Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Epigenomic landscape of the human dorsal root ganglion: sex differences and transcriptional regulation of nociceptive genes.

Gene expression is influenced by chromatin architecture via controlled access of regulatory factors to DNA. To better understand regulation of gene expression in the human dorsal root ganglion (hDRG) we used bulk and spatial transposase-accessible chromatin technology followed by sequencing (ATAC-seq). We detected a total of 3005 differentially accessible chromatin regions (DARs) between sexes using bulk ATAC-seq. DARs in female hDRG mapped mainly to the X chromosome. In males, DARs were found in autosomal genes. We also found differential transcription factor binding motifs within DARs. EGR1/3 and SP1/4 were abundant in females, and JUN, FOS and other AP-1 family members in males. With the aim of dissecting the open chromatin profile in hDRG neurons, we used spatial ATAC-seq. Consistent with our bulk ATAC-seq data, most of the DARs in female hDRG were located in X chromosome genes. Neuron cluster showed higher chromatin accessibility in GABAergic, glutamatergic, and interferon-related genes in females, and in Ca2+-signaling-related genes in males. Sex differences in open chromatin transcription factor binding sites in neuron-proximal barcodes were consistent with the bulk data, having EGR1 transcription factor activity in females and AP-1 family members in males. Accordingly, we showed higher expression of EGR1 in female hDRG compared to male with in-situ hybridization. Our findings point to epigenomic sex differences in the hDRG that likely underlie divergent transcriptional responses that determine mechanistic sex differences in pain.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's disease microglia.

Several genetic risk factors for Alzheimer's disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells1. However, the relationship between lipid metabolism in glia and Alzheimer's disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer's disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer's disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aß induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer's disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer's disease.

Cell-type-resolved somatic mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around anatomic origins of specific brain cell subtypes and lineage relationships within the human forebrain. Thus, direct observation in the mature human brain is critical for a complete understanding of the structural organization and cellular origins. Here, we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific Mosaic Variant Barcode Analysis. From four hemispheres from two different human neurotypical donors, we identified 287 and 780 mosaic variants (MVs), respectively that were used to deconvolve clonal dynamics. Clonal spread and allelic fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted compared with resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome-transcriptome analysis at both a cell-type-specific and single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of MVs across 17 locations within one parietal lobe reveals restrictions of clonal spread in the anterior-posterior axis precedes that of the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus cell-type resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Immune mechanisms of depression in rheumatoid arthritis.

Depression is a common and disabling comorbidity in rheumatoid arthritis that not only decreases the likelihood of remission and treatment adherence but also increases the risk of disability and mortality in patients with rheumatoid arthritis. Compelling data that link immune mechanisms to major depressive disorder indicate possible common mechanisms that drive the pathology of the two conditions. Preclinical evidence suggests that pro-inflammatory cytokines, which are prevalent in rheumatoid arthritis, have various effects on monoaminergic neurotransmission, neurotrophic factors and measures of synaptic plasticity. Neuroimaging studies provide insight into the consequences of inflammation on the brain (for example, on neural connectivity), and clinical trial data highlight the beneficial effects of immune modulation on comorbid depression. Major depressive disorder occurs more frequently in patients with rheumatoid arthritis than in the general population, and major depressive disorder also increases the risk of a future diagnosis of rheumatoid arthritis, further highlighting the link between rheumatoid arthritis and major depressive disorder. This Review focuses on interactions between peripheral and central immunobiological mechanisms in the context of both rheumatoid arthritis and major depressive disorder. Understanding these mechanisms will provide a basis for future therapeutic development, not least in depression.

Mechanisms underlying HIV-associated cognitive impairment and emerging therapies for its management.

