<p>Reactive oxygen species (ROS) are critical for maintaining cellular homeostasis and function when produced in physiological ranges. Important sources of cellular ROS include NADPH oxidases (Nox), which are evolutionarily conserved multi-subunit transmembrane proteins. Nox-mediated ROS regulate a variety of biological processes including stem cell proliferation and differentiation, calcium signaling, cell migration, and immunity. ROS participate in intracellular signaling by introducing post-translational modifications to proteins and thereby altering their functions. The central nervous system (CNS) expresses different Nox isoforms during both development and adulthood. There is now emerging evidence that Nox-derived ROS also control neuronal development and pathfinding. Our lab has recently shown that retinal ganglion cells (RGCs) from <i>nox2</i> mutant zebrafish exhibit pathfinding errors. However, whether Nox could act downstream of receptors for axonal growth and guidance cues is presently unknown. To investigate this question, we conducted a detailed characterization of the zebrafish <i>nox2</i> mutants that were previously established in our group. Abnormal axon projections were found throughout the CNS of the <i>nox2 </i>mutant zebrafish. Anterior commissural axons failed proper fasciculation, and aberrant axon projections were detected in the dorsal longitudinal fascicle of the spinal cord. We showed that the major brain regions are intact and that the early development of CNS is not significantly altered in <i>nox2 </i>mutants. Hence, the axonal deficits in <i>nox2</i> mutants are not due to general developmental problems, and Nox2 plays a role in axonal pathfinding and targeting. Next, we investigated whether Nox2 could act downstream of slit2/Robo2-mediated guidance during RGC pathfinding. We found that slit2-mediated RGC growth cone collapse was abolished in <i>nox2 </i>mutants <i>in vitro</i>. Further, ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms through Nox2 activation. Finally, we investigated the possible relationship between slit2/Robo2 and Nox2 signaling <i>in vivo</i>. <i>Astray/nox2</i> double heterozygous mutant larvae exhibited decreased tectal area as opposed to individual heterozygous mutants, suggesting both Nox2 and Robo2 are required for the establishment of retinotectal connections. Our results suggest that Nox2 is part of a signal transduction pathway downstream of slit2/Robo2 interaction regulating axonal guidance cell-autonomously in developing zebrafish retinal neurons.</p>

How do organisms attain the capacity to regenerate a structure, entire body, or not to regenerate? These are fundamental questions in biology for understanding how replicative systems are evolved to renew, age, and/or die. One outstanding question in regenerative biology that attracts attention is how and why the human central nervous system fails to regenerate after injury. Nervous system injuries are characterized by axonal damage and loss of synaptic function that contribute to debilitating neuronal dysfunctions. Although the molecular underpinnings of axon regeneration are well characterized, very little is known about how and what molecular pathways modulate reformation of synapses within regenerating axons to restore function. Thus, understanding the fundamental molecular and genetic mechanisms of functional axon regeneration (FAR), restoration of both axon and synapse, for the functional recovery of the nervous system remains elusive. In Chapter I, I outline the biology of regeneration and provide evolutionary perspectives of this phenomenon. Then, I provide clinical perspectives of central nervous system regeneration and therapeutic innovations. I next introduce the regulators of axon regeneration and how C. elegans as a genetic system allows detailed characterization of axon regeneration. In Chapter II, using C. elegans as a platform, I show how axon regeneration and synaptic reformation are controlled by distinct genetic pathways. I show how Poly-ADP ribose polymerase (PARP) pathway modulates functional restoration by regulating divergent genetic pathways leading to axon regeneration and synapse restoration. Finally, in Chapter III, I summarize the model of axon regeneration, evolutionary perspectives, and epistemic limitations of C. elegans axon regeneration.

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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&lt;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

A cell's composition is not uniform, but is comprised of many molecular gradients to compartmentalize functions into specialized subcellular domains. This organization is called polarity–the asymmetry of morphology and composition. Though it's a feature of nearly all prokaryotic and eukaryotic cells, polarity is plastic and highly dynamic, and is continuously instructed by the crosstalk between extracellular cues and internal effector pathways. One of the master regulators of polarity is the Par complex, canonically comprised of Cdc42, Par6, Par3 and atypical protein kinase C (aPKC). The Par complex defines the apical domain of epithelia and the neuronal axon, directs cell migration and the assembly of cell junctions, and restricts other polarity complexes to their respective domains. We have identified a novel polarity protein that counteracts the activities of the Par complex in cells. PKMζ, a truncated isoform of aPKC normally found in neurons, competes with full-length aPKC for substrate interactions. This competition results in the disruption of the canonical Par complex and its instruction of cell polarity, manifesting as a block in axon specification in developing neurons, or as a loss of the apical-basal axis of epithelial polarity. By eliminating PKMζ's ability to compete with aPKC for interaction with Par3, the effect on polarity is mitigated, while RNAi-mediated reduction of Par3 levels similarly rescues PKMζ-associated defects. We further report that PKMζ is aberrantly transcribed in certain epithelial cancers, and its expression correlates with grade. Malignant epithelial phenotypes are driven by PKMζ's Par3-dependent disruption of polarity, and its Par3- independent promotion of anoikis resistance. We demonstrate that PKMζ, as the catalytic fragment of aPKC, is surprisingly competent to influence polarity independently of its kinase activity, while other aPKC isoforms require their catalytic function to permit apical development. Together, this body of work presents PKMζ as an endogenous inhibitor of Par complex function, whose presence provides bistability to the dynamics of symmetry-breaking.

