Matthew Ramer

Inflammation, while necessary for repairing damage and preventing infection, can be pathological when it occurs at an inappropriate location or for a prolonged period; making the attenuation of inflammation an outstanding clinical need. Electrophiles are chemical species that readily accept an electron pair from nucleophiles to form a covalent bond and in the cellular context can initiate a stress state by interfering with basal function. Activating Transcription Factor 3 (ATF3) is upregulated in states of cellular stress but also has an orchestrating role in negatively regulating canonical pro-inflammatory pathways. In Chapter 1 I hypothesize that by upregulating ATF3 via exogenous electrophile treatment excessive inflammation can be attenuated. I use two electrophilic compounds to test this: 1) DMF, a cell permeable electrophile and 2) GDS, a novel compound (the adduct of reduced glutathione and DMF) – I theorize that GDS functions as a buffered electrophile that maintains a precise and stable amount of electrophile available to the cell. In Chapter 2 using cultured macrophages I demonstrate GDS’s buffered electrophile reaction dynamics, how it establishes a state of electrophilic stress resulting in ATF3 upregulation through putative succination of NRF2, its inflammation attenuatory effect, and how it differs from DMF. In Chapter 3 I use transcriptomics, epigenomics, and metabolomics to characterize the phenotype of GDS treated macrophages, interrogate the contribution of ATF3 to these altered phenotype, and comprehensively contrast its effect to that of DMF. In Chapter 4 I use GDS in vivo for the first time, establish its tolerability, and demonstrate its therapeutic efficacy in a mouse model of multiple sclerosis. Finally, in Chapter 5 I describe buffered electrophiles’ properties mathematically, propose the utility and future development of novel buffered electrophiles, and end by revisiting the hypothesis.

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Demyelination, which impairs electrical signal transmission in the nervous system, occurs during aging and following spinal cord injury (SCI). Chronically, it contributes to axonal damage and degeneration. The functional relevance of remyelination following SCI in aging individuals remains poorly understood. I first employed a fate-mapping approach in uninjured young (3 month) and aged (18 month) mice genetically modified to express green fluorescent protein in platelet-derived growth factor receptor alpha (PDGFRα)-positive cells, a marker for oligodendrocyte progenitor cells (OPCs). Contrary to the expectation, aging did not affect the density of PDGFRα+ or oligodendrocyte (OL), nor did it influence baseline locomotor or cognitive function. We then examined how remyelination failure impacts recovery in aged mice following moderate thoracic SCI. We deleted the myelin regulatory factor (Myrf) from PDGFRα+ cells, thereby inhibiting OPC differentiation into OLs and halting new OL formation. While PDGFRα+ cell density and proliferation remained unchanged in Myrf knockout mice, OL accumulation was significantly reduced in both young (3-5 month) and aged (15-18 month) cohorts. Following SCI, Myrf ICKO mice had fewer total axons and more unmyelinated axons than controls and displayed greater functional deficits in aged mice. Notably, young mice exhibited no behavioral functional deficits despite impaired remyelination, while aged Myrf ICKO mice showed pronounced locomotor and cognitive impairments. Together, these findings support remyelination therapies as a promising strategy for older individuals.

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Neural plasticity allows us to form new memories and learn new behaviours. After neurotrauma, it allows adaptation to and, to a limited extent, recovery of lost function. Even following severe injuries such as spinal cord injury (SCI), most of which spare some ascending and descending circuitry, there is a degree of functional restoration that reflects reorganization of remaining neural pathways. Understanding and exploiting mechanisms of spontaneous plasticity is an avenue to generating new approaches to a cure for SCI. To date, most of the experimental work on functional recovery following SCI has focused on descending projections that subserve voluntary movement and locomotion; however, these are initiated and informed by (and are thus dependent upon) sensory input from the body to the brain via “spinofugal” projections. Here I have exploited the developmental expression of Hoxb8, which is restricted to the spinal cord, to genetically label ascending projections to the cerebellum. A detailed anatomical analysis of four reporter lines identified many unexpected Hoxb8-lineage neuronal and non-neuronal cell types in targeted and untargeted transgenic strains, but because of the near-exclusive restriction of reporter expression to spinofugal projections, Hoxb8FlpOᵀᵈᵀ mice were selected as the best model for assessing the distribution of spinocerebellar projections and their possible structural plasticity. I used a cervical (C3) lateral hemisection model to document the natural history of sensorimotor performance over the first month following injury, employing established and novel behavioural measures. These revealed that many behavioural parameters changed spontaneously with time during the post-injury period, while several did not (i.e. were stable deficits), or did not change with injury at all. Since some of the behaviours which recovered (e.g. those which depend on grasp) normally require cerebellar function, I asked whether plasticity of spinocerebellar projections might have contributed. I found that at one month after hemisection, remaining spinocerebellar projections had increased in density (i.e. had sprouted), size, and content of presynaptic machinery, implicating these as possible substrates of functional recovery. I also identified transcriptomic changes in the partially-deafferented cerebellum expected as a consequence of axonal degeneration, and others which suggest how these might lead to spontaneous compensatory plasticity.

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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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Spinal cord injury (SCI) interrupts communication between the brain and peripheral organs resulting in profound and long-lasting effects, including clinically important dysfunction of the pelvic viscera (PV). Sensory and autonomic peripheral neurons innervating the PV are contained in the dorsal root ganglia (DRG) and pelvic ganglia (PG), respectively. Previous studies have identified changes in these neurons after SCI, but questions remain about the relationship between injury level and changes in peripheral targets and ganglia. In this dissertation, I addressed these questions using male Wistar rats with a high thoracic transection (T3x), which eliminates the majority of supraspinal connections to sympathetic preganglionics (including those innervating the splanchnic bed and adrenal glands), or a high lumbar transection (L2x), which preserve these connections but directly damage neurons innervating the pelvic peripheral ganglia and PV. I examined gene expression changes in DRGs and PGs one month post-T3x using RNA sequencing and found indications for unexpected neuron-target interactions, including changes in growth factor signaling and cell communication. In the PG, decreased expression of tyrosine hydroxylase (TH) after T3x was supported by atrophy of sympathetic (TH-positive) neurons. SCI results in bladder hypertrophy, and though L2x resulted in increased bladder weights compared to both T3x and naïve animals, the expression of TH in the PG decreased and TH-positive neuron hypertrophy was only transient. These results indicate a more complex relationship between target size and neurotrophism than generally accepted. Examination of PV changes after high and low SCI revealed different patterns of bladder activity. Two days after injury, there was augmented bladder activity at low intravesical pressures in L2x compared to T3x and naïve animals. I found that disrupting signal transmission through the PG did not change the bladder activity patterns, however, bilateral adrenalectomy concurrent to L2x resulted in bladder activity patterns that more closely resembled the T3x injury. Further to this, circulating catecholamine levels were higher in animals with intact innervation to the adrenal gland, implicating adrenal function in bladder changes after SCI. The findings in this thesis highlight the importance of studying injury level both from the perspective of both local circuitry and systemic changes.

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No abstract available.

No abstract available.