Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology.

Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.

Nano-CUT&Tag for multimodal chromatin profiling at single-cell resolution.

The ability to comprehensively analyze the chromatin state with single-cell resolution is crucial for understanding gene regulatory principles in heterogenous tissues or during development. Recently, we developed a nanobody-based single-cell CUT&Tag (nano-CT) protocol to simultaneously profile three epigenetic modalities-two histone marks and open chromatin state-from the same single cell. Nano-CT implements a new set of secondary nanobody-Tn5 fusion proteins to direct barcoded tagmentation by Tn5 transposase to genomic targets labeled by primary antibodies raised in different species. Such nanobody-Tn5 fusion proteins are currently not commercially available, and their in-house production and purification can be completed in 3-4 d by following our detailed protocol. The single-cell indexing in nano-CT is performed on a commercially available platform, making it widely accessible to the community. In comparison to other multimodal methods, nano-CT stands out in data complexity, low sample requirements and the flexibility to choose two of the three modalities. In addition, nano-CT works efficiently with fresh brain samples, generating multimodal epigenomic profiles for thousands of brain cells at single-cell resolution. The nano-CT protocol can be completed in just 3 d by users with basic skills in standard molecular biology and bioinformatics, although previous experience with single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is beneficial for more in-depth data analysis. As a multimodal assay, nano-CT holds immense potential to reveal interactions of various chromatin modalities, to explore epigenetic heterogeneity and to increase our understanding of the role and interplay that chromatin dynamics has in cellular development.

Co-expression of PADI isoforms during progenitor differentiation enables functional diversity.

Protein isoforms, generated through alternative splicing or promoter usage, contribute to tissue function. Here, we characterize the expression of predicted Padi3α and Padi3ß isoforms in hair follicles and describe expression of Padi2ß, a hitherto unknown PADI2 isoform, in the oligodendrocyte lineage. Padi2ß transcription is initiated from a downstream intronic promoter, generating an N-terminally truncated, unstable, PADI2ß. By contrast to the established role of the canonical PADI2 (PADI2α) (Falcao et al. 2019 Cell Rep. 27, 1090-1102.e10. (doi:10.1016/j.celrep.2019.03.108)), PADI2ß inhibits oligodendrocyte differentiation, suggesting that PADI2 isoforms exert opposing effects on oligodendrocyte lineage progression. We localize Padi3α and Padi3ß to developing hair follicles and find that both transcripts are expressed at low levels in progenitor cells, only to increase in expression concomitant with differentiation. When expressed in vitro, PADI3α and PADI3ß are enriched in the cytoplasm and precipitate together. Whereas PADI3ß protein stability is low and PADI3ß fails to induce protein citrullination, we find that the enzymatic activity and protein stability of PADI3α is reduced in the presence of PADI3ß. We propose that PADI3ß modulates PADI3α activity by direct binding and heterodimer formation. Here, we establish expression and function of Padi2 and Padi3 isoforms, expanding on the mechanisms in place to regulate citrullination in complex tissues. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Brain matters: unveiling the distinct contributions of region, age, and sex to glia diversity and CNS function.

The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are often differentially affected in human neurodegenerative diseases across CNS region, age and sex. We hypothesize that this selective vulnerability is underpinned by physiological variation in white matter glia. Using single nucleus RNA sequencing of human post-mortem white matter samples from the brain, cerebellum and spinal cord and subsequent tissue-based validation we found substantial glial heterogeneity with tissue region: we identified region-specific oligodendrocyte precursor cells (OPCs) that retain developmental origin markers into adulthood, distinguishing them from mouse OPCs. Region-specific OPCs give rise to similar oligodendrocyte populations, however spinal cord oligodendrocytes exhibit markers such as SKAP2 which are associated with increased myelin production and we found a spinal cord selective population particularly equipped for producing long and thick myelin sheaths based on the expression of genes/proteins such as HCN2. Spinal cord microglia exhibit a more activated phenotype compared to brain microglia, suggesting that the spinal cord is a more pro-inflammatory environment, a difference that intensifies with age. Astrocyte gene expression correlates strongly with CNS region, however, astrocytes do not show a more activated state with region or age. Across all glia, sex differences are subtle but the consistent increased expression of protein-folding genes in male donors hints at pathways that may contribute to sex differences in disease susceptibility. These findings are essential to consider for understanding selective CNS pathologies and developing tailored therapeutic strategies.

Spatial epigenome-transcriptome co-profiling of mammalian tissues.

Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context1-5. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag.

