Biology subjects

Castelo-Branco, G.

Publications and source records attributed to Castelo-Branco, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Fatty acid binding protein-8 (FABP8/PMP2) reveals molecular heterogeneity of myelin sheaths in the human CNS

Myelin is classically viewed as a uniform axon-insulating membrane, yet its molecular composition may differ between species and even within one species. Fatty acid binding protein-8 (FABP8/PMP2) was previously identified in CNS myelin of humans but not mice. Here we show that FABP8/PMP2 is a defining feature of CNS myelin in humans and old-world-monkeys, but absent from CNS myelin in other mammals, indicating evolutionary neofunctionalization of this lipid-binding protein in the primate lineage. In the human CNS, FABP8/PMP2 marks a subset of myelin sheaths that preferentially ensheath large-diameter axons, revealing sheath-to-sheath molecular heterogeneity correlated with axonal geometry. Chromatin is accessible at the PMP2/Pmp2 gene locus in oligodendrocytes of humans but not mice. Human oligodendrocytes intrinsically express FABP8/PMP2 when transplanted into mouse brains, demonstrating species-specific competence independent of environmental cues. Humanized transgenic mice expressing FABP8/PMP2 in oligodendrocytes form morphologically normal but developmentally transiently thicker myelin sheaths, and show elevated cholesterol content in purified myelin. Because FABP8/PMP2 binds cholesterol, we propose that its emergence in primate CNS myelin contributes to the cholesterol enrichment of human myelin. Thus, CNS myelin protein composition is evolvable and modular, with relevance for myelin lipids and morphology, and previously unrecognized complexity in neuron-glia co-adaptation. Main points- Fatty acid binding protein 8 (FABP8/PMP2) is present in CNS myelin of humans and old-world monkeys - PMP2 defines sheath-to-sheath heterogeneity in the human CNS - PMP2-immunopositive myelin ensheaths large-diameter axons - Human oligodendrocytes intrinsically express PMP2 upon transplantation into mice - Humanized PMP2-transgenic mice show thicker myelin and altered myelin lipid composition

neuroscience↗

bioRxiv2026

KaroSpace: a rapid-access framework for interactive exploration of multi-sample spatial omics data

Spatial omics technologies enable high-resolution mapping of molecular and cellular organization within tissues, yet interactive exploration of these data remains challenging due to computational bottlenecks, and reliance on proprietary software infrastructures. We present KaroSpace, a framework for cell-centric exploration of multi-sample and multi-modality spatial omics data, agnostic to upstream analysis pipelines and operating on preprocessed spatial features. By combining a lightweight design with flexible deployment options, KaroSpace allows immediate interactive spatial data exploration, straightforward implementation to the broader scientific community, supports transparent data sharing, and complements existing computational workflows for hypothesis generation and collaborative analysis.

neuroscience↗

bioRxiv2026

Neuronal synapses are stabilised by activity-dependent vesicular release from oligodendrocyte precursor cells

Activity-dependent regulation of synapses has long been attributed to interactions between neurons alone. Here, using in vivo imaging in the zebrafish visual system we show that one type of glia called the oligodendrocyte precursor cell (OPC) closely interacts with pre-synapses of retinal ganglion cell (RGC) axons, and exhibits activity-dependent Vamp2/3-mediated vesicular release. Critically, blocking OPC vesicle release using botulinum toxin, and silencing OPC activity integration through dampening calcium signalling, destabilises RGC pre-synapses. These observations suggest that OPCs provide a non-neuronal pathway for activity-dependent feedback to shape circuit wiring.

