Contrasting somatic mutation patterns in aging human neurons and oligodendrocytes.

Characterizing somatic mutations in the brain is important for disentangling the complex mechanisms of aging, yet little is known about mutational patterns in different brain cell types. Here, we performed whole-genome sequencing (WGS) of 86 single oligodendrocytes, 20 mixed glia, and 56 single neurons from neurotypical individuals spanning 0.4-104 years of age and identified >92,000 somatic single-nucleotide variants (sSNVs) and small insertions/deletions (indels). Although both cell types accumulate somatic mutations linearly with age, oligodendrocytes accumulated sSNVs 81% faster than neurons and indels 28% slower than neurons. Correlation of mutations with single-nucleus RNA profiles and chromatin accessibility from the same brains revealed that oligodendrocyte mutations are enriched in inactive genomic regions and are distributed across the genome similarly to mutations in brain cancers. In contrast, neuronal mutations are enriched in open, transcriptionally active chromatin. These stark differences suggest an assortment of active mutagenic processes in oligodendrocytes and neurons.

Mild respiratory COVID can cause multi-lineage neural cell and myelin dysregulation.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.

Meningeal lymphatic function promotes oligodendrocyte survival and brain myelination.

The precise neurophysiological changes prompted by meningeal lymphatic dysfunction remain unclear. Here, we showed that inducing meningeal lymphatic vessel ablation in adult mice led to gene expression changes in glial cells, followed by reductions in mature oligodendrocyte numbers and specific lipid species in the brain. These phenomena were accompanied by altered meningeal adaptive immunity and brain myeloid cell activation. During brain remyelination, meningeal lymphatic dysfunction provoked a state of immunosuppression that contributed to delayed spontaneous oligodendrocyte replenishment and axonal loss. The deficiencies in mature oligodendrocytes and neuroinflammation due to impaired meningeal lymphatic function were solely recapitulated in immunocompetent mice. Patients diagnosed with multiple sclerosis presented reduced vascular endothelial growth factor C in the cerebrospinal fluid, particularly shortly after clinical relapses, possibly indicative of poor meningeal lymphatic function. These data demonstrate that meningeal lymphatics regulate oligodendrocyte function and brain myelination, which might have implications for human demyelinating diseases.

Treg cell-derived osteopontin promotes microglia-mediated white matter repair after ischemic stroke.

The precise mechanisms underlying the beneficial effects of regulatory T (Treg) cells on long-term tissue repair remain elusive. Here, using single-cell RNA sequencing and flow cytometry, we found that Treg cells infiltrated the brain 1 to 5 weeks after experimental stroke in mice. Selective depletion of Treg cells diminished oligodendrogenesis, white matter repair, and functional recovery after stroke. Transcriptomic analyses revealed potent immunomodulatory effects of brain-infiltrating Treg cells on other immune cells, including monocyte-lineage cells. Microglia depletion, but not T cell lymphopenia, mitigated the beneficial effects of transferred Treg cells on white matter regeneration. Mechanistically, Treg cell-derived osteopontin acted through integrin receptors on microglia to enhance microglial reparative activity, consequently promoting oligodendrogenesis and white matter repair. Increasing Treg cell numbers by delivering IL-2:IL-2 antibody complexes after stroke improved white matter integrity and rescued neurological functions over the long term. These findings reveal Treg cells as a neurorestorative target for stroke recovery.

Disease-associated oligodendroglia: a putative nexus in neurodegeneration.

Neural cells in our central nervous system (CNS) have long been thought to be mere targets of neuroinflammatory events in neurodegenerative diseases such as multiple sclerosis (MS) or Alzheimer's disease. While glial populations such as microglia and astrocytes emerged as active responders and modifiers of pathological environments, oligodendroglia and neurons have been associated with altered homeostasis and eventual cell death. The advent of single-cell and spatial omics technologies has demonstrated transitions of CNS-resident glia, including oligodendroglia, into disease-associated (DA) states. Anchored in recent findings of their roles in MS, we propose that DA glia constitute key nexus of disease progression, with DA oligodendroglia contributing to the modulation of neuroinflammation in certain neurodegenerative diseases, constituting novel putative pharmacological targets for such pathologies.

SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination.

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

Post-injury born oligodendrocytes incorporate into the glial scar and contribute to the inhibition of axon regeneration.

Failure of central nervous system projection neurons to spontaneously regenerate long-distance axons underlies irreversibility of white matter pathologies. A barrier to axonal regenerative research is that the axons regenerating in response to experimental treatments stall growth before reaching post-synaptic targets. Here, we test the hypothesis that the interaction of regenerating axons with live oligodendrocytes, which were absent during developmental axon growth, contributes to stalling axonal growth. To test this hypothesis, first, we used single cell RNA-seq (scRNA-seq) and immunohistology to investigate whether post-injury born oligodendrocytes incorporate into the glial scar after optic nerve injury. Then, we administered demyelination-inducing cuprizone and stimulated axon regeneration by Pten knockdown (KD) after optic nerve crush. We found that post-injury born oligodendrocyte lineage cells incorporate into the glial scar, where they are susceptible to the demyelination diet, which reduced their presence in the glial scar. We further found that the demyelination diet enhanced Pten KD-stimulated axon regeneration and that localized cuprizone injection promoted axon regeneration. We also present a resource for comparing the gene expression of scRNA-seq-profiled normal and injured optic nerve oligodendrocyte lineage cells.

Glial Control of Cortical Neuronal Circuit Maturation and Plasticity.

The brain is a highly adaptable organ that is molded by experience throughout life. Although the field of neuroscience has historically focused on intrinsic neuronal mechanisms of plasticity, there is growing evidence that multiple glial populations regulate the timing and extent of neuronal plasticity, particularly over the course of development. This review highlights recent discoveries on the role of glial cells in the establishment of cortical circuits and the regulation of experience-dependent neuronal plasticity during critical periods of neurodevelopment. These studies provide strong evidence that neuronal circuit maturation and plasticity are non-cell autonomous processes that require both glial-neuronal and glial-glial cross talk to proceed. We conclude by discussing open questions that will continue to guide research in this nascent field.

Extracellular Vesicle-Mediated Neuron-Glia Communications in the Central Nervous System.

Communication between neurons and glia significantly influences the development maturation, plasticity, and disease progressions of the nervous system. As a new signaling modality, extracellular vesicles display a diverse role for robust functional regulation of neurons through their protein and nucleic acid cargoes. This review highlights recent breakthroughs in the research of signaling mechanisms between glia and neurons mediated by extracellular vesicles that are important for neural development, axonal maintenance, synaptic functions, and disease progression in the mammalian nervous system. We will discuss the biological roles of extracellular vesicles released from neurons, astroglia, microglia, and oligodendroglia in the nervous system and their implications in neurodegenerative disorders.

Distinct chemical structures inhibit the CEMIP hyaluronidase and promote oligodendrocyte progenitor cell maturation.

Growing evidence supports pathogenic roles for chronically elevated hyaluronidase activity in numerous conditions. Elevated expression of one such hyaluronidase, the Cell Migration Inducing and hyaluronan binding Protein (CEMIP), has been implicated in the pathogenesis and progression of several cancers as well as demyelinating diseases in the central nervous system (CNS). Developing effective and selective CEMIP inhibitors could therefore have efficacy in treating a variety of conditions where CEMIP is chronically elevated. Using two distinct screens for novel hyaluronidase inhibitors, we identified two synthetic thiocarbamates and one plant-derived flavonoid, sulfuretin, that effectively blocked CEMIP activity in live cells, including a tumorigenic cell line and primary cultures of oligodendrocyte progenitor cells (OPCs). None of these agents influenced cell proliferation, but they had differential dose-dependent and cell type specific effects on cell survival. Furthermore, we found that each of these agents could promote oligodendrocyte maturation by OPCs in the presence of high molecular weight (>2 Mda) hyaluronan, the accumulation of which is linked to the inhibition of OPC maturation and remyelination failure in demyelinating diseases. These findings indicate that CEMIP can be inhibited through distinct chemical interactions, and that CEMIP inhibitors have potential efficacy for treating demyelinating diseases or other conditions where CEMIP is elevated.

