Doctor of Philosophy in Neuroscience (PhD) 
Prevention of growth cone collapse to promote spinal cord regeneration
Postdoctoral Researcher ("Research Scientist")
RIKEN Brain Science Institute
The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.
The damage inflicted by spinal cord injury (SCI) occurs in two phases. The primary injury is a mechanical insult to the spinal cord, resulting in permanent loss of cells and tissue structure. Multiple mechanisms of secondary injury extend this damage. One such mechanism is thought to be progressive loss of myelin, such that axons that survive primary injury are rendered dysfunctional by conduction block. As a result, myelin repair has emerged as a major research focus. The goals of this research were: i) to evaluate the extent of spontaneous repair by endogenous glia, and ii) to determine whether transplantation in a clinically relevant scenario can improve the outcome of SCI. In Chapter 2, I characterized the source and extent of spontaneous myelin repair in experimental SCI. I systematically assessed the cellular origin of new myelin and myelinating cells in transgenic mice, by genetically labeling multiple lineages prior to SCI. Contrary to prevailing dogma (that endogenous myelin repair was limited), we found that ~30% of myelinated axons at the injury epicentre were ensheathed de novo (since injury) at three months after SCI. In addition, the majority of myelinating Schwann cells (SCs) in the injured spinal cord were derived from oligodendrocyte precursor cells (OPCs) and infiltration of peripheral myelinating SCs made only a small contribution. In Chapter 3, I investigated the potential for improving spontaneous repair (and the outcome of SCI) through glial transplantation. Skin-derived precursor cells directed to a SC fate (SKP-SCs) were transplanted at the site of chronic SCI. At 21 weeks after transplantation (29 weeks-post SCI), SKP-SCs contained thousands of growing/regenerating axons, which were myelinated by either transplanted or endogenous SCs. The presence of endogenous SCs was increased after SKP-SC transplantation. Rats that received SKP-SCs had higher functional motor scores and displayed less bladder wall thickening (a hallmark of bladder dysfunction following SCI) compared to controls. These data contribute to our understanding of the endogenous glial repair response after SCI, both in the absence of treatment and following a clinically relevant cell transplantation. These endogenous repair mechanisms might be exploited and augmented to develop novel treatments for SCI.
Spinal cord injury (SCI) results in paralysis due in part to the inability of central nervous system(CNS) axons to regenerate following their transection. However, after anatomically incompleteSCI, partial spontaneous recovery can often occur. I studied the regeneration and plasticity ofdescending pathways involved in forelimb motor function following SCI to better understand themechanisms underlying axon regeneration failure and spontaneous motor recovery.In Chapter 2, I developed an injury model in adult mice that results in complete axotomy of therubrospinal tract and sustained deficits in forelimb motor function. I found that when a leftdorsolateral funiculus crush injury was instigated at vertebral level C4, there were sustaineddeficits in left forelimb function while when the same injury was instigated at vertebral level C6,there was spontaneous recovery in left forelimb function to baseline levels.In Chapter 3, I used the injury model developed in Chapter 2 in conditional PTEN KO mice totest the hypotheses that 1) PTEN deletion promotes rubrospinal axonal regeneration followingSCI and 2) Aging significantly diminishes the regenerative capacity of PTEN deleted rubrospinalneurons. I found that when PTEN was deleted within rubrospinal neurons in 4 week old mice,there was significant rostral axon growth and regeneration past the lesion site to ~1 mm relativeto controls. However, when PTEN was deleted within rubrospinal neurons in 7-8 month oldmice, while rostral axon growth occurred, there was no caudal regeneration. Thus, there is anage-dependent decline in regeneration of CNS neurons.In Chapter 4, I used optogenetic and chemogenetic tools to assess motor cortical plasticity inadult mice. I found that following ablation of the dorsal corticospinal tract, the motor cortex isable to re-establish output to the limbs and the minor dorsolateral corticospinal tract representing3% of direct spinal cord transmission is able to partially mediate spontaneous recovery.Taken together, these data demonstrate an age-related decline in axon regeneration in the adultmammalian CNS and show that a minor corticospinal pathway is necessary for spontaneousrecovery following SCI.
