Biology subjects

Venkatesh, I.

Publications and source records attributed to Venkatesh, I..

14 recordsLinked to original sources

bioRxiv2026

KiMA: Kinematic Motion Analysis for Spinal Cord Injury Research

Accurate quantification of locomotor recovery is essential for evaluating therapeutic outcomes in spinal cord injury (SCI) models. Manual scoring systems remain observer-dependent, and commercial gait-analysis platforms are costly and proprietary. Markerless pose-estimation tools such as DeepLabCut generate accurate body-part coordinates, but converting these coordinates into biologically meaningful locomotor parameters typically requires custom programming and multiple external tools. We developed KiMA (Kinematic Motion Analysis), an open-source, browser-based suite for integrated analysis of rodent gait and hindlimb kinematics. KiMA accepts DeepLabCut coordinate files and performs automated coordinate parsing, stick-figure reconstruction, frame-by-frame movement inspection, and single- and multi-sample analysis, with dedicated workflows for ladder and rung analysis, footfall detection, and CatWalk gait analysis. The platform quantifies joint angles (metatarsophalangeal, ankle, knee, hip, and pelvic), stride length, stride width, cadence, stance and swing durations, paw-contact events, swing clearance, and locomotor symmetry, and supports cohort-level comparisons, correlation analysis, principal component analysis, and export of processed datasets and publication-quality figures. Because KiMA runs entirely within a standard web browser, it requires no software installation or local programming environment, supporting cross-platform accessibility and data privacy. By unifying gait quantification, visualization, and multivariate analysis in a single interface, KiMA lowers the computational barrier to markerless locomotor analysis and helps researchers detect subtle functional recovery after SCI.

bioinformatics↗

bioRxiv2026

Combinatorial Control of Corticospinal Axon Growth by Retinoic Acid Receptors

Regeneration of central nervous system (CNS) axons depends on Transcription Factors (TFs) that reactivate developmental growth programs, yet most such factors remain unknown. By intersecting developmental chromatin binding with pro-growth gene networks, we identified two retinoic acid receptor transcription factors, RARA and RARG, whose occupancy at growth-associated genes is progressively lost as neurons mature. Restoring both factors together increased neurite outgrowth beyond either alone in two independent systems, the Neuro-2a cell line and primary cortical neurons. In vivo, the same combination drove cross-midline sprouting after pyramidotomy and long-tract regeneration after thoracic spinal cord crush, with concordant recovery of hindlimb gait and grip strength. Interestingly, neither receptor alone was sufficient, hinting at combinatorial regulation. Single-nucleus transcriptomics delineated that only the combination reactivated relevant cytoskeletal and gene-expression programs, while genome-wide binding maps showed that RARA and RARG partition the regulatory landscape, with RARG dominating promoters and RARA occupying distal enhancers, so that neither receptor reconstitutes the developmental growth state alone. Intriguingly, this cooperative requirement was specific to the CNS: in peripheral sensory neurons, RARG alone was sufficient and RARA was dispensable. These data identify RARA and RARG as novel cooperative regulators of regenerative axon growth in mammalian CNS and PNS neurons and potential targets for therapeutic intervention.

neuroscience↗

bioRxiv2026

Microbiota-sensitive glial and metabolic programs define a critical window in early postnatal brainstem development

