Biology subjects

Velmeshev, D.

Publications and source records attributed to Velmeshev, D..

8 recordsLinked to original sources

bioRxiv2025

Dissecting mammalian cortical circuit development at single-cell resolution using inducible barcoded rabies virus

Highly organized circuits of connected neurons enable diverse brain functions. Improper development of these circuits is associated with neurodevelopmental disorders, and understanding how circuits are formed is crucial for unraveling the mechanisms of these diseases. We currently have an incomplete picture of how specific brain circuits develop and how they are affected in disease, because we lack methods to study them at scale and with single-cell resolution. Monosynaptic rabies tracing is the gold standard method to study circuit architecture. However, it suffers from cellular toxicity, low throughput, lack of control over the timing of labeling, and the inability to access the molecular profiles of individual neurons. To address these issues, we developed an inducible barcoded rabies virus (ibRV) to enable temporal-controlled labeling of synaptic circuits followed by high-throughput single-cell genomics readout. ibRV allows for dissecting neuronal circuit changes over time at single-cell and spatial resolution. We applied ibRV to study the development of specific mouse cortical circuits during late prenatal and postnatal life using single-cell genomics and unbiased spatial transcriptomics as readouts. We characterized and quantified developmental connectivity patterns and molecular cascades that underlie their formation. Additionally, we constructed functional in silico circuit models that enable interrogation of circuit function and dysfunction at specific developmental stages. Our study provides novel tools for circuit analysis and can provide new insights into the mechanisms of mammalian brain development.

neuroscience↗

bioRxiv2025

Circuit-specific gene editing for precision modulation of neuronal activity with CRISPR-rabies virus

CRISPR gene editing has revolutionized our ability to study and manipulate specific genes, enabling novel insights into gene function and potential therapies for brain disorders. Recent advances in cell-type-specific regulatory elements and viral delivery systems have made precise in vivo gene editing possible. However, neurons with similar molecular profiles can belong to different circuits, complicating efforts to manipulate circuit function and behavior. To address this, we developed CRISPR-rabies virus (CRV), which leverages the trans-synaptic spread of rabies virus to enable gene editing within anatomically defined neural circuits. By pairing CRV with cell type-specific Cas9 expression, we achieved targeted gene modifications in specific circuits. We demonstrate that CRV can modulate sodium and potassium channel expression in parvalbumin interneurons, thereby effectively regulating synaptic transmission of pyramidal neurons in the CA3 region of the hippocampus. Its compatibility with 3'-capture single-cell RNA-seq allows simultaneous circuit perturbation and molecular profiling. In summary, CRV allows precise circuit-level gene modulation, providing a platform for studying gene function in neural circuits and developing novel gene therapies for brain disorders.

neuroscience↗

bioRxiv2024

Single molecule array measures of LRRK2 kinase activity in serum link Parkinson's disease severity to peripheral inflammation

BackgroundLRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinsons disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. MethodsCapitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. ResultspT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil activation, antigenic responses, and the suppression of platelet activation. ConclusionsThe extracellular ratio of pT73-Rab10 to total Rab10 in serum is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics to mitigate associated deleterious immunological responses.

neuroscience↗

bioRxiv2023

Single cell analysis of dup15q syndrome reveals developmental and postnatal molecular changes in autism

Duplication 15q (dup15q) syndrome is the most common genetic cause of autism spectrum disorder (ASD). Due to a higher genetic and phenotypic homogeneity compared to idiopathic autism, dup15q syndrome provides a well-defined setting to investigate ASD mechanisms. Previous bulk gene expression studies identified shared molecular changes in ASD. However, how cell type specific changes compare across different autism subtypes and how they change during development is largely unknown. In this study, we used single cell and single nucleus mRNA sequencing of dup15q cortical organoids from patient iPSCs, as well as post-mortem patient brain samples. We find cell-type specific dysregulated programs that underlie dup15q pathogenesis, which we validate by spatial resolved transcriptomics using brain tissue samples. We find degraded identity and vulnerability of deep-layer neurons in fetal stage organoids and highlight increased molecular burden of postmortem upper-layer neurons implicated in synaptic signaling, a finding shared between idiopathic ASD and dup15q syndrome. Gene co-expression network analysis of organoid and postmortem excitatory neurons uncovers modules enriched with autism risk genes. Organoid developmental modules were involved in transcription regulation via chromatin remodeling, while postmortem modules were associated with synaptic transmission and plasticity. The findings reveal a shifting landscape of ASD cellular vulnerability during brain development.

neuroscience↗

bioRxiv2022

Single-cell analysis of prenatal and postnatal human cortical development

We analyze more than 700,000 single-nucleus RNA-seq profiles from 106 donors during prenatal and postnatal developmental stages and identify lineage-specific programs that underlie the development of specific subtypes of excitatory cortical neurons, interneurons, glial cell types and brain vasculature. By leveraging single-nucleus chromatin accessibility data, we delineate enhancer-gene regulatory networks and transcription factors that control commitment of specific cortical lineages. By intersecting our results with genetic risk factors for human brain diseases, we identify the cortical cell types and lineages most vulnerable to genetic insults of different brain disorders, especially autism. We find that lineage-specific gene expression programs upregulated in female cells are especially enriched for the genetic risk factors of autism. Our study captures the molecular progression of cortical lineages across human development. One Sentence SummarySingle-cell transcriptomic atlas of human cortical development identifies lineage and sex-specific programs and their implication in brain disorders.

neuroscience↗

bioRxiv2022

Chemico-genetic Analysis of Native Autism Proteomes Reveals Shared Biology Predictive of Functional Modifiers

One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving high specificity spatial interactomes compared to prior methods. The resulting native proximity interactomes are enriched for human genes dysregulated in the brain of autistic individuals and reveal unexpected and highly significant interactions with other lower-confidence autism risk gene products, positing new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology.

neuroscience↗

bioRxiv2021

Molecular diversity and lineage commitment of human interneuron progenitors

Cortical interneurons are indispensable for proper function of neocortical circuits. Changes in interneuron development and function are implicated in human disorders, such as autism spectrum disorder and epilepsy. In order to understand human-specific features of cortical development as well as the origins of neurodevelopmental disorders it is crucial to identify the molecular programs underlying human interneuron development and subtype specification. Recent studies have explored gene expression programs underlying mouse interneuron specification and maturation. We applied single-cell RNA sequencing to samples of second trimester human ganglionic eminence and developing cortex to identify molecularly defined subtypes of human interneuron progenitors and immature interneurons. In addition, we integrated this data from the developing human ganglionic eminences and neocortex with single-nucleus RNA-seq of adult cortical interneurons in order to elucidate dynamic molecular changes associated with commitment of progenitors and immature interneurons to mature interneuron subtypes. By comparing our data with published mouse single-cell genomic data, we discover a number of divergent gene expression programs that distinguish human interneuron progenitors from mouse. Moreover, we find that a number of transcription factors expressed during prenatal development become restricted to adult interneuron subtypes in the human but not the mouse, and these adult interneurons express species- and lineage-specific cell adhesion and synaptic genes. Therefore, our study highlights that despite the similarity of main principles of cortical interneuron development and lineage commitment between mouse and human, human interneuron genesis and subtype specification is guided by species-specific gene programs, contributing to human-specific features of cortical inhibitory interneurons.

neuroscience↗

bioRxiv2021

Tropism of SARS-CoV-2 for Developing Human Cortical Astrocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.

neuroscience↗