Pregnenolone (P5) is a neurosteroid produced in the brain. It improves cognitive function and protects against cannabis intoxication as well as spinal cord injury. P5 activates CLIP1, which helps microtubule polymerization at its growing end; however, the significance of P5 activation of CLIP1 in the brain is still unknown. Here we examined the roles of P5 in cultured neurons and in zebrafish cerebellum. We show that P5 promotes neurite outgrowth and facilitates axon development of cultured cerebellar granule neurons. P5 also changes the morphology of axon growth cone and promotes dynamic microtubule invasion into the distal part of filopodia at the growth cone. We have used CRISPR to disrupt clip1a in zebrafish, disrupting the ability of P5 to change microtubule dynamics and growth cone morphology, as well as to reorganize cytoskeleton. In vivo, P5 accelerated cerebellum development in WT but not clip1a mutant zebrafish, and expression of exogenous CLIP1 in clip1a mutant promoted cerebellum development in response to P5. Thus, we have delineated the pathway by which P5 promotes cerebellum development by activating CLIP1 to promote microtubule dynamics leading to increased microtubule penetration into the growth cone and accelerated neurite outgrowth. This study reveals the mechanism by which P5 and CLIP1 function to promote neural development.
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