This F31 diversity pre-doctoral training fellowship resubmission proposes a novel method for augmenting microtubules to promote axon regeneration following injury. This proposal is submitted to the National Institute of Neurological Disorders and Stroke because the goal of the fellowship is to enhance the understanding of axonal microtubules and their function following nerve injury. Cytoskeleton reorganization is paramount for growth cone advancement and pathfinding. Microtubules are responsible for giving strength to filopodia as well as engorgement of the growth cone for axonal advancement and elongation. Therefore, microtubule- associated proteins are potential therapeutic targets for regeneration of axons after injury. Microtubules in the axon have a stable domain and a labile domain. Fidgetin is a microtubule-severing protein that selectively severs in the labile domain of the microtubule. Fidgetin knockdown from embryonic rat cortical neurons in vitro increases the length of axons and the number of neurites, which buoys fidgetin's candidacy as a target for promoting axon regeneration. For the proposed studies, fidgetin shRNA is packaged into AAV5 [the adeno- associated virus serotype with preference for dorsal root ganglia (DRG) neurons] so that adult rat primary cultures of DRGs can be transduced and fidgetin levels thereby reduced. Dispersed DRG cells are then plated on dried-spots of aggrecan, a chondroitin sulfate proteoglycan that comprises the glial scar, to simulate the acute-injury milieu. Knocking down fidgetin in DRG primary cultures results in more cells (both glial and neuronal) growing on the spot and more axons crossing the aggrecan border. The hypothesis is that the favorable effects are due to the elongation of the labile microtubule domains, which will be confirmed by microtubule assays routine in the laboratory. After completing this cell culture work for the first specific aim, the second aim will assess the effects of fidgetin knockdown in a relevant in vivo model of peripheral nerve injury, a DRG crush injury proximal to the dorsal root entry zone (DREZ). Behavioral analyses and c-Fos staining will be conducted to assess functional recovery and re-innervation, respectively. The AAV5 has a GFP reporter that can be used to assess regeneration of axons beyond the DREZ. While conducting the proposed research, the fellowship candidate will benefit from guidance from a dedicated mentorship team, as well as a panoply of training experiences in the neurosciences, cellular and molecular biology, scientific ethics, presentation skills, and career development.
Because little can be done clinically to enhance repair of the injured adult neuronal axons, novel approaches are required. Experimentally heightening the levels of labile microtubules restores the injured adult axon to a status more akin to a growing juvenile axon and supplies the growth cone with greater dynamic microtubule mass to aid regeneration. This can be accomplished by reducing fidgetin levels in the neuron.
