Biology subjects

Gokhale, A.

Publications and source records attributed to Gokhale, A..

14 recordsLinked to original sources

bioRxiv2026

Distinct signaling mechanisms and proteome phenotypes are elicited by compartment-specific genetic defects of copper homeostasis

Impairments to the complex machinery regulating copper homeostasis lead to neurodevelopmental diseases, demonstrating the importance of copper for neuronal health and maintenance. The exact mechanisms by which the brain responds to copper deficiency following disruptions to the copper transporters ATP7A and CTR1 in conditions such as Menkes disease remain unclear, though failure to supply complex IV of the respiratory chain with copper is suspected to account for substantial pathology. Here, we studied mechanisms of copper deficiency using systems biology approaches to contrast isogenic CTR1- and COX17-deficient cells, which model copper deficiency at the level of the whole cell or complex IV, respectively. Multiomics approaches revealed distinct signaling mechanisms elicited by compartment-specific genetic defects of copper homeostasis, spanning multiple organelles and biological functions. Specifically, COX17 KO cells exhibited elevated AMPK activity and blunted mTOR activity relative to CTR1-null cells. Manipulating mTOR activity elicited inverse effects on survival in CTR1-deficient cells and flies as compared to their COX17-deficient counterparts. Increased mTOR activity and downstream protein synthesis is adaptive in models of copper deficiency but deleterious in COX17-deficient cells and flies. We propose that mTOR activation represents a resilience mechanism that fails following sustained copper deficiency and impairments to mitochondrial respiration. SignificanceO_LIComparative proteomics reveals distinct molecular mechanisms downstream of compartmentalized copper deficiency C_LIO_LICOX17- and CTR1-deficient cells exhibit distinct patterns of AMPK/mTOR pathway activity C_LIO_LImTOR and downstream S6K activity is protective in cellular copper deficiency but not in compartmentalized mitochondrial copper deficiency C_LI

cell biology↗

bioRxiv2026

Suppressive Genetic Interactions Between Haploinsufficient Mitochondrial Genes Encoded in the 22q11.2 Microdeletion Locus Define Brain and Cardiac Phenotypes

Genomic copy number variations, such as the 22q11.2 microdeletion syndrome, cause pleiotropic disorders that affect diverse organ systems and disrupt neurodevelopment. Deletions of the 22q11.2 locus reduce the dosage of up to 46 protein coding genes, raising questions about the identity of haploinsufficient genes and their genetic interactions contributing to 22q11.2 phenotypes. Here, we dissect functional and molecular relationships between two genes encoded within the 22q11.2 locus: the mitochondrial ribosomal protein gene MRPL40 and the mitochondrial citrate transporter SLC25A1. We show that a MRPL40 null mutation disrupts mitochondrial translation, impairs respiration, and affects multiple components of the SLC25A1 interactome including factors required for lipid metabolism, mitochondrial ribosome subunits, and the mitochondrial RNA processing machinery. In silico coessentiality network analysis revealed correlated and anticorrelated fitness interactions linking MRPL40 and SLC25A1 to mitochondrial translation, intermediate carbon metabolism, and interferon signaling. We determined that Mrpl40-null mutations are embryonic lethal in mice, but Mrpl40-/+ mice are viable and displayed embryonic cardiac development and adult behavioral phenotypes. Similarly, Slc25a1+/- animals showed embryonic cardiac developmental defects but lacked the adult behavioral phenotypes observed in Mrpl40-/+ mice. Surprisingly, transheterozygotic Slc25a1+/-;Mrpl40-/+ mice suppressed or mitigated cardiac development, behavioral, and brain transcriptome phenotypes observed in single heterozygotic animals. These results reveal that MRPL40 and SLC25A1 are haploinsufficient genes within the 22q11.2 locus that genetically and biochemically interact to define tissue development and physiology. Our findings provide a framework for understanding the complexity and type of gene dosage interactions within the 22q11.2 deletion syndrome locus.

neuroscience↗

bioRxiv2025

Cis-TERN: a graph-based method to integrate single-cell chromatin accessibility with gene expression data to infer the pseudotime ordering of cells