People living with HIV are affected by the chronic consequences of neurocognitive impairment (NCI) despite antiretroviral therapies that suppress viral replication, improve health and extend life. Furthermore, viral suppression does not eliminate the virus, and remaining infected cells may continue to produce viral proteins that trigger neurodegeneration. Comorbidities such as diabetes mellitus are likely to contribute substantially to CNS injury in people living with HIV, and some components of antiretroviral therapy exert undesirable side effects on the nervous system. No treatment for HIV-associated NCI has been approved by the European Medicines Agency or the US Food and Drug Administration. Historically, roadblocks to developing effective treatments have included a limited understanding of the pathophysiology of HIV-associated NCI and heterogeneity in its clinical manifestations. This heterogeneity might reflect multiple underlying causes that differ among individuals, rather than a single unifying neuropathogenesis. Despite these complexities, accelerating discoveries in HIV neuropathogenesis are yielding potentially druggable targets, including excessive immune activation, metabolic alterations culminating in mitochondrial dysfunction, dysregulation of metal ion homeostasis and lysosomal function, and microbiome alterations. In addition to drug treatments, we also highlight the importance of non-pharmacological interventions. By revisiting mechanisms implicated in NCI and potential interventions addressing these mechanisms, we hope to supply reasons for optimism in people living with HIV affected by NCI and their care providers.

Genomic Mosaicism of the Brain: Origin, Impact, and Utility.

Genomic mosaicism describes the phenomenon where some but not all cells within a tissue harbor unique genetic mutations. Traditionally, research focused on the impact of genomic mosaicism on clinical phenotype-motivated by its involvement in cancers and overgrowth syndromes. More recently, we increasingly shifted towards the plethora of neutral mosaic variants that can act as recorders of cellular lineage and environmental exposures. Here, we summarize the current state of the field of genomic mosaicism research with a special emphasis on our current understanding of this phenomenon in brain development and homeostasis. Although the field of genomic mosaicism has a rich history, technological advances in the last decade have changed our approaches and greatly improved our knowledge. We will provide current definitions and an overview of contemporary detection approaches for genomic mosaicism. Finally, we will discuss the impact and utility of genomic mosaicism.

RANK ligand converts the NCoR/HDAC3 co-repressor to a PGC1ß- and RNA-dependent co-activator of osteoclast gene expression.

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.

APOE4/4 is linked to damaging lipid droplets in Alzheimer's microglia.

Several genetic risk factors for Alzheimer's Disease (AD) implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells. However, the relationship between lipid metabolism in glia and AD pathology remains poorly understood. Through single-nucleus RNA-sequencing of AD brain tissue, we have identified a microglial state defined by the expression of the lipid droplet (LD) associated enzyme ACSL1 with ACSL1-positive microglia most abundant in AD patients with the APOE4/4 genotype. In human iPSC-derived microglia (iMG) fibrillar Aß (fAß) induces ACSL1 expression, triglyceride synthesis, and LD accumulation in an APOE-dependent manner. Additionally, conditioned media from LD-containing microglia leads to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for AD with microglial LD accumulation and neurotoxic microglial-derived factors, potentially providing novel therapeutic strategies for AD.

Human microglia maturation is underpinned by specific gene regulatory networks.

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.

An in vivo neuroimmune organoid model to study human microglia phenotypes.

Microglia are specialized brain-resident macrophages that play crucial roles in brain development, homeostasis, and disease. However, until now, the ability to model interactions between the human brain environment and microglia has been severely limited. To overcome these limitations, we developed an in vivo xenotransplantation approach that allows us to study functionally mature human microglia (hMGs) that operate within a physiologically relevant, vascularized immunocompetent human brain organoid (iHBO) model. Our data show that organoid-resident hMGs gain human-specific transcriptomic signatures that closely resemble their in vivo counterparts. In vivo two-photon imaging reveals that hMGs actively engage in surveilling the human brain environment, react to local injuries, and respond to systemic inflammatory cues. Finally, we demonstrate that the transplanted iHBOs developed here offer the unprecedented opportunity to study functional human microglia phenotypes in health and disease and provide experimental evidence for a brain-environment-induced immune response in a patient-specific model of autism with macrocephaly.

TREM2-independent microgliosis promotes tau-mediated neurodegeneration in the presence of ApoE4.