Paediatric brain tumours are increasingly recognised as diseases of disrupted development, arising when lineage progression programs, that normally govern neural and glial maturation, become stalled or dysregulated. Diffuse midline glioma (DMG), a highly aggressive paediatric brainstem tumour, emerges during early childhood in the pons, a region undergoing rapid postnatal growth characterized by oligodendrocyte precursor cell (OPC) proliferation and differentiation. However, the environmental factors that shape these developmental trajectories remain poorly defined. Here, using germ-free and conventionally colonized mice, we investigated whether early-life microbiota influences transcriptional and metabolic programs in the developing brainstem during this critical developmental window. Bulk RNA sequencing revealed pronounced microbiota-associated transcriptional differences at postnatal day 2 (P2), but not at P8, identifying a temporally restricted period during which microbial colonization is associated with pathways linked to oligodendrocyte lineage progression, myelination, and neuroimmune signalling. Transcriptional analyses further identified altered expression of genes associated with CD11c microglia, a developmental microglial subtype implicated in regulating oligodendrocyte maturation. Untargeted metabolomic profiling revealed parallel microbiota-associated differences in pathways related to mitochondrial function, redox balance, and methyl-donor metabolism. Integrated multi-omics analyses identified coordinated networks linking glial lineage programs with metabolites involved in cellular metabolism and epigenetic regulation. Notably, several of these transcriptional and metabolic programs overlap with gene signatures reported in diffuse midline glioma, suggesting that microbiota-sensitive developmental pathways intersect with cellular states relevant to paediatric brainstem tumour biology.

neuroscience↗

bioRxiv2026

Rewiring of the three-dimensional genome encodes regenerative potential in the adult central nervous system

The failure of adult central nervous system neurons to regenerate after injury has been attributed to transcriptional and epigenetic barriers, but whether three-dimensional genome organization constitutes an independent regulatory layer encoding regenerative potential remains unknown. Here we present the first genome-wide map of chromatin compartments, topologically associating domains, and loops across postnatal development, adult homeostasis, and spinal cord injury in the mouse motor cortex. Postnatal maturation progressively consolidates a growth-restrictive three-dimensional architecture, and spinal cord injury alone partially reverses this consolidation, re-engaging neonatal gene programs through reorganized but functionally recapitulative architecture despite minimal transcriptional activation. This reversion is directed rather than stochastic, preferentially targeting pro-growth gene networks, and reveals a latent three-dimensional memory of developmental growth states in the adult cortical genome. Strikingly, NR2F6, a transcription factor that promotes corticospinal axon regeneration, extends this reversion beyond the neonatal state toward an earlier embryonic chromatin configuration, a depth of developmental plasticity that injury alone cannot reach. These findings establish three-dimensional genome topology as a regulatory layer encoding regenerative potential in adult cortical neurons, demonstrating that successful CNS regeneration requires accessing embryonic rather than merely neonatal chromatin states, and reframing regenerative failure as a topological problem with new therapeutic targets.

neuroscience↗

bioRxiv2025

PATZ1 remodels the nucleosome landscape to promote chromatin accessibility in injured neurons

Adult central nervous system neurons fail to regenerate after injury, in part due to epigenetic constraints that maintain a growth-restrictive chromatin state. We previously showed that the transcription factor PATZ1 reprograms chromatin accessibility in injured corticospinal neurons to promote axon regeneration, but the underlying mechanism remained unclear. Here we use nucleosome-resolution profiling to reveal that PATZ1 induces widespread nucleosome eviction at regeneration-associated gene loci. PATZ1 treatment dramatically reduces both nucleosome occupancy and fuzziness, indicating active chromatin remodeling rather than passive destabilization. This remodeling occurs preferentially at distal regulatory elements, where PATZ1 drives a greater than 3-fold expansion of H3K27ac-marked active enhancers. Nucleosome eviction at these sites precedes enhancer activation, establishing a mechanistic link between chromatin remodeling and transcriptional reprogramming. Our findings demonstrate that targeted nucleosome eviction at enhancers underlies PATZ1-mediated chromatin reorganization, providing mechanistic insight into how epigenetic barriers to CNS regeneration can be overcome.

neuroscience↗

bioRxiv2025

Targeting RARβ With a Stable Retinoic Acid Mimic Promotes Neurite Outgrowth, Neurogenesis, and Behavioral Recovery