Probing histone modifications at a single-cell level in thousands of cells has been enabled by technologies such as single-cell CUT&Tag. Here we describe nano-CUT&Tag (nano-CT), which allows simultaneous mapping of up to three epigenomic modalities at single-cell resolution using nanobody-Tn5 fusion proteins. Multimodal nano-CT is compatible with starting materials as low as 25,000-200,000 cells and has significantly higher sensitivity and number of fragments per cell than single-cell CUT&Tag. We use nano-CT to simultaneously profile chromatin accessibility, H3K27ac, and H3K27me3 in juvenile mouse brain, allowing for discrimination of more cell types and states than unimodal single-cell CUT&Tag. We also infer chromatin velocity between assay for transposase-accessible chromatin (ATAC) and H3K27ac in the oligodendrocyte lineage and deconvolute H3K27me3 repressive states, finding two sequential waves of H3K27me3 repression at distinct gene modules during oligodendrocyte lineage progression. Given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in complex biological systems at the single-cell level.

Spatial profiling of chromatin accessibility in mouse and human tissues.

Cellular function in tissue is dependent on the local environment, requiring new methods for spatial mapping of biomolecules and cells in the tissue context1. The emergence of spatial transcriptomics has enabled genome-scale gene expression mapping2-5, but the ability to capture spatial epigenetic information of tissue at the cellular level and genome scale is lacking. Here we describe a method for spatially resolved chromatin accessibility profiling of tissue sections using next-generation sequencing (spatial-ATAC-seq) by combining in situ Tn5 transposition chemistry6 and microfluidic deterministic barcoding5. Profiling mouse embryos using spatial-ATAC-seq delineated tissue-region-specific epigenetic landscapes and identified gene regulators involved in the development of the central nervous system. Mapping the accessible genome in the mouse and human brain revealed the intricate arealization of brain regions. Applying spatial-ATAC-seq to tonsil tissue resolved the spatially distinct organization of immune cell types and states in lymphoid follicles and extrafollicular zones. This technology progresses spatial biology by enabling spatially resolved chromatin accessibility profiling to improve our understanding of cell identity, cell state and cell fate decision in relation to epigenetic underpinnings in development and disease.

Cell-type-specific cis-eQTLs in eight human brain cell types identify novel risk genes for psychiatric and neurological disorders.

To date, most expression quantitative trait loci (eQTL) studies, which investigate how genetic variants contribute to gene expression, have been performed in heterogeneous brain tissues rather than specific cell types. In this study, we performed an eQTL analysis using single-nuclei RNA sequencing from 192 individuals in eight brain cell types derived from the prefrontal cortex, temporal cortex and white matter. We identified 7,607 eGenes, a substantial fraction (46%, 3,537/7,607) of which show cell-type-specific effects, with strongest effects in microglia. Cell-type-level eQTLs affected more constrained genes and had larger effect sizes than tissue-level eQTLs. Integration of brain cell type eQTLs with genome-wide association studies (GWAS) revealed novel relationships between expression and disease risk for neuropsychiatric and neurodegenerative diseases. For most GWAS loci, a single gene co-localized in a single cell type, providing new clues into disease etiology. Our findings demonstrate substantial contrast in genetic regulation of gene expression among brain cell types and reveal potential mechanisms by which disease risk genes influence brain disorders.

Spatial and temporal heterogeneity in the lineage progression of fine oligodendrocyte subtypes.

BACKGROUND: Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations. RESULTS: We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS. CONCLUSIONS: Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.

Developmental landscape of human forebrain at a single-cell level identifies early waves of oligodendrogenesis.

Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.

Spatial-CUT&Tag: Spatially resolved chromatin modification profiling at the cellular level.

Spatial omics emerged as a new frontier of biological and biomedical research. Here, we present spatial-CUT&Tag for spatially resolved genome-wide profiling of histone modifications by combining in situ CUT&Tag chemistry, microfluidic deterministic barcoding, and next-generation sequencing. Spatially resolved chromatin states in mouse embryos revealed tissue-type-specific epigenetic regulations in concordance with ENCODE references and provide spatial information at tissue scale. Spatial-CUT&Tag revealed epigenetic control of the cortical layer development and spatial patterning of cell types determined by histone modification in mouse brain. Single-cell epigenomes can be derived in situ by identifying 20-micrometer pixels containing only one nucleus using immunofluorescence imaging. Spatial chromatin modification profiling in tissue may offer new opportunities to study epigenetic regulation, cell function, and fate decision in normal physiology and pathogenesis.

Epigenomic priming of immune genes implicates oligodendroglia in multiple sclerosis susceptibility.

