and calpain activation,
microtubule fragmentation and degradation of neurofilaments and membrane proteins (Yan et
al., 2010). We identified C/EBP and BCL 7 as two genes that play a role in neurite outgrowth
following axotomy in Lymnaea. However, it is not clear whether these genes are involved in
regeneration or degeneration of the axon. In order to determine which process BCL 7 and C/EBP
are involved in, an approach that may be used is to inhibit regeneration and measure the rate of
degeneration after axotomy. For example, anisomycin, a protein synthesis inhibitor and inhibitor
of regeneration, can be applied to the injured cells and the rates of degeneration can be measure
in C/EBP or BCL 7 knockdown cells and compared to control cells. If the rate of degeneration is
greater in the knockdown cells this will suggest that the gene of interest may play a role in
inhibiting degeneration. Whereas if there is no difference in the rates of degeneration it suggests
the gene plays a role in the regeneration process.
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Figure 15. Cytoskeletal profile following axotomy
(A-1)Live-cell image of cultured Lymnaea neurons pre-axotomy (A-2) Live-cell image of
cultured Lymnaea neurons after axotomy, arrow indicates site of transection.
(B-1)Immunostaining neurons 1.5 hours after axotomy. Immunolabelling with β-tubulin (green)
and actin staining with phalloidin (red). (B-2) Magnification of actin and β-tubulin localization
in the distal neurite (B-3) Magnification of actin and β-tubulin localization at the transection site
between the proximal and distal neurite. (In collaboration with Nasrin Nejatbakhsh and Andrew
Barszczyk)
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Figure 15
92
Figure 16. Schematic illustrating local and global effects of axotomy Following axotomy retrograde signals trigger a local response at the growth cone and a long
distance response at the nucleus, regulating gene transcription. In the proximal neurite, C/EBP
functions as a transcription factor and results in the transcription of regenerative-associated
genes, such as β-tubulin and GAP-43, via a cAMP/PKA/CREB dependent signaling pathway
(Nadeau et al., 2005). Axon injury also activates an ERK-dependent pathway that results in the
phosphorylation of C/EBP and further activation of pro-regenerative genes (Sung et al., 2001).
At the growth cone of the proximal neurite BCL 7 may regulate cytosokeletal dynamics through
a Ca2+
/CaM dependent mechanism to promote neurite outgrowth. In the distal end local protein
synthesis increases after axonal injury and plays an important role in the regenerative process
(Willis and Twiss, 2006;Wang et al., 2010). In the distal neurite the DLK-1 pathway regulates
C/EBP mRNA stability following axotomy (Yan et al., 2009). Local regulation and protein
synthesis of C/EBP mRNA and BCL 7 mRNA in distal neurites is important for outgrowth.
93
Figure 16
94
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