neuroscience↗

bioRxiv2025

Anoctamin-2-specific T Cells Link Epstein-Barr Virus to Multiple Sclerosis

Multiple sclerosis (MS) occurs when the central nervous system (CNS) is damaged by misguided adaptive immune responses, likely caused by a combination of environmental factors in genetically susceptible individuals. A known prerequisite for disease is Epstein-Barr virus (EBV) infection, and previous studies have demonstrated elevated Epstein-Barr virus nuclear antigen 1 (EBNA1) antibodies which cross-react with the calcium-activated chloride channel anoctamin-2 (ANO2) in persons with MS (pwMS). ANO2-reactive antibodies have been associated with greater neuroaxonal damage in MS, although their exact effector function is still uncertain. Here, we demonstrate that ANO2 is also the target of IFN{gamma}-producing CD4+ T cells, which are more frequent in untreated and natalizumab-treated pwMS compared to control individuals. Immunisation of SJL/J mice with either ANO2 or EBNA1 elicited cross-reactive CD4+ T cell and antibody responses in vivo. Pre-immunisation of young mice with ANO2 worsened proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), which in older mice included atypical clinical phenotype, immune infiltration into the brain and reduced survival. EAE exacerbation was recapitulated with the adoptive co-transfer of ANO2 and myelin antigen-specific CD4+ T cells, and ANO2-specific T cells alone could induce the cell death of ANO2-expressing glial cells in vitro. T cell clones with cross-reactivity to both EBNA1 and ANO2 antigens could be isolated from natalizumab-treated pwMS. Single cell sequencing of EBNA1 and ANO2-specific T cell receptors (TCR) from four pwMS revealed a significant overlap between their antigen-specific expanded TCR repertoires within donors and transcriptomic analysis showed cross-reactive T cells to have predominantly activated and cytotoxic phenotypes. In summary, we report the first mechanistic evidence that EBNA1 CD4+ T cells can target the MS-associated autoantigen ANO2, thereby establishing a link between EBV infection and development of autoimmune neuroinflammatory disease.

immunology↗

bioRxiv2025

Oligodendroglia as functional effectors of Multiple Sclerosis risk variants

Multiple sclerosis (MS) is a neuroinflammatory disease for which a large number of non-coding single nucleotide polymorphisms (SNPs) have been associated with disease risk/susceptibility. Immune cells have been suggested as the principal functional effector cell types of these common variants. Here, we identify 76 MS-associated SNPs whose loci present accessible chromatin in homeostatic and diseased oligodendroglia (OLG), including both oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (MOLs). By applying high-throughput functional genomics, we found that a subset of these SNPs led to variant- and cell-specific regulatory effects in human induced pluripotent stem cell-derived oligodendroglia. Phenotypic profiling of these variants indicated that rs483180:PHGDH interfered with human OPC proliferation via long-range chromatin interactions with the S100A6 locus, while variants at rs2248137:CYP24A1 impaired oligodendrocyte differentiation by regulating BCAS1 expression. In addition, variants at rs1415069:DIPK1A enhanced secretion of the cytokine CCL2 by oligodendroglia, suggesting that these variants might be implicated in OLG-driven immune cell recruitment in MS. These findings position oligodendroglia as important drivers of MS pathogenesis through modulation of both their cell-intrinsic oligodendroglial function and intercellular communication by non-coding MS risk variants.

cell biology↗

bioRxiv2025

Dual role of GABAB receptor in oligodendrocyte function and immune modulation in experimental multiple sclerosis

GABAB receptors (GABABR) mediate the actions of the inhibitory neurotransmitter GABA in the central nervous system, regulating key processes such as synaptic activity, interneuron communication and excitation-inhibition balance in the brain. Recent studies using the GABABR agonist baclofen have revealed a critical role for these receptors in promoting oligodendroglial differentiation in both health and disease, highlighting their potential as therapeutic targets in demyelinating diseases such as multiple sclerosis (MS). In this study, we identify a dual role for oligodendroglial GABABR in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Conditional deletion of the GABAB1 subunit in NG2+ cells ameliorates acute disease symptoms while inducing an immune-like phenotype in oligodendrocytes. This immunomodulatory role is further supported by pharmacological activation of GABABR in oligodendrocytes, which reduces the expression of MHC class II in these cells. Notably, baclofen treatment after EAE symptom onset attenuates the course of the disease while enhancing oligodendrocyte progenitor cell differentiation and suppressing T cell infiltration into demyelinating lesions. Moreover, prophylactic baclofen administration delays disease onset and further decreases immune cell recruitment into the spinal cord, underscoring its potent immunomodulatory effect. These data demonstrate that GABABR signaling exerts context-dependent effects on both oligodendrocyte lineage progression and neuroinflammatory responses. Importantly, these compelling findings validate baclofen, a drug already approved for MS-associated spasticity, as a promising candidate for therapies targeting both inflammation and remyelination, advancing our understanding of glial-immune interactions in demyelinating diseases and supporting the translational potential of GABABR modulation in MS.