Injury to the adult central nervous system triggers minimal axonal regeneration, partly due to the limited activation of intrinsic growth programs. Retinoic acid (RA) signaling has been shown to promote modest regenerative responses, but its clinical utility is restricted by poor biochemical stability and short-lived receptor engagement. Here, we report a tribenzamide-based small molecule DM04, that recapitulates RA-like transcriptional and phenotypic effects and enhances regenerative outcomes, independent of canonical RARE-dependent transactivation. DM04 promoted neurite outgrowth in primary neurons, upregulated canonical RA-responsive genes, and supported neural induction from human iPSCs. In a murine spinal injury model, DM04 treatment improved motor recovery. Transcriptomic analysis revealed shared target gene activation between RA and DM04, along with unique enrichment of extracellular matrix remodeling pathways. These findings establish DM04 as a small molecule based approach with dual efficacy in injury and developmental contexts, acting through a mechanism distinct from canonical RAR transactivation, and highlight its promise as a candidate for further preclinical investigation in neural repair.

neuroscience↗

bioRxiv2025

Deep Neurite Analysis Tool (DeNAT): A machine-learning framework for precise automated neurite outgrowth measurement

Accurate quantification of neurite sprouting after injury is a critical step in axon regeneration research. Yet it remains a major bottleneck, as the current gold standard relies on manual counting by multiple blinded observers. This process is slow, labor-intensive, and prone to variability. While some software can measure total neurite length, they arent made to specifically measure new growth in complicated images from real-life injury models, like the thoracic crush and pyramidotomy model. Existing software can measure total neurite length in culture, but it is not designed to capture new growth in complex images from injury models, such as thoracic crush or pyramidotomy. Crucially, these tools lack the ability to selectively analyze growth within user-defined regions, a key requirement for injury paradigms. To address this need, we developed the Deep Neurite Analysis Tool (DeNAT), an accessible deep-learning-based platform that automatically measures neurite outgrowth after injury. DeNAT allows users to define regions of interest to specifically quantify sprouting in images from common spinal cord injury paradigms. We benchmarked its performance against manual scoring and conventional automated approaches. DeNAT achieved 87 percent accuracy in detecting neurite sprouts relative to manual counts, while reducing variability and labor. By combining user-guided region selection with automated deep learning analysis, DeNAT offers an accurate, reproducible, and efficient solution for measuring neurite outgrowth in injury models.

bioinformatics↗

bioRxiv2025

Nuclear Receptor Transcription factors promote axon regeneration in the Adult Corticospinal Tract

Transcription factors are potent levers for neural repair, but which factors govern regenerative capacity in the corticospinal tract remains largely unknown. By intersecting developmental RNA-seq with ATAC-seq footprinting, we identified two nuclear-receptor transcription factors, NR2F1 and NR2F6, neither previously linked to CNS axon growth, whose chromatin occupancy at pro-growth enhancers is progressively lost as neurons mature. Forced expression of either factor significantly increased neurite outgrowth in single-neuron tracing assays, and each drove strong cross-midline sprouting after pyramidotomy and long-tract CST regeneration after complete thoracic crush, with concordant recovery of hip-rise kinematics and grip strength. Parallel multi-omic profiling (CUT&RUN, snRNA-seq and Ribo-seq) of both factors together with NR2F6 Hi-C revealed distinct mechanisms: NR2F1 reactivated chromatin-remodeling and cytoskeletal programs, whereas NR2F6, via a conserved corepressor domain, re-occupied developmental enhancers, reorganized three-dimensional chromatin architecture into new topologically associating domains, and imposed a transient translational down-shift in which growth-relevant modules were selectively preserved through translational buffering. Together, these data identify NR2F nuclear receptors as regulators of corticospinal regeneration, acting through enhancer redeployment, translational reprogramming and three-dimensional genome reorganization.

neuroscience↗

bioRxiv2025

PATZ1 Reinstates a Growth-Permissive Chromatin Landscape in Adult Corticospinal Neurons After Injury