Microglia in brain development and regeneration.

It has recently emerged that microglia, the tissue-resident macrophages of the central nervous system, play significant non-innate immune roles to support the development, maintenance, homeostasis and repair of the brain. Apart from being highly specialized brain phagocytes, microglia modulate the development and functions of neurons and glial cells through both direct and indirect interactions. Thus, recognizing the elements that influence the homeostasis and heterogeneity of microglia in normal brain development is crucial to understanding the mechanisms that lead to early disease pathogenesis of neurodevelopmental disorders. In this Review, we discuss recent studies that have elucidated the physiological development of microglia and summarize our knowledge of their non-innate immune functions in brain development and tissue repair.

ALCAM on human oligodendrocytes mediates CD4 T cell adhesion.

Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the CNS. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 Th17 cells can interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we used single cell RNA sequencing, flow cytometry and immunofluorescence studies on CNS tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) in multiple sclerosis, in experimental autoimmune encephalomyelitis and in controls, although their regulation differs between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of MCAM but not of ALCAM at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live imaging, immunofluorescence and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking ALCAM but not MCAM limited prolonged interactions between human primary oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferring significant protection of oligodendrocytic processes. In conclusion, we showed that human oligodendrocytes express MCAM and ALCAM, which are differently modulated by inflammation and T cell contact. We found that ALCAM is a ligand for Th17-polarized cells, contributing to their capacity to adhere and induce damage to human oligodendrocytes, and therefore could represent a relevant target for neuroprotection in multiple sclerosis.

BCAS1-positive oligodendrocytes enable efficient cortical remyelination in multiple sclerosis.

Remyelination is a crucial regenerative process in demyelinating diseases, limiting persisting damage to the central nervous system (CNS). It restores saltatory nerve conduction and ensures trophic support of axons. In multiple sclerosis (MS) patients, remyelination has been observed in both white and grey matter and found to be more efficient in the cortex. Brain-enriched myelin-associated protein 1 (BCAS1) identifies oligodendrocyte lineage cells in the stage of active myelin formation in development and regeneration. Other than in the white matter, BCAS1+ oligodendrocytes are maintained at high densities in the cortex throughout life. Here, we investigated cortical lesions in human biopsy and autopsy tissue from patients with MS in direct comparison to demyelinating mouse models and demonstrate that following a demyelinating insult BCAS1+ oligodendrocytes in remyelinating cortical lesions shift from a quiescent to an activated, internode-forming morphology co-expressing myelin-associated glycoprotein (MAG), necessary for axonal contact formation. Noteworthy, activated BCAS1+ oligodendrocytes are found at early time points of experimental demyelination amidst ongoing inflammation. In human tissue, activated BCAS 1+ oligodendrocytes correlate with the density of myeloid cells, further supporting their involvement in an immediate regenerative response. Furthermore, studying the microscopically normal appearing non demyelinated cortex in patients with chronic MS, we find a shift from quiescent BCAS1+ oligodendrocytes to mature, myelin-maintaining oligodendrocytes, suggesting oligodendrocyte differentiation and limited replenishment of BCAS1+ oligodendrocytes in long-standing disease. We also demonstrate that part of perineuronal satellite oligodendrocytes are BCAS1+ and contribute to remyelination in human and experimental cortical demyelination. In summary, our results provide evidence from human tissue and experimental models that BCAS1+ cells in the adult cortex represent a population of pre-differentiated oligodendrocytes that rapidly react after a demyelinating insult thus enabling immediate myelin regeneration. In addition, our data suggest that limited replenishment of BCAS1+ oligodendrocytes may contribute to the remyelination failure observed in the cortex in chronic MS.

Deficiency of galactosyl-ceramidase in adult oligodendrocytes worsens disease severity during chronic experimental allergic encephalomyelitis.