Pseudotime ordering methods computationally arrange individually sequenced cells along a continuous trajectory to infer their progression through biological processes. Chromatin accessibility at regulatory elements is pivotal for orchestrating the timing of gene expression during cellular differentiation. However, the sparsity inherent in single-cell data, along with the discrete grouping imposed by clustering-based approaches, often obscures subtle cell-state transitions and intermediate populations. Here, we introduce Cis-TERN (Cis-Temporally Encoded Regulation Network), a graph-based method that integrates single-cell measurements of gene expression (scRNA-seq) and chromatin accessibility (scATAC-seq) to infer a pseudotime ordering of cells. Exploiting the fact that distal cis chromatin accessibility often precedes gene activation, Cis-TERN builds a directed bipartite graph between scRNA-seq and scATAC-seq cells with edge weights determined by either proximal or distal accessibility. Through network diffusion, Cis-TERN assigns continuous pseudotime scores to all cells. Cis-TERN infers cellular pseudotime without the need for prior biological knowledge, such as specifying a root cell type, thereby enabling unbiased reconstruction of differentiation from data alone. We show that our scores align with known lineages in healthy and tumor-derived hematopoietic cells. Additionally, Cis-TERN is able to trace individual cancer cells back to progenitor cell types. Our approach establishes a framework for leveraging chromatin accessibility and gene expression jointly to learn the timing of cell state progression.

bioinformatics↗

bioRxiv2025

Brain Mecp2 Gene Dosage and Gene Therapy Shape Multi-Omic Signatures and Biomarkers in Rett Syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Like other genetic neurodevelopmental disorders, it lacks protein biomarkers to evaluate disease and therapeutic outcomes. We present a strategy to define putative biomarkers of MeCP2 dysfunction in brain with potential to delineate mechanisms and monitor therapeutic interventions. This strategy relies on a library of proteins responsive to Mecp2 gene dosage and correlated with molecular and clinical outcomes after AAV9-mediated MECP2 gene therapy in Mecp2-KO mice. Gene rescue restored MeCP2 in brain, improved clinical phenotypes, and reverted transcriptome and proteome abnormalities. We identified 327 shared proteins among 1852 cortical and hippocampal proteins responsive to Mecp2/MECP2. Of these, 119 also displayed Mecp2/MECP2-dependent transcript changes. Both the Mecp2-responsive proteome and transcript-protein pairs were enriched in synaptic and metabolic pathways, including central carbon and NAD+ metabolism. We used this therapy-responsive protein library to guide selection of candidate cerebrospinal fluid (CSF) biomarkers in RTT. CSF composition from neurotypical and RTT groups was analyzed using ultrasensitive nucleic acid-based multiplexed ELISA. Twenty-eight proteins were altered in RTT, nine overlapping with Mecp2 dosage- and therapy-sensitive proteins. Multivariate regression linked several candidates to Mecp2/MeCP2 abundance and phenotypic improvement in mice. This paradigm provides a rigorous molecular systems-level framework integrating genetics, preclinical gene therapy, and clinical metrics to define robust cross-species putative biomarkers and possible mechanisms in RTT, with potential applicability to other neurodevelopmental disorders. One Sentence SummaryGenetic Identification of cross-species biomarkers and mechanisms in Rett Syndrome

