Biology subjects

Bauman, A. J.

Publications and source records attributed to Bauman, A. J..

4 recordsLinked to original sources

bioRxiv2023

Distinct roles of Bdnf I and Bdnf IV transcript variant expression in hippocampal neurons

Brain-derived neurotrophic factor (Bdnf) plays a critical role in brain development, dendritic growth, synaptic plasticity, as well as learning and memory. The rodent Bdnf gene contains nine 5' non-coding exons (I-IXa), which are spliced to a common 3' coding exon (IX). Transcription of individual Bdnf variants, which all encode the same BDNF protein, is initiated at unique promoters upstream of each non-coding exon, enabling precise spatiotemporal and activity-dependent regulation of Bdnf expression. Although prior evidence suggests that Bdnf transcripts containing exon I (Bdnf I) or exon IV (Bdnf IV) are uniquely regulated by neuronal activity, the functional significance of different Bdnf transcript variants remains unclear. To investigate functional roles of activity-dependent Bdnf I and IV transcripts, we used a CRISPR activation (CRISPRa) system in which catalytically-dead Cas9 (dCas9) fused to a transcriptional activator (VPR) is targeted to individual Bdnf promoters with single guide RNAs (sgRNAs), resulting in transcript-specific Bdnf upregulation. Bdnf I upregulation is associated with gene expression changes linked to dendritic growth, while Bdnf IV upregulation is associated with genes that regulate protein catabolism. Upregulation of Bdnf I, but not Bdnf IV, increased mushroom spine density, volume, length, and head diameter, and also produced more complex dendritic arbors in cultured rat hippocampal neurons. In contrast, upregulation of Bdnf IV, but not Bdnf I, in the rat hippocampus attenuated contextual fear expression. Our data suggest that while Bdnf I and IV are both activity-dependent, BDNF produced from these promoters may serve unique cellular, synaptic, and behavioral functions.

neuroscience↗

bioRxiv2020

A Cre-dependent CRISPR/dCas9 activation system for gene expression regulation in neurons

Site-specific genetic and epigenetic targeting of distinct cell populations is a central goal in molecular neuroscience and is crucial to understand the gene regulatory mechanisms that underlie complex phenotypes and behaviors. While recent technological advances have enabled unprecedented control over gene expression, many of these approaches are focused on selected model organisms and/or require labor-intensive customizations for different applications. The simplicity and modularity of CRISPR-based systems have transformed this aspect of genome editing, providing a variety of possible applications and targets. However, there are currently few available tools for cell-selective CRISPR regulation in neurons. Here, we designed, validated, and optimized CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems for Cre recombinase-dependent gene regulation. Unexpectedly, CRISPRa systems based on a traditional double-floxed inverted open reading frame (DIO) strategy exhibited leaky target gene induction in the absence of Cre. Therefore, we developed an intron-containing Cre-dependent CRISPRa system (SVI-DIO-dCas9-VPR) that alleviated leaky gene induction and outperformed the traditional DIO system at endogenous genes in both HEK293T cells and rat primary neuron cultures. Using gene-specific CRISPR sgRNAs, we demonstrate that SVI-DIO-dCas9-VPR can activate highly inducible genes (GRM2, Tent5b, and Fos) as well as moderately inducible genes (Sstr2 and Gadd45b) in a Cre-specific manner. Furthermore, to illustrate the versatility of this tool, we created a parallel CRISPRi construct that successfully inhibited expression from of a luciferase reporter in HEK293T cells only in the presence of Cre. These results provide a robust framework for Cre-dependent CRISPR-dCas9 approaches across different model systems, and will enable cell-specific targeting when combined with common Cre driver lines or Cre delivery via viral vectors.

neuroscience↗

bioRxiv2020

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective promoter human synapsin 1 enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.

neuroscience↗

bioRxiv2019

A dopamine-induced gene expression signature regulates neuronal function and cocaine response

Drug addiction is a worldwide health problem, with overdose rates of both psychostimulants and opioids currently on the rise in many developed countries. Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. However, even with well-studied drugs such as cocaine, drug-induced transcriptional responses remain poorly understood due to the cellular heterogeneity of the NAc and complex drug actions via multiple neurotransmitter systems. Here, we leveraged high-throughput single-nucleus RNA-sequencing to create a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type-specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified specific neuronal subpopulations that are activated by cocaine, and defined an immediate early gene expression program that is upregulated following cocaine experience in vivo and dopamine (DA) receptor activation in vitro. To characterize the neuronal response to this DA-mediated gene expression signature, we engineered a large-scale CRISPR/dCas9 activation strategy to recreate this program. Multiplexed induction of this gene program initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Taken together, these results define the genome-wide transcriptional response to cocaine with cellular precision, and demonstrate that drug-responsive gene programs are sufficient to initiate both physiological and behavioral adaptations to drugs of abuse.

neuroscience↗