ABSTRACT:Innate immunity is increasingly recognized as a driver of neurodegeneration, although pathogenic mechanisms are incompletely understood. Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplastic disorder caused by activating somatic mutations in MAPK pathway genes, most commonly BRAFV600E, in myeloid precursors. A subset of patients with LCH develop progressive neurodegeneration (LCH-ND). We generated a human induced pluripotent stem cell (iPSC) model from patients with somatic hematologic mosaicism for BRAFV600E. Brain macrophages/microglia from LCH iPSCs exhibit unique disease-specific pathogenic features. Stepwise differentiation identified hematopoietic progenitors as hyperproliferative, whereas brain macrophages were apoptosis resistant. Through application of cerebral organoids and a humanized murine xenotransplantation model, we identify marked heterogeneity of differentiation potential within clonal BRAFV600E lines in vivo. This model phenocopied human-specific phenotypes, including dense basal ganglia foci of abnormal macrophages, marked neurodegeneration with astrogliosis, and progressive ataxia. This approach will allow for preclinical testing of therapeutics for LCH-ND.

Alcohol use disorder (AUD) is a complex polygenic disease. Rodent models of alcohol dependence have been instrumental in modeling various aspects of dependence. Single-nucleus transcriptomics has enabled the profiling of cell-type-specific changes in gene expression in both human AUD and animal models. In this study, we identified shared dysregulated transcriptomic networks (TN), comprising gene co-expression modules and gene regulatory networks (GRNs) in a mouse model of alcohol dependence and individuals with AUD. Through cell-type-specific TN analysis, we identified translationally relevant, conserved dependence dysregulated molecular signatures. We identified conserved dependence-upregulated gene co-expression modules in astrocytes and oligodendrocytes, with hub genes Slc1a3 and Pde4b, respectively. These genes are linked to alcohol dependence mechanisms, such as glutamate signaling, a well-established target of alcohol’s effects, and PDE4, whose inhibition has been shown to reduce alcohol intake in preclinical and clinical studies. We then integrated publicly available human and mouse GRN data to identify upstream regulators of alcohol-dysregulated gene signatures in each cell type. This approach revealed a set of transcription factors (TFs), including Mef2a, Mef2c, Jund, Nr3c1, and Zeb1, that were upstream of most dysregulated genes in both the mouse and human datasets and have established relevance to addiction biology, representing promising targets for translational research. Collectively, these findings demonstrate the utility of cross-species, cell-type-specific network analysis for uncovering conserved molecular mechanisms in alcohol dependence. The identification of shared dysregulated networks, cell type homology, and upstream regulators provides a foundation for developing translationally relevant targeting strategies that can be tested in animal models.

BACKGROUND:Alcohol use disorder (AUD) is a prevalent neuropsychiatric disorder that is a major global health concern, affecting millions of people worldwide. Previous studies of AUD used underpowered single-cell analysis or bulk homogenates of postmortem brain tissue, which obscure gene expression changes in specific cell types. Therefore, we sought to conduct the largest-to-date single-nucleus RNA sequencing (snRNA-seq) postmortem brain study in AUD to elucidate transcriptomic pathology with cell type-specific resolution. METHODS:Here, we performed snRNA-seq and high-dimensional network analysis of 73 postmortem samples from individuals with AUD (n = 36, nnuclei = 248,873) and neurotypical control individuals (n = 37, nnuclei = 210,573) in the dorsolateral prefrontal cortex from both male and female donors. Additionally, we performed analysis for cell type-specific enrichment of aggregate genetic risk for AUD as well as integration of the AUD proteome for secondary validation. RESULTS:We identified 32 distinct cell clusters and found widespread cell type-specific transcriptomic changes across the cortex in AUD, particularly affecting glial populations. We found the greatest dysregulation in novel microglial and astrocytic subtypes that accounted for the majority of differential gene expression and coexpression modules linked to AUD. Differential gene expression was secondarily validated by integration of a publicly available AUD proteome. Finally, analysis for aggregate genetic risk for AUD identified subtypes of glia as potential key players not only affected by but also causally linked to the progression of AUD. CONCLUSIONS:These results highlight the importance of cell type-specific molecular changes in AUD and offer opportunities to identify novel targets for treatment on the single-nucleus level.