neuroscience↗

bioRxiv2025

Multi-omics Profiling of the Lateral Ventricle Choroid Plexus Reveals Developmental Cellular Remodeling, Early Immune Gene Activation, and a Novel Epithelial Subtype

Healthy brain development and function highly depend on the choroid plexus. Temporal alterations in the cellular landscape and gene expression of choroid plexus cells can alter immune cell trafficking in the brain and cerebrospinal fluid composition, ultimately impacting brain dynamics. Here, we performed a comprehensive multi-omics analysis--including bulk and single-cell transcriptomics and epigenomics--of the lateral ventricle choroid plexus across early postnatal and adult stages in mice and rats. We uncovered striking changes in the choroid plexus cellular composition from neonatal to adult stages, accompanied by transcriptional remodeling of all main cell types. Immune cells were markedly increased in adulthood and immune cell profiling revealed an altered cell-type diversity through time. Surprisingly, we observed an early gene activation of host-defense genes in all choroid plexus main cell types, beginning in the neonatal period and progressively increasing into young adulthood. Moreover, some genes induced in epithelial cells in response to inflammation were found to be epigenetically primed, despite not being transcriptionally active. Epithelial cells exhibited subtype diversity and plasticity, with distinct gene expression programs and chromatin accessibility profiles emerging over time. Notably, we identified a novel epithelial cell subtype with unique gene markers suggesting a specialized function potentially linked to neuro-signaling. Ligand-receptor interaction analysis revealed a progressive remodeling of cellular crosstalk networks during choroid plexus maturation, suggesting dynamic intercellular signaling as the tissue develops. Our study offers a comprehensive atlas of transcriptional activity and chromatin accessibility in choroid plexus cells, providing a valuable resource to guide future efforts in targeting gene expression at the choroid plexus for therapeutical purposes.

neuroscience↗

bioRxiv2025

G-quadruplex profiling in complex tissues using single-cell CUT&Tag

G-quadruplexes (G4) are non-canonical DNA structures that gained increasing attention for their potential roles in gene regulation, with implications in neurodegenerative diseases and cancer. Despite their biological significance, G4 structures have not been studied systematically across tissues and cell types. In this study, we employ G4 single-cell CUT&Tag (G4 scCUT&Tag) to characterize G4 landscapes in postnatal mouse brain cells, leveraging single-cell analytical approaches commonly used in scRNA-Seq and scATAC-Seq datasets. Using conventional single-cell omics workflows to process and explore our data, we distinguished different cell lineages based on G4 heterogeneity and established that a subset of lineage-specific genes show unique promoter G4s. Multi-omics integration with scRNA-Seq gene expression profiles, using both a covariance-based technique (canonical correlation analysis) and a transfer learning-based approach, enabled a more detailed annotation of cell types. These integrations not only revealed significant correlation of G4 and gene expression signals, but demonstrated that G4 scCUT&Tag enables detailed examination of G4 heterogeneity in complex tissues and supports integrative analysis of G4 profiles with other omics layers, offering new insights into the epigenomic landscapes of the developing brain.

