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Parkinson’s disease, the second most common neurodegenerative disorder,is characterized by loss of nigrostriatal dopaminergic neurons. To date,there is no defined cause and cure for the disease. An ideal treatmentstrategy is to replace the lost neurons by transplanting fetal dopaminergicneurons to the brain of parkinsonian patients. Clinical trials have beenperformed and the outcome was variable where one significant obstaclewas the limited graft reinnervation of the host brain. To study this issue,organotypic tissue culture can be utilized to monitor dopaminergic nervefiber outgrowth in vitro and their association with astrocytes. Using thisculture technique, dopaminergic nerve fibers appear in twomorphologically and temporally different types. The early appearing nervefibers are formed in the absence of astrocytes, reach long distances, andare called non-glial-associated tyrosine hydroxylase (TH) -positive nervefibers. After a few days, the second sequence of nerve fibers, the glialassociatedTH-positive nerve fibers, are formed, and their growth arelimited to the presence of astrocytes, that migrate and form a monolayersurrounding the plated tissue. The aim of this thesis was to study theinteraction between nerve fiber formation and astrocytes with a specialfocus on the long-distance growing nerve fibers. Ventral mesencephalic(VM) organotypic slice cultures from embryonic day (E) 12, E14, and E18were incubated for 14, 21, 28, and 35 days in vitro (DIV). The resultsrevealed that the two morphologically different processes were found incultures from the younger stages, while no non-glial-associated growthwas found in cultures of tissue from E18. Instead neurons had migratedonto the migrating astrocytes. Astrocytes migrated longer distances intissue from older stages, and the migration reached a plateau at 21 DIV.Co-cultures of E14 VM tissue pieces and cell suspension of matureastrocytes promoted migration of neurons, as seen in E18 cultures. Thus,9the maturity of the astrocytes was an important factor for nerve fiberoutgrowth. Hence, targeting molecules secreted by astrocytes might bebeneficial for regeneration. Chondroitin sulfate proteoglycan (CSPG), amember of proteoglycan family, is produced by the astrocytes and has adual role of being permissive during development and inhibitory afterbrain injury in adult brain. Cultures were treated with chondroitinase ABC(ChABC) or methyl-umbelliferyl-β-D-xyloside (β-xyloside) in twodifferent protocols, early and late treatments. The results from the earlytreated cultures showed that both compounds inhibited the outgrowth ofnerve fibers and astrocytic migration in cultures from E14 tissue, while β-xyloside but not ChABC promoted the non-glial-associated growth incultures derived from E18 fetuses. In addition, β-xyloside but not ChABCinhibited neuronal migration in E18 cultures. Taken together, β-xylosideappeared more effective than ChABC in promoting nerve fiber growth.Another potential candidate, integrin-associated protein CD47, was studiedbecause of its role in synaptogenesis, which is important for nerve fibergrowth. Cultures from E14 CD47 knockout (CD47-/-) mice were plated andcompared to their wildtypes. CD47-/- cultures displayed a massive and longnon-glial-associated TH-positive nerve fiber outgrowth despite theirnormal astrocytic migration. Blocking either signal regulatory protein-α(SIRPα) or thrombospondin-1 (TSP-1), which bind to CD47, had nogrowth promoting effect. In conclusion, to promote nerve growth, youngertissue can grow for longer distances than older tissue, and inhibiting CSPGproduction promotes nerve growth in older tissue, while gene deletion ofCD47 makes the astrocytes permissive for a robust nerve fiber growth.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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

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