MEF2C encodes a transcription factor that is critical in nervous system development. Here, to examine disease-associated functions of MEF2C in human microglia, we profiled microglia differentiated from isogenic MEF2C-haploinsufficient and MEF2C-knockout induced pluripotent stem cell lines. Complementary transcriptomic and functional analyses revealed that loss of MEF2C led to a hyperinflammatory phenotype with broad phagocytic impairment, lipid accumulation, lysosomal dysfunction and elevated basal inflammatory cytokine secretion. Genome-wide profiling of MEF2C-bound sites coupled with the active regulatory landscape enabled inference of its transcriptional functions and potential mechanisms for MEF2C-associated cellular functions. Transcriptomic and epigenetic approaches identified substantial overlap with idiopathic autism datasets, suggesting a broader role of human microglial MEF2C dysregulation in idiopathic autism. In a mouse xenotransplantation model, loss of MEF2C led to morphological, lysosomal and lipid abnormalities in human microglia in vivo. Together, these studies reveal mechanisms by which reduced microglial MEF2C could contribute to the development of neurological diseases.

Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by a GAA repeat expansion in the frataxin (FXN) gene, leading to a severe reduction of the mitochondrial FXN protein, crucial for iron metabolism. While microglial inflammation is observed in FRDA, it remains unclear whether immune dysfunction is a primary disease mediator or a secondary reactionary phenotype. Utilizing patient-derived induced pluripotent stem cells (iPSCs), we report an intrinsic microglial phenotype of stark mitochondrial defects, iron overload, lipid peroxidation, and lysosomal abnormalities. These factors drive a pro-inflammatory state that contributes to neuronal death in co-culture systems. In a murine xenograft model, transplanted human FRDA microglia accumulate in white matter and the Purkinje cell layer, resulting in Purkinje neuron loss in otherwise healthy brains. Notably, CRISPR/Cas9-mediated correction of the GAA repeat reverses microglial defects and mitigates neurodegeneration. Here, we suggest that microglial dysfunction serve as a disease driver and a promising therapeutic target in FRDA.

Elevated brain levels of the essential metals manganese (Mn), copper, or iron induce motor disease. However, mechanisms of metal-induced motor disease are unclear and treatments are lacking. Elucidating the mechanisms of Mn-induced motor disease is particularly important because occupational and environmental Mn overexposure is a global public health problem. To address this, here we combined unbiased transcriptomics and metabolomics with functional studies in a mouse model of human environmental Mn exposure. Transcriptomics unexpectedly revealed that Mn exposure up-regulated expression of metabolic pathways in the brain and liver. Notably, genes in the kynurenine pathway of tryptophan metabolism, which produces neuroactive metabolites that impact neurological function, were up-regulated by Mn. Subsequent unbiased metabolomics revealed that Mn treatment altered kynurenine pathway metabolites in the brain and liver. Functional experiments then demonstrated that pharmacological inhibition of the first and rate-limiting step of the kynurenine pathway fully rescued Mn-induced motor deficits. Finally, elevated Mn directly activates hypoxia-inducible factor (HIF) transcription factors, and additional mechanistic assays identified a role for HIF1, but not HIF2, in regulating expression of hepatic kynurenine pathway genes under physiological or Mn exposure conditions, suggesting that Mn-induced HIF1 activation may contribute to the dysregulation of the kynurenine pathway in Mn toxicity. These findings (1) identify the upregulation of the kynurenine pathway by elevated Mn as a fundamental mechanism of Mn-induced motor deficits; (2) provide a pharmacological approach to treat Mn-induced motor disease; and (3) should broadly advance understanding of the general principles underlying neuromotor deficits caused by metal toxicity.