developmental biology↗

bioRxiv2024

TAF1-dependent transcriptional dysregulation underlies multiple sclerosis

A major conceptual and clinical challenge in multiple sclerosis (MS) is understanding the mechanisms that drive the central nervous system (CNS)-resident neuroinflammation and neurodegeneration underneath disease progression. Genome-wide association studies (GWAS) have implicated RNA polymerase II (RNAPII) promoter-proximal pausing in oligodendrocyte pathology, but the causal mechanisms remain unclear. Here we find that the C-terminal region of TAF1, a core component of the general transcription factor TFIID, is underdetected in progressive MS brains, which can be explained by endoproteolysis due to extralysosomal cathepsin B (CTSB). Mice lacking the C-terminal TAF1 domain (Taf1d38) exhibit MS-like brain transcriptomic signature, alongside CNS-resident inflammation, progressive demyelination, and motor disability. Mechanistically, C-terminal TAF1 interacts with MS-linked factors that cooperate to regulate RNAPII pausing, particularly affecting oligodendroglial myelination genes. These findings uncover a previously unrecognized transcriptional mechanism underlying MS progression and establish a tractable in vivo model for therapeutic development.

neuroscience↗

bioRxiv2024

Spatial dynamics of mammalian brain development and neuroinflammation by multimodal tri-omics mapping

The ability to spatially map multiple layers of the omics information over different time points allows for exploring the mechanisms driving brain development, differentiation, arealization, and alterations in disease. Herein we developed and applied spatial tri-omic sequencing technologies, DBiT ARP-seq (spatial ATAC-RNA-Protein-seq) and DBiT CTRP-seq (spatial CUT&Tag- RNA-Protein-seq) together with multiplexed immunofluorescence imaging (CODEX) to map spatial dynamic remodeling in brain development and neuroinflammation. A spatiotemporal tri-omic atlas of the mouse brain was obtained at different stages from postnatal day P0 to P21, and compared to the regions of interest in the human developing brains. Specifically, in the cortical area, we discovered temporal persistence and spatial spreading of chromatin accessibility for the layer-defining transcription factors. In corpus callosum, we observed dynamic chromatin priming of myelin genes across the subregions. Together, it suggests a role for layer specific projection neurons to coordinate axonogenesis and myelination. We further mapped the brain of a lysolecithin (LPC) neuroinflammation mouse model and observed common molecular programs in development and neuroinflammation. Microglia, exhibiting both conserved and distinct programs for inflammation and resolution, are transiently activated not only at the core of the LPC lesion, but also at distal locations presumably through neuronal circuitry. Thus, this work unveiled common and differential mechanisms in brain development and neuroinflammation, resulting in a valuable data resource to investigate brain development, function and disease.

neuroscience↗

bioRxiv2024

Single-nuclei histone modification profiling of the adult human central nervous system unveils epigenetic memory of developmental programs

The adult human central nervous system (CNS) is remarkably complex, with neural cells displaying extensive transcriptional heterogeneity. However, how different layers of epigenetic regulation underpin this heterogeneity is poorly understood. Here, we profile the adult human CNS from distinct regions, for chromatin accessibility at the single-nuclei level. In addition, we simultaneously co-profiled the histone modifications H3K27me3 and H3K27ac at the single nuclei-level, providing their first map in all major human CNS cell types. We unveil primed chromatin signatures at HOX loci in spinal cord-derived human oligodendroglia (OLG) but not microglia. These signatures were reminiscent of developmental OLG but were decoupled from robust gene expression. Moreover, using high-resolution Micro-C, we show that induced pluripotent stem cell (iPS) derived human OLGs exhibit a HOX chromatin architecture compatible with the primed chromatin in adult OLGs, and bears a strong resemblance not only to OLG developmental architecture, but also high-grade pontine gliomas. Thus, adult OLG retain epigenetic memory from developmental states, which might enable them to promptly transcribe Hox genes, in contexts of regeneration, but also make them susceptible to gliomagenesis.

neuroscience↗

bioRxiv2024

Large-scale characterization of cell niches in spatial atlases using bio-inspired graph learning