neuroscience↗

bioRxiv2025

Loss of cep57 function induces G1 arrest and microcephaly

Faithful chromosome segregation and coordinated cell-cycle progression are essential for maintaining genome stability and preserving progenitor populations during vertebrate embryogenesis. Although pathogenic CEP57 variants are associated with developmental disorders including aneuploidy and microcephaly, the molecular mechanisms by which CEP57 coordinates these processes remain poorly understood. Here, we identify Cep57 as a previously unrecognized regulator of chromosome segregation, G1/S transition, centrosome integrity, and genome surveillance during early zebrafish embryogenesis. Cep57 localizes to both the nucleus and centrosomes of zebrafish blastulae, indicating probable dual functions in nuclear cell cycle regulation and centrosome organization. Our results show that Cep57 associates with Rad21, Smc1, and Smc3, revealing an unexpected functional link between Cep57 and the cohesin complex. Loss of Cep57 results in Rad21 depletion, destabilization of the cohesin complex, chromosome segregation errors resulting in supernumerary nuclei, and disruption of pericentriolar material organization, demonstrating a critical role in maintaining mitotic fidelity. We further show that Cep57 interacts with Geminin and promotes an Rb1-dependent G1/S checkpoint, whereas Cep57 deficiency causes widespread cell cycle dysregulation, genome instability, and apoptosis. Quantitative proteomic analyses reveal robust activation of DNA damage and checkpoint signaling pathways, consistent with engagement of genome surveillance mechanisms following Cep57 loss. Importantly, these cellular abnormalities precede extensive neural apoptosis, depletion of neuroprogenitor populations, and the emergence of microcephaly-associated phenotypes. Collectively, our findings establish Cep57 as a cohesin-associated integrator of chromosome segregation, cell cycle progression, and genome stability that is essential for neural progenitor maintenance during vertebrate embryogenesis, thereby expanding its functional repertoire far beyond its canonical role in centrosome regulation.

developmental biology↗

bioRxiv2025

Mitochondrially Transcribed dsRNA Mediates Manganese-induced Neuroinflammation

Manganese is an essential trace element required for various biological functions, but in excess is neurotoxic and leads to significant health concerns. The mechanisms underlying manganese neurotoxicity remain poorly understood. Neuropathological studies of affected brain regions reveal astrogliosis, neuronal loss, and neuroinflammation. Here, we present a novel manganese-dependent mechanism linking mitochondrial dysfunction to neuroinflammation. We found that manganese disruption of the mitochondrial transcriptome processing results in the accumulation of double stranded RNA (dsRNA). This dsRNA is released into the cytoplasm, where it activates the cytosolic sensor MDA5, triggering type I interferon responses and inflammatory cytokine production. This mechanism is evident in 100 day human cerebral organoids, where manganese-increased mitochondrial dsRNA and induced inflammatory responses in mature astrocytes. Similarly, we observed an increase in mitochondrial dsRNA content, the activation of an inflammatory transcriptome and the production of cytokines in female and male mouse brains carrying mutations in the Slc30a10 gene, a model for human hypermanganesemia with dystonia 1 disorder. These findings highlight a previously unrecognized role for mitochondrial dsRNA in manganese-induced neuroinflammation and provide insights into the molecular pathogenesis of manganism. We propose that this mitochondrial dsRNA-induced inflammatory pathway could be active in other neurological diseases caused by environmental or genetic factors. Significance StatementEnvironmental exposures and genetic defects that perturb manganese homeostasis are an underappreciated cause of neurodegeneration and neuroinflammation. We describe a new paradigm for inducible neuroinflammation, where manganese disruption of mitochondrial transcriptome processing leads to the accumulation of mitochondrial double-stranded RNA (dsRNA), which activate antiviral responses in the cytoplasm driving type I interferon dependent inflammation. This manganese-dsRNA axis is induced in cell lines in vitro and a subpopulation of mature astrocytes in exposed human cerebral organoids. Brain cortex of mice deficient in the manganese efflux transporter Slc30a10, a genetic model of chronic manganese accumulation, show dsRNA accumulation, and up-regulation of type I interferon response and astrogliosis markers, supporting a role for this pathway in neurotoxicity and parkinsonism.

neuroscience↗

bioRxiv2024

Adaptive protein synthesis in genetic models of copper deficiency and childhood neurodegeneration

