Molecular genetic mechanisms of spontaneous spinal cord regeneration

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ABSTRACT In mammals, spinal cord injury frequently leads to irreversible damage mainly due to the very limited capacity of injured central nervous system (CNS) axons to reconnect with their preinjury targets. Functional regeneration requires injured CNS axons to extend over long distances and reconnect with their original synaptic targets, however even in animal models current treatment strategies produce only modest levels of recovery. Despite enormous progress over the past decades, our knowledge and understanding of the fundamental molecular pathways and mechanisms that contribute to the process of spinal cord regeneration has left many fundamental questions unanswered. For example, are growth rates of regenerating axons uniform, are they preprogramed and invariable or are they modulated as they extend towards and into the injury site? And if so, what mechanisms and genes regulate and tune regenerating growth rates? In contrast to mammals, non-mammalian vertebrates including zebrafish have retained a remarkable capacity for spontaneous CNS regeneration. We have developed a laser-based axotomy approach to study spinal cord regeneration in larval zebrafish at single axon resolution in otherwise intact animals. From a candidate screen we identified the Cadherin EGF LAG receptor celsr3 to play a critical role in CNS regeneration. Our preliminary data reveal that in wild type animals regenerating M-ell axons switch to 3 fold higher growth rates once they cross the injury site. Celsr3 mutant M-cell axons respond to injury and grow across the injury site at growth rates indistinguishable from wildtype siblings, but then fail to increase their growth rates and frequently stall prematurely at about 25% of pre-injury length. Thus, our preliminary results identified a genetic entry point into the fundamental yet understudied question of whether and if so through which molecular mechanisms regenerating spinal cord axons regulate their growth rates along their regenerative path as their environment changes. Finally, we find that Celsr3 is also required for optic nerve regeneration but is dispensable for peripheral nerve regeneration, strongly suggesting that Celsr3 plays a selective role in CNS axon regeneration. The experiments in this proposal will (1) determine cellular and molecular mechanism by which Celsr3 growth rates selectively of regenerating CNS axons; (2) identify the molecular signaling cascade through which celsr3 promotes regeneration; and (3) Identify additional entry points into pathways that promote spontaneous spinal cord regeneration. Combined, our results are expected to make significant contributions to fundamental mechanisms that promote spontaneous spinal cord regeneration in vivo, and lay the foundation for a comprehensive analysis of spontaneous spinal cord regeneration. Although spontaneous spinal cord regeneration is largely absent in mammals, mechanisms of spontaneous spinal cord regeneration might be masked and thus undetectable by the presence and dominance of growth inhibitory mechanism. Our studies therefore complement studies in mammalian models that focus predominantly on strategies to overcome growth inhibition.