Spatial omics allow us to identify and analyze communities of cells coordinating specific functions within a tissue. While these communities, defined as cell niches, are fundamentally shaped by interactions between spatially neighboring cells, we lack computational frameworks that can leverage spatial omics data to quantitatively characterize niches based on cell interaction events. To address this, we introduce NicheCompass, a graph deep learning method designed based on the principles of cellular communication. NicheCompass not only identifies cell niches, but also learns and informs about the signaling events shaping the identity of these niches. Unlike existing methods, it uniquely characterizes niches by quantifying their activity of spatial gene programs which represent diverse mechanisms of cell-cell communication and transcriptional regulation, thereby uncovering the underlying cellular processes constituting each niche. We showcase a comprehensive workflow encompassing data integration, niche identification, and functional interpretation, and demonstrate that, with its biologically informed design, NicheCompass outperforms existing methods. NicheCompass is broadly applicable to spatial transcriptomics data, which we illustrate by mapping the architecture of diverse tissues during mouse embryonic development, and delineating basal (KRT14) and luminal (KRT8) tumor niches in human breast cancer. We further introduce fine-tuning-based spatial reference mapping, revealing an SPP1+ macrophage-dominated tumor niche in non-small cell lung cancer patients. Additionally, we extend NicheCompass to multimodal spatial profiling of gene expression and chromatin accessibility, identifying and characterizing distinct white matter niches in the mouse brain. Finally, we apply NicheCompass to a whole mouse brain spatial atlas with 8.4 million cells demonstrating its scalability and ability to build foundational, interpretable spatial representations for entire organs. Overall, NicheCompass provides a novel approach to the challenge of identifying and analyzing niches, and suggests a more rigorous niche definition grounded in the quantitative characterization of underlying cellular processes.

bioinformatics↗

bioRxiv2023

Distinct transcriptomic and epigenomic responses of mature oligodendrocytes during disease progression in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a chronic demyelinating autoimmune disease that targets mature oligodendrocytes (MOLs) and their myelin. MOLs are transcriptionally heterogeneous and can transition to immune-like states in the context of MS. However, the intricacies of their dynamics throughout disease progression remain poorly understood. Here, we employed simultaneous single-cell multiome ATAC and RNA sequencing targeting oligodendroglia (OLGs) from the experimental autoimmune encephalomyelitis (EAE) MS mouse model at different stages of the disease course. We found that the transition to immune OLG states appear already at the early stages of EAE and persist to the late stages of the disease. Interestingly, transcription factor activity suggested immunosuppression in MOLs at early stages of EAE and we also observed a transitory activation of a regenerative program in MOLs at this stage. Importantly, different MOLs exhibit a differential responsiveness to EAE, with MOL2 exhibiting a stronger transcriptional immune response than MOL5/6. Moreover, we observed divergent responses at the epigenetic level of MOL2 and MOL5/6 during disease evolution. Thus, our single-cell multiomic resource highlights dynamic and distinct responses of OLG subpopulations to the evolving environment in EAE, which might modulate their response to regenerative therapeutic interventions in MS.

neuroscience↗

bioRxiv2023

Oligodendrocytes and neurons contribute to amyloid-β deposition in Alzheimer's disease

In Alzheimers disease (AD), amyloid-{beta} (A{beta}) is thought to be of neuronal origin. However, in single-cell RNAseq datasets from mouse and human, we found transcripts of amyloid precursor protein (APP) and the amyloidogenic-processing machinery equally abundant in oligodendrocytes (OLs). By cell-type-specific deletion of Bace1 in a humanized knock-in AD model, APPNLGF, we demonstrate that almost a third of cortical A{beta} deposited in plaques is derived from OLs. However, excitatory projection neurons must provide a threshold level of A{beta} production for plaque deposition to occur and for oligodendroglial A{beta} to co-aggregate. Indeed, very few plaques are deposited in the absence of neuronally-derived A{beta}, although soluble A{beta} species are readily detected, especially in subcortical white matter. Our data identify OLs as a source of A{beta} in vivo and further underscore a non-linear relationship between cellular A{beta} production and resulting plaque formation. Ultimately, our observations are relevant for therapeutic strategies aimed at disease prevention in AD.

neuroscience↗

bioRxiv2023

Single cell-resolution in situ sequencing elucidates spatial dynamics of multiple sclerosis lesion and disease evolution

