Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons

A complex of stomatin-family proteins and acid-sensing (proton-gated) ion channel (ASIC) family members participate in sensory transduction in invertebrates and vertebrates. Here, we have examined the role of the stomatin-family protein stomatin-like protein-3 (STOML3) in this process. We demonstrate that STOML3 interacts with stomatin and ASIC subunits and that this occurs in a highly mobile vesicle pool in dorsal root ganglia (DRG) neurons and Chinese hamster ovary cells. We identify a hydrophobic region in the N-terminus of STOML3 that is required for vesicular localization of STOML3 and regulates physical and functional interaction with ASICs. We further characterize STOML3-containing vesicles in DRG neurons and show that they are Rab11-positive, but not part of the early-endosomal, lysosomal or Rab14-dependent biosynthetic compartment. Moreover, uncoupling of vesicles from microtubules leads to incorporation of STOML3 into the plasma membrane and increased acid-gated currents. Thus, STOML3 defines a vesicle pool in which it associates with molecules that have critical roles in sensory transduction. We suggest that the molecular features of this vesicular pool may be characteristic of a ‘transducosome’ in sensory neurons

A unique role for DNA (hydroxy)methylation in epigenetic regulation of human inhibitory neurons.

Brain function depends on interaction of diverse cell types whose gene expression and identity are defined, in part, by epigenetic mechanisms. Neuronal DNA contains two major epigenetic modifications, methylcytosine (mC) and hydroxymethylcytosine (hmC), yet their cell type-specific landscapes and relationship with gene expression are poorly understood. We report high-resolution (h)mC analyses, together with transcriptome and histone modification profiling, in three major cell types in human prefrontal cortex: glutamatergic excitatory neurons, medial ganglionic eminence-derived γ-aminobutyric acid (GABA)ergic inhibitory neurons, and oligodendrocytes. We detected a unique association between hmC and gene expression in inhibitory neurons that differed significantly from the pattern in excitatory neurons and oligodendrocytes. We also found that risk loci associated with neuropsychiatric diseases were enriched near regions of reduced hmC in excitatory neurons and reduced mC in inhibitory neurons. Our findings indicate differential roles for mC and hmC in regulation of gene expression in different brain cell types, with implications for the etiology of human brain diseases

DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper- and Hypomethylation and a Younger Epigenetic Age.

We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose (N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based "epigenetic age" of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC

DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper- and Hypomethylation and a Younger Epigenetic Age

We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose (N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based “epigenetic age” of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC

DNA Methylation Profiling of Human Prefrontal Cortex Neurons in Heroin Users Shows Significant Difference between Genomic Contexts of Hyper- and Hypomethylation and a Younger Epigenetic Age

We employed Illumina 450 K Infinium microarrays to profile DNA methylation (DNAm) in neuronal nuclei separated by fluorescence-activated sorting from the postmortem orbitofrontal cortex (OFC) of heroin users who died from heroin overdose (N = 37), suicide completers (N = 22) with no evidence of heroin use and from control subjects who did not abuse illicit drugs and died of non-suicide causes (N = 28). We identified 1298 differentially methylated CpG sites (DMSs) between heroin users and controls, and 454 DMSs between suicide completers and controls (p < 0.001). DMSs and corresponding genes (DMGs) in heroin users showed significant differences in the preferential context of hyper and hypo DM. HyperDMSs were enriched in gene bodies and exons but depleted in promoters, whereas hypoDMSs were enriched in promoters and enhancers. In addition, hyperDMGs showed preference for genes expressed specifically by glutamatergic as opposed to GABAergic neurons and enrichment for axonogenesis- and synaptic-related gene ontology categories, whereas hypoDMGs were enriched for transcription factor activity- and gene expression regulation-related terms. Finally, we found that the DNAm-based “epigenetic age” of neurons from heroin users was younger than that in controls. Suicide-related results were more difficult to interpret. Collectively, these findings suggest that the observed DNAm differences could represent functionally significant marks of heroin-associated plasticity in the OFC

Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation

Abstract A repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we investigate single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem motor and frontal cortices from C9-ALS, C9-FTD, and control donors. C9-ALS donors present pervasive alterations of gene expression with concordant changes in chromatin accessibility and histone modifications. The greatest alterations occur in upper and deep layer excitatory neurons, as well as in astrocytes. In neurons, the changes imply an increase in proteostasis, metabolism, and protein expression pathways, alongside a decrease in neuronal function. In astrocytes, the alterations suggest activation and structural remodeling. Conversely, C9-FTD donors have fewer high-quality neuronal nuclei in the frontal cortex and numerous gene expression changes in glial cells. These findings highlight a context-dependent molecular disruption in C9-ALS and C9-FTD, indicating unique effects across cell types, brain regions, and diseases

Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation

A repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we investigate single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem motor and frontal cortices from C9-ALS, C9-FTD, and control donors. C9-ALS donors present pervasive alterations of gene expression with concordant changes in chromatin accessibility and histone modifications. The greatest alterations occur in upper and deep layer excitatory neurons, as well as in astrocytes. In neurons, the changes imply an increase in proteostasis, metabolism, and protein expression pathways, alongside a decrease in neuronal function. In astrocytes, the alterations suggest activation and structural remodeling. Conversely, C9-FTD donors have fewer high-quality neuronal nuclei in the frontal cortex and numerous gene expression changes in glial cells. These findings highlight a context-dependent molecular disruption in C9-ALS and C9-FTD, indicating unique effects across cell types, brain regions, and diseases

Neuronal and glial 3D chromatin architecture informs the cellular etiology of brain disorders.

Cellular heterogeneity in the human brain obscures the identification of robust cellular regulatory networks, which is necessary to understand the function of non-coding elements and the impact of non-coding genetic variation. Here we integrate genome-wide chromosome conformation data from purified neurons and glia with transcriptomic and enhancer profiles, to characterize the gene regulatory landscape of two major cell classes in the human brain. We then leverage cell-type-specific regulatory landscapes to gain insight into the cellular etiology of several brain disorders. We find that Alzheimer's disease (AD)-associated epigenetic dysregulation is linked to neurons and oligodendrocytes, whereas genetic risk factors for AD highlighted microglia, suggesting that different cell types may contribute to disease risk, via different mechanisms. Moreover, integration of glutamatergic and GABAergic regulatory maps with genetic risk factors for schizophrenia (SCZ) and bipolar disorder (BD) identifies shared (parvalbumin-expressing interneurons) and distinct cellular etiologies (upper layer neurons for BD, and deeper layer projection neurons for SCZ). Collectively, these findings shed new light on cell-type-specific gene regulatory networks in brain disorders