Rare inherited diseases caused by mutations in the copper transporters SLC31A1 (CTR1) or ATP7A induce copper deficiency in the brain, causing seizures and neurodegeneration in infancy through poorly understood mechanisms. Here, we used multiple model systems to characterize the molecular mechanisms by which neuronal cells respond to copper deficiency. Targeted deletion of CTR1 in neuroblastoma cells produced copper deficiency that was associated with a metabolic shift favoring glycolysis over oxidative phosphorylation. Proteomic and transcriptomic analysis of CTR1 KO cells revealed simultaneous upregulation of mTORC1 and S6K signaling and reduced PERK signaling. Patterns of gene and protein expression and pharmacogenomics show increased activation of the mTORC1-S6K pathway as a pro-survival mechanism, ultimately resulting in increased protein synthesis. Spatial transcriptomic profiling of Atp7aflx/Y :: Vil1Cre/+ mice identified upregulated protein synthesis machinery and mTORC1-S6K pathway genes in copper-deficient Purkinje neurons in the cerebellum. Genetic epistasis experiments in Drosophila demonstrated that copper deficiency dendritic phenotypes in class IV neurons are partially rescued by increased S6k expression or 4E-BP1 (Thor) RNAi, while epidermis phenotypes are exacerbated by Akt, S6k, or raptor RNAi. Overall, we demonstrate that increased mTORC1-S6K pathway activation and protein synthesis is an adaptive mechanism by which neuronal cells respond to copper deficiency. SignificanceO_LICopper deficiency is present in rare conditions such as Menkes disease and CTR1 deficiency and in more common diseases like Alzheimers. The mechanisms of resilience and ultimate susceptibility to copper deficiency and associated pathology in the brain remain unknown. C_LIO_LIWe demonstrate that in a human cell line, Drosophila, and the mouse cerebellum, copper-deficient neuronal cells exhibit increased protein synthesis through mTORC1 activation and decreased PERK (EIF2AK3) activity. C_LIO_LIUpregulation of protein synthesis facilitates resilience of neuronal cells to copper deficiency, including partial restoration of dendritic arborization. Our findings offer a new framework for understanding copper deficiency-related pathology in neurological disorders. C_LI

neuroscience↗

bioRxiv2023

Systemic Metabolic and Mitochondrial Defects in Rett Syndrome Models

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.

neuroscience↗

bioRxiv2022

APOE Expression and Secretion are Modulated by Copper-Dependent and -Independent Mitochondrial Dysfunction

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimers disease risk factor apolipoprotein E (APOE) and other secretome components. This upregulation of secretory proteins was of a similar extent as modifications to the mitochondrial annotated proteome. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain, elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.

cell biology↗

bioRxiv2022

RelCoVax(R), a two antigen subunit protein vaccine candidate against SARS-CoV-2 induces strong immune responses in mice

The COVID-19 pandemic has spurred an unprecedented movement to develop safe and effective vaccines against the SARS-CoV-2 virus to immunize the global population. The first set of vaccine candidates that received emergency use authorization targeted the spike (S) glycoprotein of the SARS-CoV-2 virus that enables virus entry into cells via the receptor binding domain (RBD). Recently, multiple variants of SARS-CoV-2 have emerged with mutations in S protein and the ability to evade neutralizing antibodies in vaccinated individuals. We have developed a dual RBD and nucleocapsid (N) subunit protein vaccine candidate named RelCoVax(R) through heterologous expression in mammalian cells (RBD) and E. coli (N). The RelCoVax(R) formulation containing a combination of aluminum hydroxide (alum) and a synthetic CpG oligonucleotide as adjuvants elicited high antibody titers against RBD and N proteins in mice after a prime and boost dose regimen administered 2 weeks apart. The vaccine also stimulated cellular immune responses with a potential Th1 bias as evidenced by increased IFN-{gamma} release by splenocytes from immunized mice upon antigen exposure particularly N protein. Finally, the serum of mice immunized with RelCoVax(R) demonstrated the ability to neutralize two different SARS-CoV-2 viral strains in vitro including the Delta strain that has become dominant in many regions of the world and can evade vaccine induced neutralizing antibodies. These results warrant further evaluation of RelCoVax(R) through advanced studies and contribute towards enhancing our understanding of multicomponent subunit vaccine candidates against SARS-CoV-2.

immunology↗

bioRxiv2022

The Mitochondrial RNA Granule is Necessary for Parkinsonism-Associated Metal Toxicity

