Biology subjects

Ramani, B.

Publications and source records attributed to Ramani, B..

3 recordsLinked to original sources

bioRxiv2026

In vivo Proximity & Spatial Proteomics with CRISPR Screening Identify STXBP1 as a Protective Modifier of a-synuclein Toxicity in Dopamine Neurons.

Parkinsons disease (PD) is a disease of adults involving the loss of dopaminergic neurons after a long, asymptomatic, prodromal period. -synuclein, LRRK2, and VPS35 are linked to familial PD, however, how these mutations predispose dopamine neurons to death during the early prodromal phases remains unclear. Here, we used in vivo native proximity proteomics (iBioID) and dopaminergic neuron-specific subcellular proteomics across multiple PD models to uncover early alterations preceding neuronal loss. Our analyses identified convergent disruptions in synaptic protein abundance, indicating that presynaptic trafficking defects are early events in PD pathogenesis. Using a targeted CRISPR-based genetic screen in dopamine neurons, we demonstrated that mimicking this misregulation of STXBP1 amplifies vulnerability to -synuclein, implicating it as a previously underappreciated toxicity buffering factor. These findings highlight convergent mechanisms that sensitize dopamine neuronal degeneration and that presynaptic vesicle SNARE-complex proteins could serve as key targets for disease-modifying therapies in PD and related neurodegenerative disorders. HighlightsO_LIIn vivo native-BioID mapping of multiple Parkinsons disease (PD) protein interactomes revealed a convergent presynaptic network. C_LIO_LIiBioID analysis on mutant PD proteins (-synuclein A30P, LRRK2 G2019S, VPS35 D620N) uncovered mutation-specific shifts in local proximity networks, notably in endocytic and vesicle recycling pathways. C_LIO_LISpatial proteomics (iBioCoFrac) of dopamine neurons in vivo identified functional modules with reduced levels of key synaptic proteins in PD models. C_LIO_LIComparative proteomics using iBioCoFrac revealed synaptic vesicle regulation as a primary site of molecular convergence and early molecular signatures in dopamine neurons across multiple PD mouse models. C_LIO_LIAn in vivo CRISPR screen pinpointed the presynaptic protein Stxbp1/Munc18-1 as an -synuclein toxicity modifier in dopaminergic neurons. C_LI

neuroscience↗

bioRxiv2025

The Hsp40 co-chaperone DNAJC7 modifies polyglutamine but not polyglycine aggregation

Protein-encoding nucleotide repeat expansion diseases, including polyglutamine (polyQ) and polyglycine (polyG) diseases, are characterized by the accumulation of aggregation-prone proteins. In the polyQ diseases, including Huntingtons disease and several spinocerebellar ataxias, substantial prior evidence supports a pathogenic role for mutant polyQ-expanded protein misfolding and aggregation, with molecular chaperones showing promise in suppressing disease phenotypes in cellular and animal models. The goal of this study is to establish a scalable cell-based model to systematically evaluate genetic modifiers of protein aggregation in both polyQ and polyG diseases. We developed FRET-based reporter systems that model polyQ and polyG aggregation in human cells and used them to perform high-throughput CRISPR interference screens targeting all known molecular chaperones. In the polyQ model, the screen identified multiple Hsp70 chaperones and Hsp40 co-chaperones previously implicated in polyQ aggregation and additionally revealed the Hsp40 co-chaperone DNAJC7 as a potent and previously unrecognized suppressor of polyQ aggregation. In contrast, in a FRET-based polyG aggregation model of neuronal intranuclear inclusion disease, CRISPRi screening showed minimal overlap of chaperone modifiers of the polyQ screen. Direct knockdown of DNAJC7 also did not affect polyG aggregation, yet overexpressed DNAJC7 co-localized with both polyQ and polyG aggregates in cells and reduced their aggregation. In addition to establishing new inducible, scalable cellular models for polyQ and polyG aggregation, this work expands the role of DNAJC7 in regulating folding of disease-associated proteins.

neuroscience↗

bioRxiv2023

Scalable, cell type-selective in vivo AAV-based CRISPR screens in mice

There is a significant need for scalable CRISPR-based genetic screening methods that can be applied directly in mammalian tissues in vivo while enabling cell type-specific analysis. To address this, we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe- seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We demonstrate the utility of this approach by screening two distinct large sgRNA libraries, together targeting over 5,000 genes, in mouse brains to create a robust profile of neuron-essential genes. We validate two genes as strongly neuron-essential in both primary mouse neurons and in vivo, confirming the predictive power of our platform. By comparing results from individual mice and across different cell populations, we highlight the reproducibility and scalability of the platform and show that it is highly sensitive even for screening smaller neuronal subpopulations. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and multiplicity of infection on screen performance to establish general guidelines for large-scale in vivo screens.

genomics↗