Damage to the adult mammalian central nervous system (CNS), either by traumatic injury or disease, usually results in permanent sensory and/or motor deficits. Regeneration of neural circuits is limited both by the lack of growthpromoting molecules and by the presence of growth-inhibitory molecules in the mature brain and spinal cord. The research described here examines the therapeutic potential of viral vectors and neuronal transplants to reconstruct damaged neural pathways in the CNS. Experimental neural transplantation techniques often fall short of expectations because of limited transplant survival and insufficient neurite outgrowth to repair connections and induce behavioral recovery. These shortcomings are addressed in the current studies by virus-mediated expression of cell-specific neurotrophic and guidance molecules in the host brain prior to cell transplantation. The initial proof-of-principle studies show that viral vectors can be used to create axon-guidance pathways in the adult mammalian brain. With such pathways in place, subsequent transplantation of neurons leads to longdistance, targeted outgrowth of neurites. Application of this technique to a rat model of Parkinsons disease demonstrates that circuit reconstruction leads to functional recovery. For this study, rats were lesioned on one side of their brain with 6-hydroxydopamine to produce a hemiparkinsonian state. The motor deficit was confirmed by amphetamine-induced rotation testing and spontaneous motor asymmetry testing. The rats were then divided into experimental groups to receive lentivirus injections along a path between the substantia nigra (SN) and the striatum to express glial cell-line derived neurotrophic factor (GDNF), GDNF family receptor alpha-1 (GFR1), netrin-1 or green fluorescent protein (GFP, control). One group received combination injections of lenti-GDNF and lenti-GFR1. One week after virus injections, animals received transplants of embryonic midbrain dopaminergic neurons into their SNs. They were tested for motor asymmetry every two weeks for a total of eight weeks and then brain tissue was harvested for immunohistochemical analysis. Results demonstrate that virus-induced expression of GDNF and GFR1 supports growth of dopaminergic fibers from cells transplanted into the SN all the way to the striatum, and these animals have a significant reduction in both drug-induced and spontaneous motor asymmetry.

The compartmental hypothesis of neurodegeneration proposes that the neurone, long recognized to consist of morphologically and functionally distinct compartments, also houses distinct degeneration mechanisms for the soma, axon and nerve endings. Support for this hypothesis is provided by the phenomenon of the WldS (for Wallerian Degeneration, slow) mouse, a mutant in which axons survive several weeks after transection, rather than degenerating within 24-48 hours as in wild type mice, by virtue of expression of a chimeric Nmnat1/Ube4b protein. In this thesis I used the WldS-mouse to re-examine and extend the theory of compartmental neurodegeneration by focusing specifically on sensory axons and endings; and finally by considering a fourth compartment, the dendrites. The first part of this thesis reports that Ia afferent axons and their annulospiral endings are robustly protected from degeneration in WldS mice. Homozygous or heterozygous WldS mice crossbred with transgenic mice expressing fluorescent protein in neurones were sacrificed at various times after sciatic nerve transection. Fluorescence microscopy of whole mount preparations of lumbrical muscles in these mice revealed excellent preservation of annulospiral endings on muscle spindles for at least 10 days after axotomy. No significant difference was detected in the protection with age or gene copy-number in contrast to the protection of motor nerve terminals, which degenerate rapidly in heterozygote and aged homozygote WldS mice. In an attempt to explain the difference in motor and sensory protection by WldS, examination of three hypotheses was undertaken: a) differences in protein expression, tested by western blot and immunohistochemistry; b) differences in the degree of neuronal branching, tested through examination of g-motor axons and endings which have a degree of branching intermediate to motor and sensory neurons; and c) differences in the activity in the disconnected stumps, through primary culture of the saphenous and phrenic nerve, selected because they comprise largely pure sensory and motor axons respectively. The data suggest that none of these hypotheses provides a sufficient explanation for the difference between sensory and motor protection by WldS. The last part of this thesis attempts to extend the theory of compartmental degeneration. I examine a system for investigation of WldS-mediated protection of dendrites. In preliminary experiments retinal explants from transgenic mice expressing YFP in a subset of retinal ganglion-cell neurones were cultured. The dendritic arbours of these cells were shown to be amenable for repeated visualization and accessible to injury and monitoring of degeneration. Overall the data in this thesis suggest that the level of WldS -mediated protection conferred to an axon or axonal endings varies between different neuronal types. This has implications for the potential applications of WldS research to clinical problems. Specifically, the data imply that sensory neuropathies may benefit more than motor neuropathies from treatments based on the protective effects of WldS. These findings in sensory neurones also challenge some of the assumptions made about WldS- mediated protection of neurones, for example the extent of the age-effect on axonal endings. Further investigation of WldS-mediated protection in the CNS could give renewed impetus to attempts to discover targets for treatment in common neurodegenerative diseases. Finally, a system for investigation of dendritic degeneration has been piloted, suggesting that molecules involved in the degeneration of dendrites or in protection from this degeneration may be amenable to investigation in this system, prospectively extending the compartmental hypothesis of neuronal degeneration.