For much of human history the devastating loss of neurological functions that occurs after spinal cord injury (SCI) was thought to be irreversible, so the people afflicted with such injuries were offered no hope of effective medical treatment. Today that has changed, as advances in neurobiology and medicine over the past century have led to the development of treatments aimed specifically at repairing the injured spinal cord. The transplantation of Schwann cells (SCs) has emerged as one promising example of such a treatment, withdemonstrated efficacy in multiple animal models of SCI and encouraging preliminary results in clinical trials. Although SCs possess many of the qualities of an ideal cellular therapy, the harvest of autologous SCs from peripheral nerve (N-SCs) causes permanent nerve injury, which could be avoided by generating SCs from an alternative autologous source. One such source is skin-derived precursors (SKPs), which can be isolated from the adult mammalian dermis and differentiated into SCs (SKP-SCs) in vitro. Herein I examined the efficacy of SKP-SCs as a treatment for SCI in rodent injurymodels and compared those cells to their nerve-derived counterparts. This work provided the first demonstration of efficacy for SKP-SC therapy after thoracic contusion and showed that, much like N-SCs, SKP-SCs myelinate, promote axonal growth, and enhance functional recovery after SCI. In addition, we found evidence that SKP-SCs may have advantages over N-SCs with respect to their ability to interact favourably with spared astrocyte-rich host tissue and promote axonal growth. Subsequently we directly compared neonatal SKP-SCs and N-SCs and found that those cell types were highly similar in terms of their protein/gene expression profiles, migration and integration into astrocyte-rich domains in vitro and in vivo, and many reparative effects following transplantation into the partially crushed cervical spinal cord. Taken together our findings suggest that SKP-SCs and N-SCs have similar therapeutic efficacy, and that where differences between those two cell types exist, they consistently favour the SKP-SCs as the more favourable cell type for SCI repair. Thus, our work to-date supports the notion that SKP-SCs are a suitable alternative to N-SCs for transplantation-based central nervous system repair.
No abstract available.
Corticofugal axons projecting to the thalamus, brainstem and spinal cord must travel the same initial trajectory through the subcortical lateral and medial ganglionic eminences, and are therefore likely subject to the same sets of guidance cues. These cues direct the course of corticofugal axons bringing them to each intermediate target, until they reach the diencephalic-telencephalic boundary, where axons targeting the thalamus turn dorsally and brainstem and spinal cord targeting axons turn ventrally. Many of these guidance cues have been elucidated, yet there are still gaps in our understanding of the formation of the corticofugal projection. I found that Sema5B expression flanked the presumptive internal capsule during its formation, and was therefore ideally situated both spatially and temporally to act as an instructive cue for descending cortical axons. In Chapter 2, I show that Sema5B is not only capable of inhibiting cortical axons in vitro, but can cause misguidance of cortical axons in slice culture when placed ectopically over normally non-Sema5B expressing regions. In addition, I show that the loss of Sema5B from the neocortical VZ resulted in aberrant penetration of this normally avoided region. Therefore Sema5B is both necessary and sufficient to inhibit the corticofugal projection. Semaphorins and their plexin receptors are frequently proteolyzed to modulate the elicited responses in navigating growth cones. In Chapter 3 I show that Sema5B cleavage results in an inhibitory fragment that in heterologous cells can produce inhibitory gradients for cortical explants in collagen gel co-cultures, and collapse dissociated cortical neuronal growth cones, an effect that can be blocked with a function disrupting antibody to the cell adhesion molecule TAG-1. This thesis shows that Sema5B, a guidance cue with a hitherto unknown function, is responsible for a very important aspect of cortical development. My work leads to a final proposal that Sema5B is in fact a two-in-one protein with separable inhibitory and alternate complex functions, the implications of which are discussed thoroughly in Chapter 4.
Multiple sclerosis is the most common cause of neurologic disability in the developed world. A heterogeneous disease of unknown cause that most often presents in mid life, it is characterized by years of relapsing and remitting symptoms that eventually become progressive. The pathology of Multiple Sclerosis is characterized by resolving and non-resolving neurological deficits caused by axonal dysfunction and degeneration, which are mediated by both inflammation and demyelination. Remyelination is an intrinsic myelin repair mechanism that replaces lost myelin in response to demyelination and mitigates its pathological effects. Experimentally, remyelination has been shown to promote axon survival in inflammatory demyelination contexts, however, it is unknown if blocking remyelination will result in increased axon loss and damage. Determining the importance of remyelination for axon preservation is necessary to determine the efficacy of remyelination therapies. Assessing the effect of blocked remyelination is an important part of this picture. To determine this we used the rodent model of MS, Experimental Autoimmune Encephalomyelitis (EAE), to initiate demyelination, and blocked remyelination by inducibly deleting the gene Myelin Gene Regulatory Factor (Myrf) using the Cre-lox gene editing system. We then assessed axon survival and damage, myelination state, EAE disease severity and progression, microglial activation, and dorsal column size in the lumbar spinal cord of affected mice. We found that blocked remyelination did not affect axon survival or damage, but delayed EAE disease onset, reduced myelin in the ventral white matter and increase activation of microglia in affected areas. Strictly interpreted, our results suggest that remyelination may not be as important for axon survival as hypothesized. However, non-statistically significant trends in the results (Axon Loss p = 0.11 and Axon damage p = 0.15 in ventral/dorsal white matter combined) suggest that the lack of effect seen here may have been due to limitations and unforeseen problems encountered during these experiments. Unfortunately, these casts some doubt on our findings, but may highlight some previously unknown difficulties and effects such as delayed EAE induction and increased disease severity that may have been unintended consequences of the Myrf knock out, indicating that more investigation is warranted.
