Biology subjects

Pruitt, A.

Publications and source records attributed to Pruitt, A..

3 recordsLinked to original sources

bioRxiv2026

Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

Sex hormone signaling during neurodevelopment may act as a modulator of risk for neurodevelopmental disorders (NDDs), including autism spectrum disorders (ASDs). By conducting a systematic and multi-modal evaluation of estradiol effects across loss-of-function deletions in 36 functionally diverse, large-effect ASD/NDD genes in human induced pluripotent stem cell-derived neurons and larval zebrafish, we uncover convergent and divergent gene-by-estradiol interactions at the transcriptomic, circuit, and behavioral levels. Whereas estradiol partially ameliorates dysregulated expression signatures across all ASD/NDD gene knockouts examined, it selectively and rapidly dampens network hyperexcitability phenotypes in only a subset of ASD/NDD gene knockouts. Two genes, ASH1L and SCN2A, are particularly responsive to estradiol treatment, with dramatic rescue of molecular and cellular effects in human knockout neurons, coupled with behavioral phenotypes in larval zebrafish mutants. The seven most estradiol responsive genes show enhanced protein-protein interaction network connectivity, strong co-expression in specific postnatal time periods, and enrichment in upper-layer mature excitatory neurons. Altogether, we describe distinct estradiol rescue in different modalities, elucidating shared transcriptomic effects as well as gene-specific cellular and behavioral rescue in the context of loss of function mutations of select ASD/NDD genes.

genomics↗

bioRxiv2024

Stable primary brain cell cultures from zebrafish reveal hyperproliferation of non-neuronal cells from scn1lab mutants

Zebrafish are a popular model system for studying the genetic and neural underpinnings of perception and behavior, both in wild-type animals and in the context of disease modelling. Cultured primary neurons provide a key complementary tool for such studies, but existing protocols for culturing embryonic zebrafish primary neurons are limited by short cell survival and low neuronal purity. In this study, we set out to establish a protocol to produce long lived, pure neuronal cultures from zebrafish that could be used to study the mechanistic contributions of genes to neuronal networks. We then used these primary cultures to characterize cell proliferation and differentiation in primary neurons derived from scn1lab mutant embryos, which lack a sodium channel relevant to Dravet syndrome and autism. Using our optimized protocol, we generated cultures that proliferate, diversify, and form stable networks of neurons surviving for months. These stable cultures allowed us to perform genetic experiments, in this case revealing dramatic differences in the cellular composition of cultures derived from scn1lab mutant embryos versus their wild type siblings. Specifically, we find that loss of scn1lab promotes hyperproliferation of non-neuronal cells in mixed cultures of brain cells. In pure neuronal cultures, we find alterations in neurotransmitter subtypes consistent with known effects of scn1lab loss of function. Validating the utility of this approach, we then identify a corresponding hyperproliferation phenotype in live scn1lab mutant embryos, shedding light on potential mechanisms that may be relevant for Dravet syndrome. Significance statementMost existing embryonic zebrafish primary neuron culture protocols describe growing mixed cell types for restricted durations. Here, we report generation of zebrafish mixed type or pure neuronal cultures that are viable for over 100 days. We apply these cultures to gain new insight into scn1lab, a zebrafish orthologue of the Dravet Syndrome-associated sodium channel gene SCN1A. We report that loss of scn1lab results in hyperproliferation of non-neuronal cells, revealing an underappreciated mechanism by which mutations in SCN1A impact the structure and function of neuronal networks. Our in vitro cultures thus faithfully recapitulate in vivo neurobiology and provide a powerful platform to interrogate brain function in health and disease.

neuroscience↗

bioRxiv2024

Dynamic convergence of autism disorder risk genes across neurodevelopment

Over three hundred and seventy-three risk genes, broadly enriched for roles in neuronal communication and gene expression regulation, underlie risk for autism spectrum disorder (ASD) and developmental delay (DD). Functional genomic studies of subsets of these genes consistently indicate a convergent role in neurogenesis, but how these diverse risk genes converge on a smaller number of biological pathways in mature neurons is unclear. To uncover shared downstream impacts between neurodevelopmental disorder (NDD) risk genes, here we apply a pooled CRISPR approach to contrast the transcriptomic impacts of targeting 29 NDD loss-of-function genes across human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells, glutamatergic neurons, and GABAergic neurons. Points of convergence vary between the cell types of the brain and are greatest in mature glutamatergic neurons, where they broadly target not just synaptic and epigenetic, but unexpectedly, mitochondrial biology. The strongest convergent networks occur between NDD genes with common co-expression patterns in the post-mortem brain, biological annotations, and clinical associations, suggesting that convergence may one-day inform patient stratification and treatment. Towards this, ten out of eleven drugs tested that were predicted to reverse convergent signatures in human cells and/or arousal and sensory processing behaviors in zebrafish ameliorated at least one behavioral phenotype in vivo. Altogether, robust convergence in post-mitotic neurons represents a clinically actionable therapeutic window.

genetics↗