Primary sensory afferent outgrowth within the developing longitudinal pathway of the spinal cord is important for intrasegmental and intersegmental communication that underlies coordination and development of reflexes and contributes to sensory perception. The endogenous mechanisms that regulate primary sensory afferent extension are the primary focus of this dissertation. This dissertation tested the hypothesis that primary sensory afferent extension in the longitudinal pathway is regulated by sphingosine 1-phosphate type 1 receptor (S1P1R) and tyrosine kinase receptor B (TrkB) through a brain derived neurotrophic factor (BDNF) related mechanism. To test this hypothesis we used embryonic day five (E5) chicken embryos, as this is the developmental time point when sensory afferents are growing along the longitudinal axis of the spinal cord but have not yet turned ventrally to make connections with the grey matter of the spinal cord. Chicken embryos were removed from their in ovo environment to allow for labeling of primary afferent neurons in the thoracic 3/4 (T3/4) dorsal root ganglia (DRG). Tissue was then put into culture with or without various pharmacological agents and subsequently assayed for length of growth of the labeled primary afferent axons along the longitudinal axis of the spinal cord. Results showed both BDNF and fingolimod-p, an S1P1R agonist known to increase BDNF mRNA and protein production/secretion in cortical neurons, increased primary axon extension along the longitudinal pathway. Further, fingolimod-p increased BDNF mRNA production in DRG in this system. Conversely, inhibition of BDNF or S1PRs attenuated primary afferent axon extension along the longitudinal pathway. We found BDNF signaling to be required for fingolimod-p's effects as addition of αBDNF attenuated the effects of fingolimod-p on axon outgrowth. TrkB, the high affinity receptor for BDNF, is expressed in chicken DRG during embryonic development. We hypothesized that TrkB activation by BDNF regulates DRG axon extension in the longitudinal pathway through the PLC-γ signaling pathway. We found inhibition of TrkB and/or PLC-γ signaling pathway attenuated DRG axon extension with or without BDNF stimulation. Additional pathways associated with TrkB activation: mitogen activated kinase (MAPK) and phosphoinositide 3-kinase (PI3K) appeared to either have no effect on DRG axon extension or were involved in DRG axon extension through a mechanism that is not related to TrkB. Collectively, these studies suggest an endogenous mechanism for the regulation of DRG axon outgrowth within the longitudinal pathway. With this mechanism, DRG axon outgrowth may be enhanced or attenuated following manipulation of S1P1R, BDNF and/or TrkB. Further, these findings suggest an action through BDNF on CNS axons as a potential therapeutic effect of fingolimod-p, a treatment for relapsing remitting forms of Multiple Sclerosis

Treatment with buprenorphine represents a promising alternative for pregnant opioid addicts but there is a need to understand potential effects on nervous system development. We previously showed effects of perinatal exposure to buprenorphine on axonal caliber and myelination in 26-day-old rat corpus callosum. These changes, detected at the end of rapid brain myelination and accompanied by earlier oligodendrocyte maturation, suggested interference with mechanisms coordinating axonal growth and myelination. To better understand buprenorphine actions and to establish whether these effects extend to the spinal cord, we analyzed the corpus callosum and corticospinal tract at 16 days of age, just before the peak of myelination. Our results point to an important role of the opioid system in regulating early axo-glial interactions coordinating axonal growth and myelination. Moreover, in addition to reinforcing previous findings in the brain, we showed for the first time that these effects are also exerted in the spinal cord.