BackgroundThe failure of axon regeneration in the adult central nervous system represents a major barrier to recovery from spinal cord injury and neurodegenerative disease. Pro-growth transcription factors can promote regenerative responses, but their effects remain partial, suggesting that additional restraints must be relieved for these factors to achieve their full potential. The chromatin landscape of adult neurons has emerged as a candidate mechanism, yet we lack a developmental map of when and how this epigenetic restriction occurs, whether injury can reverse it, and how to therapeutically target it. ResultsWe assembled a comprehensive chromatin accessibility atlas spanning mouse forebrain development from embryonic day 11 through adulthood using bulk and single-nucleus ATAC-seq. This revealed progressive restriction of growth-gene promoters and enhancers across postnatal development, leaving over 95% of growth-associated regulatory elements substantially inaccessible in mature neurons. We found that the distance between injury site and neuronal cell body determines the magnitude of chromatin reopening: intracortical lesions proximal to motor cortex soma triggered ten-fold greater enhancer reactivation compared to distal thoracic spinal cord crush. Motif analysis identified PATZ1, a chromatin-remodeling transcription factor, as correlated with this proximity effect. Viral delivery of PATZ1 to adult cortex converted the limited epigenomic response to distal injury into a profile approaching that of proximal injury, selectively reopening enhancers at growth-associated loci and depositing active H3K27ac marks. Hi-C analysis demonstrated that PATZ1 additionally reorganizes higher-order chromatin architecture, inducing compartment switching at growth loci and remodeling topologically associating domain boundaries. Integration with single-nucleus transcriptomics revealed that while PATZ1 selectively opens chromatin at growth genes, transcriptional output and axon regeneration remain modest, indicating that combinatorial approaches pairing epigenetic priming with pro-growth transcription factors may be required for functional repair. ConclusionsThis study provides a developmental timeline of chromatin closure at regeneration-associated genes and identifies PATZ1 as a molecular tool capable of reversing this epigenetic barrier in adult neurons. Our findings indicate that chromatin accessibility functions as a gatekeeping mechanism that must be addressed before transcription factor-based therapies can achieve their full effect, establishing epigenetic priming as a targetable component of CNS repair strategies.

neuroscience↗

bioRxiv2024

Injury distance limits the transcriptional response to spinal injury

The ability of neurons to sense and respond to damage is fundamental to homeostasis and nervous system repair. For some cell types, notably dorsal root ganglia (DRG) and retinal ganglion cells (RGCs), extensive profiling has revealed a large transcriptional response to axon injury that determines survival and regenerative outcomes. In contrast, the injury response of most supraspinal cell types, whose limited regeneration constrains recovery from spinal injury, is mostly unknown. Here we employed single-nuclei sequencing in mice to profile the transcriptional responses of diverse supraspinal cell types to spinal injury. Surprisingly, thoracic spinal injury triggered only modest changes in gene expression across all populations, including corticospinal tract (CST) neurons. Moreover, CST neurons also responded minimally to cervical injury but much more strongly to intracortical axotomy, including upregulation of numerous regeneration and apoptosis-related transcripts shared with injured DRG and RGC neurons. Thus, the muted response of CST neuron to spinal injury is linked to the injurys distal location, rather than intrinsic cellular characteristics. More broadly, these findings indicate that a central challenge for enhancing regeneration after a spinal injury is the limited sensing of distant injuries and the subsequent modest baseline neuronal response.

neuroscience↗

bioRxiv2020

SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

Spatial organization of molecules and cells in complex tissue microenvironments provides essential cues during healthy growth and in disease. Novel techniques are needed for elucidation of their spatial relationships and architecture. Although a few multiplex immunofluorescence based techniques have been developed for visualization of the spatial relationships of various molecules in cells and tissues, there remains a significant need for newer methods that are rapid, easy to adapt and are gentle during the cyclic steps of fluorescence staining and de-staining. Here, we describe a novel, multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA labelled antibodies for immunofluorescence staining of cells and tissues, and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies and antibody fragments, such as Fabs, to efficiently analyse complex cells and tissues in multiple rounds of staining and de-staining. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals, while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy to use and gentle new technique for spatialomic analyses of complex microenvironments.

molecular biology↗

bioRxiv2020

Computational approaches identify novel transcription factor combinations that promote corticospinal axon growth following injury

Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a novel bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in an animal model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify novel TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics roadmap to detect TF synergy.

neuroscience↗

bioRxiv2018

Developmental chromatin restriction of pro-growth gene networks acts as an epigenetic barrier to axon regeneration in cortical neurons

Axon regeneration in the central nervous system is prevented in part by a developmental decline in the intrinsic regenerative ability of maturing neurons. This loss of axon growth ability likely reflects widespread changes in gene expression, but the mechanisms that drive this shift remain unclear. Chromatin accessibility has emerged as a key regulatory mechanism in other cellular contexts, raising the possibility that chromatin structure may contribute to the age-dependent loss of regenerative potential. Here we establish an integrated bioinformatic pipeline that combines analysis of developmentally dynamic gene networks with transcription factor regulation and genome-wide maps of chromatin accessibility. When applied to the developing cortex, this pipeline detected overall closure of chromatin in sub-networks of genes associated with axon growth. We next analyzed mature CNS neurons that were supplied with various pro-regenerative transcription factors. Unlike prior results with SOX11 and KLF7, here we found that neither JUN nor an activated form of STAT3 promoted substantial corticospinal tract regeneration. Correspondingly, chromatin accessibility in JUN or STAT3 target genes was substantially lower than in predicted targets of SOX11 and KLF7. Finally, we used the pipeline to predict pioneer factors that could potentially relieve chromatin constraints at growth-associated loci. Overall this integrated analysis substantiates the hypothesis that dynamic chromatin accessibility contributes to the developmental decline in axon growth ability and influences the efficacy of pro-regenerative interventions in the adult, while also pointing toward selected pioneer factors as high-priority candidates for future combinatorial experiments.

neuroscience↗

bioRxiv2018

KLF6 activates complementary gene modules relevant to axon growth and promotes corticospinal tract regeneration after spinal injury

Members of the KLF family of transcription factors can exert both positive and negative effects on axon regeneration in the central nervous system, but the underlying mechanisms are unclear. KLF6 and -7 share nearly identical DNA binding domains and stand out as the only known growth-promoting family members. Here we confirm that similar to KLF7, expression of KLF6 declines during postnatal cortical development and that forced re-expression of KLF6 in corticospinal tract neurons of adult female mice enhances axon regeneration after cervical spinal injury. Unlike KLF7, however, these effects were achieved with wildtype KLF6, as opposed constitutively active mutants, thus simplifying the interpretation of mechanistic studies. To clarify the molecular basis of growth promotion, RNA sequencing identified 454 genes whose expression changed upon forced KLF6 expression in cortical neurons. Network analysis of these genes revealed sub-networks of downregulated genes that were highly enriched for synaptic functions, and sub-networks of upregulated genes with functions relevant to axon extension including cytoskeleton remodeling, lipid synthesis and transport, and bioenergetics. The promoter regions of KLF6-sensitive genes showed enrichment for the binding sequence of STAT3, a previously identified regeneration-associated gene. Notably, co-expression of constitutively active STAT3 along with KLF6 in cortical neurons produced synergistic increases in neurite length. Finally, genome-wide ATAC-seq footprinting detected frequent co-binding by the two factors in pro-growth gene networks, indicating co-occupancy as an underlying mechanism for the observed synergy. These findings advance understanding of KLF-stimulated axon growth and indicate functional synergy of KLF6 transcriptional effects with those of STAT3.\n\nSIGNIFICANCE STATEMENTThe failure of axon regeneration in the CNS limits recovery from damage and disease. These findings show the transcription factor KLF6 to be a potent promoter of axon growth after spinal injury, and more importantly clarify the underlying transcriptional changes. In addition, bioinformatics analysis predicted a functional interaction between KLF6 and a second transcription factor, STAT3, and genome-wide footprinting confirmed frequent co-occupancy. Co-expression of the two factors yielded synergistic elevation of neurite growth in primary neurons. These data point the way toward novel transcriptional interventions to promote CNS regeneration.

neuroscience↗