The implications of the neuroimmune system in the pathogenesis of alcohol use disorders (AUD) have been undeniable. Understanding how chronic alcohol dysregulates inflammatory pathways in the brain leading to altered neuronal functions could provide insight into specific mechanisms and neuroadaptations that may contribute to drinking behaviors. For example, the neuroadaptations at inhibitory GABAergic synapses in the central nucleus of the amygdala (CeA) of rodents and macaques involve the recruitment of neuroimmune pathways. This study tested the hypothesis that chronic alcohol consumption dysregulates the pro-inflammatory cytokine, interleukin 6 (IL-6) in the CeA of rhesus macaques. Male and female rhesus macaques were provided continuous choice to drink either 4 % (w/v) ethanol or water for 22 h/day, every day, for more than one year. We assessed the impact of chronic ethanol drinking on the cytokine abundance, including IL-6, in the blood, and adaptive changes in the CeA GABAergic transmission and transcriptome. We observed a main effect of sex on the IL-6 circulating plasma levels at necropsy, with higher IL-6 plasma levels in females, but no main effect of ethanol nor an interaction between sex and ethanol drinking. IL-6 decreased CeA GABA release (sIPSC frequency) in both control and alcohol drinkers, however chronic ethanol drinking significantly potentiated the IL-6 effects in both sexes. While, IL-6 had no effects on the sIPSC amplitudes in the control group, we observed a main effect of ethanol drinking on IL-6-induced decrease of sIPSC amplitude in both male and female drinkers. IL-6 also significantly prolonged the kinetics (decay times) of sIPSCs in male controls and drinkers, but not in the females, regardless of drinking. These data suggest that IL-6 modulation of GABAergic transmission within the CeA via a presynaptic reduction in GABA release independent of sex, whereas postsynaptic GABA receptor mediated functions (sIPSC amplitude and decay time) show sex- and ethanol specific effects. Lastly, transcriptomic analysis of the IL-6-immune-related genes in the CeA between high and low ethanol drinkers identified several DEGs (differentially expressed genes) implicating the neural and glial processes, and extracellular matrix as a generalized inflammatory response to ethanol in the high drinkers.

The essential metal manganese (Mn) induces neuromotor disease at elevated levels. The manganese efflux transporter SLC30A10 regulates brain Mn levels. Homozygous loss-of-function mutations in SLC30A10 induce hereditary Mn neurotoxicity in humans. Our prior characterization of Slc30a10 knockout mice recapitulated the high brain Mn levels and neuromotor deficits reported in humans. But, mechanisms of Mn-induced motor deficits due to SLC30A10 mutations or elevated Mn exposure are unclear. To gain insights into this issue, we characterized changes in gene expression in the basal ganglia, the main brain region targeted by Mn, of Slc30a10 knockout mice using unbiased transcriptomics. Compared with littermates, >1000 genes were upregulated or downregulated in the basal ganglia sub-regions (i.e. caudate putamen, globus pallidus, and substantia nigra) of the knockouts. Pathway analyses revealed notable changes in genes regulating synaptic transmission and neurotransmitter function in the knockouts that may contribute to the motor phenotype. Expression changes in the knockouts were essentially normalized by a reduced Mn chow, establishing that changes were Mn dependent. Upstream regulator analyses identified hypoxia-inducible factor (HIF) signaling, which we recently characterized to be a primary cellular response to elevated Mn, as a critical mediator of the transcriptomic changes in the basal ganglia of the knockout mice. HIF activation was also evident in the liver of the knockout mice. These results: (i) enhance understanding of the pathobiology of Mn-induced motor disease; (ii) identify specific target genes/pathways for future mechanistic analyses; and (iii) independently corroborate the importance of the HIF pathway in Mn homeostasis and toxicity.

Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia.

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.

Microglia may only represent 10% of central nervous system (CNS) cells but they perform critical roles in development, homeostasis and neurological disease. Microglia are also environmentally regulated, quickly losing their transcriptomic and epigenetic signature after leaving the CNS. This facet of microglia biology is both fascinating and technically challenging influencing the study of the genetics and function of human microglia in a manner that recapitulates the CNS environment. In this review we provide a comprehensive overview of existing in vitro and in vivo methodology to study human microglia, such as immortalized cells lines, stem cell-derived microglia, cerebral organoids and xenotransplantation. Since there is currently no single method that completely recapitulates all hallmarks of human ex vivo adult homeostatic microglia, we also discuss the advantages and limitations of each existing model as a practical guide for researchers.

Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ–SMAD signaling axis.

Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state tran-scripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering re-veals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combi-natorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage-and disease-specific phenotypes.