Kornbluth, D. J. | bioRxiv Science

bioRxiv2026

In Vivo Massively Parallel Reporter Assay Reveals Sequence Determinants of mRNA Localization in Astrocytes

RNA localization and local translation are essential mechanisms to fine-tune spatiotemporal gene expression in the nervous system. However, efficiently assessing the thousands of possible sequence determinants of RNA localization is a challenge, particularly for cell types that only reach morphological maturity in vivo. Here, we developed an in vivo Massively Parallel Reporter Assay (MPRA), termed Synaptoneurosomal (SN)-MPRA to enable identification of sequence determinants of mRNA localization and local translation, and applied this to astrocytes. We evaluated multiple models of RNA localization for two locally translated astrocyte mRNAs, Glt1 and Sparc, including increased transcript abundance, "zipcode" elements, and RNA secondary structure. Our results establish a high-throughput in vivo framework for identifying cis-regulatory sequences driving RNA localization and local translation, and suggest astrocytes use diverse mechanisms to regulate subcellular gene expression. More broadly, SN-MPRA offers a versatile platform to study RNA localization in vivo, where biological context and intercellular interactions are preserved.

neuroscience↗

bioRxiv2025

Single-Cell Resolution of Individual Variation in Hypothalamic Neurons Allows Targeted Manipulation Affecting Social Motivation

Despite decades of research, connecting molecular and cellular phenotypes to complex behavioral traits remains an elusive goal1. Social motivation exhibits individual trait variation2, which we hypothesize is mediated by molecular and cellular variability across hypothalamic neurons. To test this, we generated single-nucleus RNA-sequencing profiles3,4 of >120,000 neurons from tuberal hypothalamus and adjacent thalamus in 36 mice, balanced across sex and autism-associated mutation5, with all mice assessed for social motivation2. First, we show that molecular activation patterns predict behavior across individuals: specifically, activation of paraventricular Agtr1a+ (angiotensin receptor 1a) neurons predicted reduced social behavior. Subsequent inhibition of AGTR1A with telmisartan--an FDA-approved antihypertensive6--improved social orienting. Second, we show natural variation in neuronal proportions--likely arising from stochastic developmental events7--is sufficient to shape adult behavior even among genetically-identical individuals: we identified multiple neuronal populations whose relative abundance predicted social reward-seeking behavior. Chemogenetic inhibition of one such population, Nxph4+ neurons of the postero-lateral hypothalamus8, suppressed multiple aspects of social motivation. This work establishes proof-of-principle for an approach where single-cell genomics precisely maps neural substrates governing behavior. This approach revealed that stochastic variations in neuronal architecture deterministically influence social motivation, and enabled identification of therapeutically-actionable targets with immediate translational potential for disorders with social deficits.

genetics↗