Multiple sclerosis (MS) is characterized by a targeted attack on oligodendroglia (OLG) and myelin by immune cells, which are thought to be the main drivers of MS susceptibility. We found that immune genes exhibit a primed chromatin state in single mouse and human OLG in a non-disease context, compatible with transitions to immune-competent states in MS. We identified BACH1 and STAT1 as transcription factors involved in immune gene regulation in oligodendrocyte precursor cells (OPCs). A subset of immune genes presents bivalency of H3K4me3/H3K27me3 in OPCs, with Polycomb inhibition leading to their increased activation upon interferon gamma (IFN-γ) treatment. Some MS susceptibility single-nucleotide polymorphisms (SNPs) overlap with these regulatory regions in mouse and human OLG. Treatment of mouse OPCs with IFN-γ leads to chromatin architecture remodeling at these loci and altered expression of interacting genes. Thus, the susceptibility for MS may involve OLG, which therefore constitutes novel targets for immunological-based therapies for MS.

Cell-type specialization is encoded by specific chromatin topologies.

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1-3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4-6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive 'melting' of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.

Single-cell CUT&Tag profiles histone modifications and transcription factors in complex tissues.

In contrast to single-cell approaches for measuring gene expression and DNA accessibility, single-cell methods for analyzing histone modifications are limited by low sensitivity and throughput. Here, we combine the CUT&Tag technology, developed to measure bulk histone modifications, with droplet-based single-cell library preparation to produce high-quality single-cell data on chromatin modifications. We apply single-cell CUT&Tag (scCUT&Tag) to tens of thousands of cells of the mouse central nervous system and probe histone modifications characteristic of active promoters, enhancers and gene bodies (H3K4me3, H3K27ac and H3K36me3) and inactive regions (H3K27me3). These scCUT&Tag profiles were sufficient to determine cell identity and deconvolute regulatory principles such as promoter bivalency, spreading of H3K4me3 and promoter-enhancer connectivity. We also used scCUT&Tag to investigate the single-cell chromatin occupancy of transcription factor OLIG2 and the cohesin complex component RAD21. Our results indicate that analysis of histone modifications and transcription factor occupancy at single-cell resolution provides unique insights into epigenomic landscapes in the central nervous system.

Crossing boundaries: Interplay between the immune system and oligodendrocyte lineage cells.

Oligodendrocytes and their progenitors are glial cells in the central nervous system, which have been mainly implicated with the homeostatic roles of axonal myelin ensheathment but serve as targets of the peripheral immune system attack in the context of diseases like multiple sclerosis. This view of oligodendroglia as passive bystanders with no immunological properties was first challenged in the 1980s when it was reported that the cytokine interferon γ could induce the gene expression of the major histocompatibility complexes (MHC) class I and II. While the physiological role of this induction was controversial for decades to follow, recent studies suggest that oligodendroglia survey their environment, respond to a larger array of cues and can indeed exert immunomodulatory functions, which are particularly relevant in the context of neurodegeneration and demyelinating diseases. The alternative functionality of oligodendroglia not only regulates immune cell responses, but also hinders remyelination, and might thereby be key to understanding MS disease pathology and promoting regeneration after immune-mediated demyelination.

Distinct oligodendrocyte populations have spatial preference and different responses to spinal cord injury.

Mature oligodendrocytes (MOLs) show transcriptional heterogeneity, the functional consequences of which are unclear. MOL heterogeneity might correlate with the local environment or their interactions with different neuron types. Here, we show that distinct MOL populations have spatial preference in the mammalian central nervous system (CNS). We found that MOL type 2 (MOL2) is enriched in the spinal cord when compared to the brain, while MOL types 5 and 6 (MOL5/6) increase their contribution to the OL lineage with age in all analyzed regions. MOL2 and MOL5/6 also have distinct spatial preference in the spinal cord regions where motor and sensory tracts run. OL progenitor cells (OPCs) are not specified into distinct MOL populations during development, excluding a major contribution of OPC intrinsic mechanisms determining MOL heterogeneity. In disease, MOL2 and MOL5/6 present different susceptibility during the chronic phase following traumatic spinal cord injury. Our results demonstrate that the distinct MOL populations have different spatial preference and different responses to disease.

Thyroid Disease Spectrum in the Emergency Department.

We present two cases of thyroid hormone alterations revealing clinical emergencies that require early diagnosis and prompt treatment. The first patient, a 56-year-old woman, presented in the emergency room with psychomotor agitation, disorientation and headache. She was very agitated, incapable of standing still, looked very thin, feverish, tachycardic and presented no alteration at neurological examination with negative meningeal signs. Analyses revealed a severe hyperthyroidism. She initiated propylthiouracil 100 mg 8/8 h. After six months, thyroid function was normal. The second patient, a 54-year-old woman, was transferred from the Psychiatry Department due to memory and behavior changes for the past two weeks. She presented visual and auditive hallucinations and inadequate daily behavior. Analyses revealed a severe hypothyroidism. She was medicated with levothyroxine 100 ug/day. At the third month, she presented normalized thyroid function, normal thyroid ultrasound and an increased antithyroperoxidase antibody.