In addition to tau and Aß pathologies, inflammation plays an important role in Alzheimer's disease (AD). Variants in APOE and TREM2 increase AD risk. ApoE4 exacerbates tau-linked neurodegeneration and inflammation in P301S tau mice and removal of microglia blocks tau-dependent neurodegeneration. Microglia adopt a heterogeneous population of transcriptomic states in response to pathology, at least some of which are dependent on TREM2. Previously, we reported that knockout (KO) of TREM2 attenuated neurodegeneration in P301S mice that express mouse Apoe. Because of the possible common pathway of ApoE and TREM2 in AD, we tested whether TREM2 KO (T2KO) would block neurodegeneration in P301S Tau mice expressing ApoE4 (TE4), similar to that observed with microglial depletion. Surprisingly, we observed exacerbated neurodegeneration and tau pathology in TE4-T2KO versus TE4 mice, despite decreased TREM2-dependent microgliosis. Our results suggest that tau pathology-dependent microgliosis, that is, TREM2-independent microgliosis, facilitates tau-mediated neurodegeneration in the presence of ApoE4.

Microglial pattern recognition via IL-33 promotes synaptic refinement in developing corticothalamic circuits in mice.

Microglia are critical regulators of brain development that engulf synaptic proteins during postnatal synapse remodeling. However, the mechanisms through which microglia sense the brain environment are not well defined. Here, we characterized the regulatory program downstream of interleukin-33 (IL-33), a cytokine that promotes microglial synapse remodeling. Exposing the developing brain to a supraphysiological dose of IL-33 altered the microglial enhancer landscape and increased binding of stimulus-dependent transcription factors including AP-1/FOS. This induced a gene expression program enriched for the expression of pattern recognition receptors, including the scavenger receptor MARCO. CNS-specific deletion of IL-33 led to increased excitatory/inhibitory synaptic balance, spontaneous absence-like epileptiform activity in juvenile mice, and increased seizure susceptibility in response to chemoconvulsants. We found that MARCO promoted synapse engulfment, and Marco-deficient animals had excess thalamic excitatory synapses and increased seizure susceptibility. Taken together, these data define coordinated epigenetic and functional changes in microglia and uncover pattern recognition receptors as potential regulators of postnatal synaptic refinement.

Increased post-mitotic senescence in aged human neurons is a pathological feature of Alzheimer's disease.

The concept of senescence as a phenomenon limited to proliferating cells has been challenged by growing evidence of senescence-like features in terminally differentiated cells, including neurons. The persistence of senescent cells late in life is associated with tissue dysfunction and increased risk of age-related disease. We found that Alzheimer's disease (AD) brains have significantly higher proportions of neurons that express senescence markers, and their distribution indicates bystander effects. AD patient-derived directly induced neurons (iNs) exhibit strong transcriptomic, epigenetic, and molecular biomarker signatures, indicating a specific human neuronal senescence-like state. AD iN single-cell transcriptomics revealed that senescent-like neurons face oncogenic challenges and metabolic dysfunction as well as display a pro-inflammatory signature. Integrative profiling of the inflammatory secretome of AD iNs and patient cerebral spinal fluid revealed a neuronal senescence-associated secretory phenotype that could trigger astrogliosis in human astrocytes. Finally, we show that targeting senescence-like neurons with senotherapeutics could be a strategy for preventing or treating AD.

CSF1R-Mediated Myeloid Cell Depletion Prolongs Lifespan But Aggravates Distinct Motor Symptoms in a Model of Multiple System Atrophy.

As the CNS-resident macrophages and member of the myeloid lineage, microglia fulfill manifold functions important for brain development and homeostasis. In the context of neurodegenerative diseases, they have been implicated in degenerative and regenerative processes. The discovery of distinct activation patterns, including increased phagocytosis, indicated a damaging role of myeloid cells in multiple system atrophy (MSA), a devastating, rapidly progressing atypical parkinsonian disorder. Here, we analyzed the gene expression profile of microglia in a mouse model of MSA (MBP29-hα-syn) and identified a disease-associated expression profile and upregulation of the colony-stimulating factor 1 (Csf1). Thus, we hypothesized that CSF1 receptor-mediated depletion of myeloid cells using PLX5622 modifies the disease progression and neuropathological phenotype in this mouse model. Intriguingly, sex-balanced analysis of myeloid cell depletion in MBP29-hα-syn mice revealed a two-faced outcome comprising an improved survival rate accompanied by a delayed onset of neurological symptoms in contrast to severely impaired motor functions. Furthermore, PLX5622 reversed gene expression profiles related to myeloid cell activation but reduced gene expression associated with transsynaptic signaling and signal release. While transcriptional changes were accompanied by a reduction of dopaminergic neurons in the SNpc, striatal neuritic density was increased upon myeloid cell depletion in MBP29-hα-syn mice. Together, our findings provide insight into the complex, two-faced role of myeloid cells in the context of MSA emphasizing the importance to carefully balance the beneficial and adverse effects of CSF1R inhibition in different models of neurodegenerative disorders before its clinical translation.SIGNIFICANCE STATEMENT Myeloid cells have been implicated as detrimental in the disease pathogenesis of multiple system atrophy. However, long-term CSF1R-dependent depletion of these cells in a mouse model of multiple system atrophy demonstrates a two-faced effect involving an improved survival associated with a delayed onset of disease and reduced inflammation which was contrasted by severely impaired motor functions, synaptic signaling, and neuronal circuitries. Thus, this study unraveled a complex role of myeloid cells in multiple system atrophy, which indicates important functions beyond the previously described disease-associated, destructive phenotype and emphasized the need of further investigation to carefully and individually fine-tune immunologic processes in different neurodegenerative diseases.