Prolonged manganese exposure causes manganism, a neurodegenerative movement disorder. The identity of adaptive and non-adaptive cellular processes targeted by manganese remains mostly unexplored. Here we study mechanisms engaged by manganese in genetic cellular models known to increase susceptibility to manganese exposure, the plasma membrane manganese efflux transporter SLC30A10 and the mitochondrial Parkinsons gene PARK2. We found that SLC30A10 and PARK2 mutations as well as manganese exposure compromised the mitochondrial RNA granule as well as mitochondrial transcript processing. These RNA granule defects led to impaired assembly and function of the mitochondrial respiratory chain. Notably, cells that survived a cytotoxic manganese challenge had impaired RNA granule function, thus suggesting that this granule phenotype was adaptive. CRISPR gene editing of subunits of the mitochondrial RNA granule, FASTKD2 or DHX30, as well as pharmacological inhibition of mitochondrial transcription-translation, were protective rather than deleterious for survival of cells acutely exposed to manganese. Similarly, adult Drosophila mutants with defects in the mitochondrial RNA granule component scully were safeguarded from manganese-induced mortality. We conclude that the downregulation of the mitochondrial RNA granule function is a protective mechanism for acute metal toxicity. Significance StatementMutations in the manganese efflux transporter SLC30A10 and the mitochondrial Parkinsons gene PARK2, cause neurodegeneration and increased susceptibility to toxic manganese exposure. Thus, molecular processes affected in both mutants could offer insight into fundamental mechanisms conferring susceptibility or resilience to environmental and genetic factors associated with neurodegeneration. Here we report that SLC30A10 and PARK2 mutations compromise an understudied structure, the mitochondrial RNA granule, which is required for processing polycistronic mitochondrial RNAs. Cells and Drosophila lacking mitochondrial RNA granule components were resistant to manganese exposure. We conclude that the downregulation of the mitochondrial RNA granule function is an adaptive mechanism for cells exposed to manganese.

cell biology↗

bioRxiv2020

Golgi-Dependent Copper Homeostasis Sustains Synaptic Development and Mitochondrial Content

Rare genetic diseases preponderantly affect the nervous system with phenotypes spanning from neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate conserved interactions between ATP7 paralogs with the COG complex, a Golgi complex tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, catecholaminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation or haploinsufficiency of COG complex subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses. Significance StatementMenkes and Wilson disease affect copper homeostasis and characteristically afflict the nervous system. However, their molecular neuropathology mechanisms remain mostly unexplored. We demonstrate that copper homeostasis in neurons is maintained by two factors that localize to the Golgi apparatus, ATP7 and the COG complex. Disruption of these mechanisms affect mitochondrial function and localization to synapses as well as neurotransmission and synaptic plasticity. These findings suggest a new principle of interorganelle communication whereby the Golgi apparatus and mitochondria are functionally coupled through homeostatically controlled cellular copper levels.

neuroscience↗

bioRxiv2020

Mitochondrial Proteostasis Requires Genes Encoded in a Neurodevelopmental Syndrome Locus that are Necessary for Synapse Function

Eukaryotic cells maintain proteostasis through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. Here we focus on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. We demonstrate that SLC25A1 and MRPL40, two genes present in this microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial protein translation and proteostasis. Our Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. We propose that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a novel pathogenic mechanism for neurodevelopmental disorders.

neuroscience↗

bioRxiv2018

Systems Analysis of the 22q11.2 Microdeletion Syndrome Converges on a Mitochondrial Interactome Necessary for Synapse Function and Behavior

Neurodevelopmental disorders offer insight into synaptic mechanisms. To unbiasedly uncover these mechanisms, we studied the 22q11.2 syndrome, a recurrent copy number variant, which is the highest schizophrenia genetic risk factor. We quantified the proteomes of 22q11.2 mutant human fibroblasts and mouse brains carrying a 22q11.2-like defect, Df(16)A+/-. Molecular ontologies defined mitochondrial compartments and pathways as some of top ranked categories. In particular, we identified perturbations in the SLC25A1-SLC25A4 mitochondrial transporter interactome as associated with the 22q11.2 genetic defect. Expression of SLC25A1-SLC25A4 interactome components was affected in neuronal cells from schizophrenia patients. Furthermore, hemideficiency of the Drosophila SLC25A4 orthologue, dSLC25A4-sesB, affected synapse function and impaired sleep patterns in a neuronal-specific manner. These results identify a novel synaptic role of mitochondrial inner membrane solute transporters. We propose that mitochondria are among key organelles affected by genetic defects that increase the risk of neurodevelopmental disorders.

neuroscience↗