Wolfram Tetzlaff

Spinal cord injury (SCI) is a debilitating condition with no curative treatment. In recent years metabolism has been suggested as a factor that could be altered to enhance recovery after SCI, and research from the Tetzlaff lab showed that a ketogenic diet (KD) can improve recovery after a cervical SCI in rodents. KDs are high fat, low carbohydrate diets that have been successfully used as a treatment for drug-resistant epilepsy in children. KDs produce high levels of the ketone body, beta-hydroxybutyrate (BHB), which can act as an energy source or bind and activate the cell surface receptor, hydroxycarboxylic acid receptor 2 (HCAR2). BHB levels can also be increased by oral ketone ester (KE) supplementation making KE a potential alternative to KD. In Chapter 2, the use of KD on recovery after a cervical forceps crush SCI in mice was assessed. In this injury model KD did not improve recovery of the mice. In Chapter 3, the effect of KD following a more clinically relevant T9 midline contusive SCI was investigated. We observed that KD could reduce pro-inflammatory cytokines CCL3 and CCL4 at 7 days post-injury and a scRNA-sequencing analysis of CD45+ cells at the injury site at 7 days after injury confirmed KD-mediated downregulation of many immune pathways. We also used the T9 contusion injury model with wild type and HCAR2₋/₋ mice to investigate the role of HCAR2 activation on KD-mediated effects. We found that KD could reduce the influx of CD45+CD11b+ myeloid cells into the injury site at 7 days post-injury, which required the HCAR2 receptor. Chapter 4 investigated the use of KE supplementation as a treatment following a C5 hemi-contusive injury in rats. Proteomic analysis at 2 weeks after injury showed that KE and KD appear to impact different cellular pathways. In this model, KE showed only minor improvements in behavioural recovery. Together these results suggest that KD can reduce inflammation at 7 days after injury in mice and that different paradigms of KE supplementation may be needed to see behavioral improvement following SCI in rats.

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Remyelination occurs after spinal cord injury (SCI) but its functional relevance isunclear. We assessed the necessity of myelin regulatory factor (Myrf) in remyelination aftercontusive SCI by deleting the gene from platelet-derived growth factor receptor alpha positive(PDGFRα-positive) oligodendrocyte precursor cells (OPCs) in mice prior to SCI. While OPCproliferation and density were not altered by Myrf inducible knockout after SCI, theaccumulation of new oligodendrocytes was prevented. This greatly inhibited myelin regenerationresulting in a loss of myelinated axons at the lesion epicenter. However, spontaneous locomotorrecovery after SCI was not altered by remyelination failure. In controls with functional MYRF,locomotor recovery preceded the onset of substantial oligodendrocyte myelin regeneration. Wenext assessed locomotor recovery in a severe model of SCI where fewer axons were spared. Hereanimals were still able to recover despite the inhibition of remyelination. We noticed that ionchannels were redistributed in demyelinated axons. Further testing showed knockout animalswere able to show conduction properties similar to that of control animals. Collectively, thesedata demonstrate that MYRF expression in PDGFRα-positive cell derived oligodendrocytes isindispensable for oligodendrocyte myelin regeneration following contusive SCI, thatremyelination is not required for spontaneous recovery of stepping in moderate or severeinjuries, and that demyelinated axons redistribute voltage gated ion channels and conductionproperties similar to that of unmyelinated axons.

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The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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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.

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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.

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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.

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Spinal cord injury (SCI) results in substantial oligodendrocyte death and demyelination. Remyelination is deemed critical because denuded axons not only lack the myelin necessary to achieve normal conduction velocity, but are also at increased risk of degeneration. A more rapid remyelination thus hypothesized to spare more axons from axonal degeneration, ultimately sparing neurological circuitry from the secondary damage that continues in the days and weeks following SCI. In this thesis I undertake two strategies to improve remyelination after SCI.In Chapter 2, I investigated whether transplantation of murine Platelet derived growth factor (PDGF)-responsive neural precursor cells (PRPs) could differentiate into remyelinating oligodendrocytes and improve functional recovery after SCI. Transplanted PRPs integrated into host tissue, differentiated into extensively branched mature oligodendrocytes that ensheathed multiple axons, and produced mature myelin. Thus, PRP-derived oligodendrocytes were capable of generating mature myelin sheaths on denuded CNS axons. To our surprise, although transplanted PRPs efficiently produced oligodendrocytes in the injured spinal cord, there was no significant increase in the total number of myelinated axons in PRP-transplanted versus media control animals. Likewise there was no improvement in behavioural recovery following transplantation in two separate experiments.Blocking known inhibitors of oligodendrocyte differentiation or maturation could improve remyelination. Myelin debris is present following SCI and inhibits oligodendrocyte development in vitro, and I hypothesized that myelin debris inhibits remyelination after SCI. In Chapter 3, oligodendrocyte precursor cells (OPCs) were grown in culture in the presence of myelin. Using this approach, I found that on myelin there was a robust inhibition of oligodendroglia maturation, without a corresponding increase in cell death or proliferation. To understand how myelin inhibits maturation, I measured the expression of a number of genes encoding well-characterized transcription factors that negatively regulate oligodendrocyte development. Associated with stalled maturation, I found myelin increases Inhibitor of Differentiation (ID) 2 and 4, which upon overexpression in OPCs is known to stall maturation. Thus, enhanced levels of ID2 and ID4 in oligodendroglia that are in contact with myelin provides a mechanistic understanding as to how myelin inhibits oligodendroglial maturation.

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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.

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