Background Pregnenolone (P5) is a neurosteroid that promotes microtubule polymerization. It also reduces stress and negative symptoms of schizophrenia, promotes memory, as well as recovery from spinal cord injury. P5 is the first substance in the steroid-synthetic pathway; it can be further metabolized into other steroids. Therefore, it is difficult to differentiate the roles of P5 versus its metabolites in the brain. To alleviate this problem, we synthesized and screened a series of non-metabolizable P5 derivatives for their ability to polymerize microtubules similar to P5. Results We identified compound #43 (3-beta-pregnenolone acetate), which increased microtubule polymerization. We showed that compound #43 modified microtubule dynamics in live cells, increased neurite outgrowth and changed growth cone morphology in mouse cerebellar granule neuronal culture. Furthermore, compound #43 promoted the formation of stable microtubule tracks in zebrafish developing cerebellar axons. Conclusions We have developed compound #43, a nonmetabolized P5 analog, that recapitulates P5 functions in vivo and can be a new therapeutic candidate for the treatment of neurodevelopmental diseases.

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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.