Dysferlin deficient muscular dystrophy is a devastating disease that leads to loss of mobility and quality of life in patients. Dysferlin is a 230 kD protein primarily expressed in skeletal muscle that functions in membrane resealing. Dysferlin loss of function leads to a decrease in the membrane resealing response after injury in skeletal muscle, which is thought to cause degeneration of the musculature over time. Dysferlin cDNA is 7.4 kb and exceeds AAV packaging capacity of ~ 5kb. This thesis focuses on the generation of mini dysferlin mutants that can be packaged in AAV for downstream testing of therapeutic efficacy. In addition, this thesis creates the groundwork for preclinical studies in mice that can potentially be translated to human patients. A mouse model for dysferlin deficiency was characterized and key disease phenotypes were identified. In addition, cell lines carrying a genetically encoded calcium indicator protein, gCaMP, were established to measure mini dysferlin resealing capacity and for downstream testing in vivo.

Human spinal cord injury (SCI) results in persistent functional deficits as damaged axons in the mature central nervous system (CNS) fail to regenerate after injury. This is due to both growth-inhibiting compounds, e.g., the myelin-associated growth inhibitors and the chondroitin sulfate proteoglycans (CSPGs), in the extracellular environment, and growth-limiting intrinsic factors. Unlike mammals, the primitive sea lamprey robustly recovers swimming and other locomotor behaviors after complete spinal cord transection (TX), despite the presence of homologues of the mammalian growth-inhibiting molecules. This recovery is accompanied by heterogeneous anatomical regeneration of the reticulospinal (RS) system, which, in lampreys, is the dominant descending pathway for motor control. Within the RS system, there are 18 pairs of identifiable neurons that can be classified as “good” or “bad” regenerators based on the likelihood that their axons will regenerate beyond the TX site. Most bad regenerators undergo a delayed form of caspase-mediated cell death. Because both good and bad regenerators project through the same extracellular environment, investigating their divergent responses to axotomy has the potential to reveal the key intrinsic properties that regulate axon regeneration. And, since lampreys share much of the same CNS organization and signaling pathways with higher order mammals, regeneration mechanisms discovered in lampreys may be useful therapeutic targets in humans with SCI. Lampreys do not express myelin, so the CSPGs probably form the principal extracellular inhibitory component of the injured spinal cord. Mammalian in vitro and in vivo studies suggest that CSPGs bind the LAR-family receptor protein tyrosine phosphatases (RPTPs), PTPσ and LAR, leading to growth inhibiting cytoskeletal remodeling and reduced activity of pro-survival pathways via the small GTPAse, RhoA. Intriguingly, preliminary in situ hybridization experiments with antisense riboprobes revealed that PTPσ is preferentially expressed on bad regenerator neurons. Thus, we hypothesized that differential PTPσ expression may be a key signaling determinant of regeneration. Using antisense morpholino oligomers (MOs) applied to the proximal spinal cord stump immediately after TX, we inhibited PTPσ expression among lamprey RS neurons and assessed its effects on regeneration. Contrary to our hypothesis, PTPσ deletion did not promote supraspinal regeneration or enhance behavioral recovery. Most surprisingly, we observed reduced survival of RS neurons at long timepoints post-TX among the PTPσ knockdown cohort. Western blot analysis, using pan-LAR-family receptor antibodies, indicated that the PTPσ knockdown did not affect expression of other LAR-family receptors. Although these results are the opposite of what we expected, there are several potential biological explanations that may explain why the loss of PTPσ antagonizes survival. Notably, these include interactions with the pro-regenerative PTPσ ligands, heparin sulfate proteoglycans (HSPGS), exacerbation of inflammatory processes, reduced synaptogenesis leading to loss of trophic support, and potentially off-target toxicity. These explanations remain under investigation. Notably, pilot studies involving HSPG digestion using bacterial heperainase III did not recapitulate the knockdown phenotype. Following the surprising results of PTPσ knockdown, we stepped back and considered whether simpler factors between good and bad regenerators may contribute to their divergent response to axotomy. We had long noted that bad regenerators tended to be larger than good regenerators, but generally believed this was an epiphenomenon unrelated to axon regeneration. However, a careful reexamination of primary and historic data uncovered an even stronger inverse correlation between soma cross-sectional area and regenerative ability (r = -0.92) than we had suspected. Using a similar approach, we determined that RS neuron soma size is proportional to axon caliber. Because large axons may reseal more slowly following axotomy than smaller axons, we hypothesized that inefficient axolemmal resealing after axotomy may be a key driver of the degenerative processes observed among bad regenerators. Using dye exclusion assays with 10,000 MW fluorescent dextran tracers, we assessed the rate of axolemma resealing for each of the identifiable neurons. Within 2 hours of TX, 75% of axons from small to medium sized neurons (≤ 20 x102 µm2; B5, I3, I5, mth’, M4, B6, I4, I6, M1, B2, I2) were impermeable to dye compared to only 5% of axons from the larger bad regenerator RS neurons (B1, M3, M2, B4, Mth, B3, I1). Indeed, many of these large bad regenerators remained permeable to dextran dye for more than 24 hours after injury. Importantly, approximately 65% of neurons with axons that remained dye permeable at 24 hours post-TX were positive for active caspases at +2 weeks, compared to only 10% of neurons with sealed axons (p<0.0001***). When axon resealing was artificially induced with the fusogen, polyethylene glycol (PEG), caspase activation was inhibited, suggesting that slow axolemma causatively promotes degeneration among lamprey RS neurons. Although this study did not investigate the underlying mechanisms, we suspect that prolonged influx of toxic mediators in the extracellular environment, particularly calcium, may drive the degenerative response. Together, these results demonstrate that axon regeneration and cell survival after spinal cord TX is a complex process strongly shaped by the intrinsic characteristics of the neurons themselves. Selective expression of putative inhibitory or pro-growth molecules may regulate the regeneration process in ways that can be difficult to predict a priori and with effects that vary among taxa. Because lampreys are one of the few vertebrates to recover after complete SCI, they remain an essential model organism to study true axon regeneration in the CNS. / Neuroscience