A prebiotic diet modulates microglial states and motor deficits in α-synuclein overexpressing mice.

Parkinson's disease (PD) is a movement disorder characterized by neuroinflammation, α-synuclein pathology, and neurodegeneration. Most cases of PD are non-hereditary, suggesting a strong role for environmental factors, and it has been speculated that disease may originate in peripheral tissues such as the gastrointestinal (GI) tract before affecting the brain. The gut microbiome is altered in PD and may impact motor and GI symptoms as indicated by animal studies, although mechanisms of gut-brain interactions remain incompletely defined. Intestinal bacteria ferment dietary fibers into short-chain fatty acids, with fecal levels of these molecules differing between PD and healthy controls and in mouse models. Among other effects, dietary microbial metabolites can modulate activation of microglia, brain-resident immune cells implicated in PD. We therefore investigated whether a fiber-rich diet influences microglial function in α-synuclein overexpressing (ASO) mice, a preclinical model with PD-like symptoms and pathology. Feeding a prebiotic high-fiber diet attenuates motor deficits and reduces α-synuclein aggregation in the substantia nigra of mice. Concomitantly, the gut microbiome of ASO mice adopts a profile correlated with health upon prebiotic treatment, which also reduces microglial activation. Single-cell RNA-seq analysis of microglia from the substantia nigra and striatum uncovers increased pro-inflammatory signaling and reduced homeostatic responses in ASO mice compared to wild-type counterparts on standard diets. However, prebiotic feeding reverses pathogenic microglial states in ASO mice and promotes expansion of protective disease-associated macrophage (DAM) subsets of microglia. Notably, depletion of microglia using a CSF1R inhibitor eliminates the beneficial effects of prebiotics by restoring motor deficits to ASO mice despite feeding a prebiotic diet. These studies uncover a novel microglia-dependent interaction between diet and motor symptoms in mice, findings that may have implications for neuroinflammation and PD.

Human microglia phenotypes in the brain associated with HIV infection.

Cognitive impairment in individuals infected with HIV is highly prevalent despite life-long antiretroviral therapy. A growing line of evidence suggests that the human brain serves as a sanctuary for HIV persistence. Microglia, the innate immune cells of the brain parenchyma, may serve as a reservoir for HIV and drive the pathogenesis of HIV-associated neurocognitive disorders. Here, we highlight recent advances in understanding microglia diversity in HIV regarding their epigenome, transcriptome, and function.

Warburg-like metabolic transformation underlies neuronal degeneration in sporadic Alzheimer's disease.

The drivers of sporadic Alzheimer's disease (AD) remain incompletely understood. Utilizing directly converted induced neurons (iNs) from AD-patient-derived fibroblasts, we identified a metabolic switch to aerobic glycolysis in AD iNs. Pathological isoform switching of the glycolytic enzyme pyruvate kinase M (PKM) toward the cancer-associated PKM2 isoform conferred metabolic and transcriptional changes in AD iNs. These alterations occurred via PKM2's lack of metabolic activity and via nuclear translocation and association with STAT3 and HIF1α to promote neuronal fate loss and vulnerability. Chemical modulation of PKM2 prevented nuclear translocation, restored a mature neuronal metabolism, reversed AD-specific gene expression changes, and re-activated neuronal resilience against cell death.