Characterization and optimization of neurosphere culture as in vitro screening tool against valproic acid-induced neurodevelopmental toxicity

Valproic acid (VPA) is an antiepileptic drug known to cause autism when consumed during pregnancy. Autism is a complex neurodevelopmental disorder of early-onset, highly variable in its clinical presentation. In spite of well validated animal model for autism, screening compounds is time consuming, laborious and challenging. In utero VPA exposure in rodents leads to autism-like behavioral defects in their offspring. Therefore, we attempted to establish a VPA-induced neurodevelopmental toxicity model employing neural stem cells/neurospheres (in vitro). In this study, we investigated the defects in neurospheres with direct exposure to VPA. Neuronal precursor cells were collected from time pregnant Sprague Dawley rats and allowed to generate free floating neurospheres. Neurospheres were treated with VPA (0.5mM, 1mM, and 2mM) for 7 days with daily observation and were investigated for cytotoxicity, proliferation, gene expression, and differentiation pattern. Since the marketed drugs available for autism only rescues symptoms with no effect on molecular phenotypes of autism, the model system was validated using two known neuroprotective herbal drugs Withania somnifera and Bacopa monnieri. The VPA exposure in neurospheres did not cause any significant alteration in LDH release, indicating no cytotoxicity of VPA (up to 2mM), however, a decrease in proliferation of neurospheres with significant alteration in the expression level of high-risk genes for autism was observed with VPA treatment. VPA treated neurospheres showed poor differentiation with decreased neurite outgrowth. The neuroprotective herbal drugs were able to rescue the neurospheres against VPA toxicity in terms of proliferation and differentiation. The study was substantiated with an established zebrafish in vivo model. Thus, this study provides insight towards developing an in vitro system for the preliminary drug screening against VPA induced neurodevelopmental toxicity and autism. However, further exploration of the mechanism might be needed to validate the utility of neurospheres for target-specific screening.