Pregnenolone reorganizes cytoskeleton to promote neuron development via CLIP1

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. Significance Statement1. We have elucidated the mechanism of pregnenolone (P5) actionP5 enhances brain functions, but its mode of action was unclear. Here we show that P5 activates CLIP1 to promote microtubule dynamics at the growth cone and to accelerate neural development. 2. We have generated a zebrafish model of CLIP1 deficiencyCLIP1 deficiency causes intellectual disability and defective neural development. Our zebrafish model can be used to study mechanisms related to this disease and other microtubule defects. 3. We point to therapeutic intervention of neurological diseases using P5P5 is beneficial to the brain. We elucidate the mechanism of P5 action, thus accelerate the development of therapeutics using P5 and its derivatives.