Traumatic injury to the adult mammalian central nervous system (CNS) commonly results in permanent functional impairment due to the inability of injured CNS neurons to mount an effective regenerative response. Injury to the spinal cord is associated with a range of sensory, motor, and autonomic deficits, the most severe of which is complete paralysis. At a histological level, injury induced pathophysiological processes have been thoroughly characterized for the tissue area immediately surrounding the lesion epicentre, however remote to the lesion these changes are less well described. Previous studies have demonstrated that various perturbations, including traumatic injury, demyelination, artificial neural stimulation, neurodegeneration, and social experience, among others, induce robust oligodendrocyte precursor cells (OPC) responses, which function as endogenous precursors for myelinating oligodendrocytes. De novo myelination in the adult CNS has been implicated in motor learning, memory, and optimization of neural network function, thought to represent a potent form of neural plasticity. The demonstration of robust OPC proliferation and oligodendrogenesis in models of dorsal rhizotomy, axotomy, and axon degeneration, combined with the robust OPC responses characteristic of SCI lesion epicentres, lead us to hypothesize that contusion SCI would induce significant responses in resident OPC populations remote to the lesion site (specifically comprised of OPC proliferation, oligodendrogenesis, and new myelination). This may be functionally relevant to myelin plasticity on spared motor and sensory tracts remote to the lesion. To test this hypothesis, we conducted an in vivo study employing transgenic mouse lines (i.e. PDGFRα-CreERT:ROSA26-YFP and PDGFRα-CreERT:ROSA26-mGFP) that enabled the visualization and fate-mapping of OPCs and their progeny in the adult murine spinal cord following a moderate (70 Kdyne) T9-T10 thoracic contusion injury. Consistent with our predictions, we observed robust OPC proliferation and oligodendrogenesis remote to the lesion in both the cervical and lumbar spinal cord. Surprisingly, this did not manifest as increased new myelination, attributed (at least in part) to an observed maturation impairment of newly-formed oligodendrocytes.
One major avenue towards repair of the damaged mammalian nervous system is an enhancement of the process known as remyelination. This process restores to damaged neurons the protective sheath that is critical not only for their survival but also for the conduction of their electrical signals. However, remyelination is inhibited after many types of nervous system damage as well as in degenerative diseases of the nervous system. This inhibition functions primarily through prevention of the capacity of progenitor cells to become remyelinating cells. This work aims to address the role that the p75 neurotrophin receptor (p75) – an important signalling protein that stands at the intersection of several key signalling cascades – plays in mediating this inhibition of remyelination. The intention of this work is to investigate p75 as a potential therapeutic target for clinical interventions aimed at enhancing remyelination.
A high cervical injury to the rubrospinal tract (RST) generally results in cellular and nuclear atrophy of the rubral neurons, abortive axonal regeneration and eventual retraction from the forming injurious scar and enlarging cavity. The neuronal cell body response to axonal injury plays an important role in the failure of central nervous system (CNS) neurons to regenerate. Adult mammalian CNS neurons fail to re-express a variety of genes and signalling factors, such as cAMP, after axotomy that are seen at increased levels in regenerating peripheral neurons or in developing CNS neurons. By mimicking the sustained increase in cAMP levels, prior to or during an upper cervical (C3/4) crush injury of the dorsolateral funiculus in adult male Sprague-Dawley rats using the membrane permeant analogue dibutyryl cAMP (db-cAMP) near the vicinity of rubrospinal neurons, will enhance the regenerative cell body response in order to promote axonal sprouting or regeneration to and across the site of a spinal cord injury. Two experimental groups, acute and pre-treatment, were used. Both groups received either 25mM db-cAMP (treatment) or vehicle (control) solutions delivered via 14 day mini osmotic-pumps. The pretreated group had db-cAMP infused beginning one week prior to injury for two weeks while the acutely treated group received concurrent treatment with injury. The rubrospinal tract was anterogradedly traced with biotinylated dextran amine (BDA) for quantification purposes. Camera lucida like reconstructions (CLLRs) were created for enhanced visualization and density calculation of the RST. Changes in behaviour were assessed using the vertical exploration test. Application of db-cAMP to the RN in both the acute and pretreated groups increased the number of labelled rubrospinal fibres in the gray matter and proximal to the site of injury in comparison to the controls. Db-cAMP treatment did not reduce lesion sizes nor were there any visibly traced fibres caudal to the site of injury in any of the treatment groups. Unexpectedly, pre-treatment of db-cAMP conferred no advantages over acute treatment. Behavioural analysis of spontaneous forelimb usage as indicated by the vertical exploration test revealed no significant differences between any of the db-cAMP and control groups.