Multiple sclerosis (MS) is a neurological disease characterised by multifocal lesions and smouldering pathology. While single-cell analyses have provided insights into neuropathology, cellular processes underlying MS remain poorly understood. We modelled the cellular dynamics of MS by examining temporal and regional rates of disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model. By performing single-cell spatial expression profiling using In situ sequencing, we annotated disease neighbourhoods during lesion evolution and found centrifugal propagation of active lesions. We demonstrated that disease-associated (DA) glia are dynamic and induced independently of lesions, preceding their formation. Single-cell spatial analysis of human archival MS spinal cord confirmed differential distribution of DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identification of 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 disease evolution.

cell biology↗

bioRxiv2022

Single nuclei RNAseq stratifies multiple sclerosis patients into three distinct white matter glia responses

The lack of understanding of the cellular and molecular basis of clinical and genetic heterogeneity in progressive multiple sclerosis (MS) has hindered the search for new effective therapies. Here, to address this gap, we analysed 632,000 single nuclei RNAseq profiles of 156 brain tissue samples, comprising white matter (WM) lesions, normal appearing WM, grey matter (GM) lesions and normal appearing GM from 54 MS patients and 26 controls. We observed the expected changes in overall neuronal and glial numbers previously described within the classical lesion subtypes. We found highly cell type-specific gene expression changes in MS tissue, with distinct differences between GM and WM areas, confirming different pathologies. However, surprisingly, we did not observe distinct gene expression signatures for the classical different WM lesion types, rather a continuum of change. This indicates that classical lesion characterization better reflects changes in cell abundance than changes in cell type gene expression, and indicates a global disease effect. Furthermore, the major biological determinants of variability in gene expression in MS WM samples relate to individual patient effects, rather than to lesion types or other metadata. We identify four subgroups of MS patients with distinct WM glial gene expression signatures and patterns of oligodendrocyte stress and/or maturation, suggestive of engagement of different pathological processes, with an additional more variable regenerative astrocyte signature. The discovery of these patterns, which were also found in an independent MS patient cohort, provides a framework to use molecular biomarkers to stratify patients for optimal therapeutic approaches for progressive MS, significantly advances our mechanistic understanding of progressive MS, and highlights the need for precision-medicine approaches to address heterogeneity among MS patients.

neuroscience↗

bioRxiv2022

Marked regional glial heterogeneity in the human white matter of the central nervous system

The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are commonly affected in neurodegenerative diseases. However, these often uniquely human diseases differentially affect white matter regions, at various ages and between males and females, and we hypothesised that this is secondary to physiological variation in white matter glia with region, age and sex. Using single nucleus RNA sequencing of healthy human post-mortem samples, we find marked glial heterogeneity with tissue region (primary motor cortex, cerebellum, cervical spinal cord), with tissue-specific cell populations of oligodendrocyte precursor cells and astrocytes, and a spinal cord-enriched oligodendrocyte type that appears human-specific. Spinal cord microglia but not astrocytes show a more activated phenotype compared to brain. These regional effects, with additional differentially expressed genes with age and sex in all glial lineages, help explain pathological patterns of disease - essential knowledge for therapeutic strategies.

neuroscience↗

bioRxiv2022

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

Probing epigenomic marks such as histone modifications at a single cell level in thousands of cells has been recently enabled by technologies such as scCUT&Tag. Here we developed a multimodal and optimized iteration of scCUT&Tag called nano-CT (for nano-CUT&Tag) that allows simultaneous probing of three epigenomic modalities at single-cell resolution, using nanobody-Tn5 fusion proteins. nano-CT is compatible with starting materials as low as 25 000 cells and has significantly higher resolution than scCUT&Tag, with a 16-fold increase in the number of fragments per cells. We used nano-CT to simultaneously profile chromatin accessibility, H3K27ac and H3K27me3 in a complex tissue - juvenile mouse brain. The obtained multimodal dataset allowed for discrimination of more cell types/states that scCUT&Tag, and inference of chromatin velocity between ATAC and H3K27ac in the oligodendrocyte (OL) lineage. In addition, we used nano-CT to deconvolute H3K27me3 repressive states and infer two sequential waves of H3K27me3 repression at distinct gene modules during OL lineage progression. Thus, given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in different biological systems at single cell level.

molecular biology↗