During a traumatic insult to the brain, tissue is subjected to large stresses at high rates which often surpass cellular thresholds leading to cell dysfunction or death. Cellular events that occur at the time of and immediately after an insult are poorly understood. Immediately following traumatic brain injury (TBI), the neuronal plasma membrane may become disrupted and potentiate detrimental pathways by allowing extracellular contents to gain access to the cytosol. In the current study, neuronal plasma membrane disruption was assessed in vivo following moderate unilateral controlled cortical impact in rats using a normally cell-impermeant fluorescent compound as a plasma membrane permeability marker. This fluorescent dye was injected into the cerebrospinal fluid and was allowed to diffuse into the brain. TBI caused a widespread acute disruption of neuronal membranes which was significantly different compared to uninjured brains. Affected cells were present in cortex and hippocampal regions. These findings were complemented by an in vitro model of TBI where membrane disruption was quantified and its mechanisms elucidated. Permeability marker(s) were added to neuronal cultures before the insult as indicators for increases in plasma membrane permeability. The percentage of cells containing the permeability marker was dependent on the molecular mass, as smaller molecules gained access to a higher percentage of cells than larger ones. Permeability increases were also positively correlated with the rate of insult. Membrane disruption was transient, evidenced by a robust resealing within the first minute after the insult. In addition, membrane resealing was found to be dependent on extracellular Ca2+, as chelation of the ion abolished a significant amount of resealing. We have also investigated the effects of mechanically-induced plasma membrane disruptions on neuronal network electrical activity. We have developed a multielectrode array system that allows the study of electrical activity before, during, and after a traumatic insult to neurons. Endogenous electrical activity of neuronal cultures presented a heterogeneous response following mechanical insult. Moreover, spontaneous firing dysfunction induced by injury outlasted the presence of membrane disruptions. This study provides a multi-faceted approach to elucidate the role of neuronal plasma membrane disruptions in TBI and its functional consequences.

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