Galactosyl-ceramidase (GALC) is a ubiquitous lysosomal enzyme crucial for the correct myelination of the mammalian nervous system during early postnatal development. However, the physiological consequence of GALC deficiency in the adult brain remains unknown. In this study, we found that mice with conditional ablation of GALC activity in post-myelinating oligodendrocytes were lethally sensitized when challenged with chronic experimental allergic encephalomyelitis (EAE), in contrast with the non-lethal dysmyelination observed in Galc-ablated mice without the EAE challenge. Mechanistically, we found strong inflammatory demyelination without remyelination and an impaired fusion of lysosomes and autophagosomes with accumulation of myelin debris after a transcription factor EB-dependent increase in the lysosomal autophagosome flux. These results indicate that the physiological impact of GALC deficiency is highly influenced by the cell context (oligodendroglial vs. global expression), the presence of inflammation, and the developmental time when it happens (pre-myelination vs. post-myelination). We conclude that Galc expression in adult oligodendrocytes is crucial for the maintenance of adult central myelin and to decrease vulnerability to additional demyelinating insults.

Perinatal loss of galactosylceramidase in both oligodendrocytes and microglia is crucial for the pathogenesis of Krabbe disease in mice.

Lysosomal galactosylceramidase (GALC) is expressed in all brain cells, including oligodendrocytes (OLs), microglia, and astrocytes, although the cell-specific function of GALC is largely unknown. Mutations in GALC cause Krabbe disease (KD), a fatal neurological lysosomal disorder that usually affects infants. To study how Galc ablation in each glial cell type contributes to Krabbe pathogenesis, we used conditional Galc-floxed mice. Here, we found that OL-specific Galc conditional knockout (CKO) in mice results in a phenotype that includes wasting, psychosine accumulation, and neuroinflammation. Microglia- or astrocyte-specific Galc deletion alone in mice did not show specific phenotypes. Interestingly, mice with CKO of Galc from both OLs and microglia have a more severe neuroinflammation with an increase in globoid cell accumulation than OL-specific CKO alone. Moreover, the enhanced phenotype occurred without additional accumulation of psychosine. Further studies revealed that Galc knockout (Galc-KO) microglia cocultured with Galc-KO OLs elicits globoid cell formation and the overexpression of osteopontin and monocyte chemoattractant protein-1, both proteins that are known to recruit immune cells and promote engulfment of debris and damaged cells. We conclude that OLs are the primary cells that initiate KD with an elevated psychosine level and microglia are required for the progression of neuroinflammation in a psychosine-independent manner.

Functional myelin in cognition and neurodevelopmental disorders.

In vertebrates, oligodendrocytes (OLs) are glial cells of the central nervous system (CNS) responsible for the formation of the myelin sheath that surrounds the axons of neurons. The myelin sheath plays a crucial role in the transmission of neuronal information by promoting the rapid saltatory conduction of action potentials and providing neurons with structural and metabolic support. Saltatory conduction, first described in the peripheral nervous system (PNS), is now generally recognized as a universal evolutionary innovation to respond quickly to the environment: myelin helps us think and act fast. Nevertheless, the role of myelin in the central nervous system, especially in the brain, may not be primarily focused on accelerating conduction speed but rather on ensuring precision. Its principal function could be to coordinate various neuronal networks, promoting their synchronization through oscillations (or rhythms) relevant for specific information processing tasks. Interestingly, myelin has been directly involved in different types of cognitive processes relying on brain oscillations, and myelin plasticity is currently considered to be part of the fundamental mechanisms for memory formation and maintenance. However, despite ample evidence showing the involvement of myelin in cognition and neurodevelopmental disorders characterized by cognitive impairments, the link between myelin, brain oscillations, cognition and disease is not yet fully understood. In this review, we aim to highlight what is known and what remains to be explored to understand the role of myelin in high order brain processes.

Versatile and automated workflow for the analysis of oligodendroglial calcium signals.

Although intracellular Ca2+ signals of oligodendroglia, the myelin-forming cells of the central nervous system, regulate vital cellular processes including myelination, few studies on oligodendroglia Ca2+ signal dynamics have been carried out and existing software solutions are not adapted to the analysis of the complex Ca2+ signal characteristics of these cells. Here, we provide a comprehensive solution to analyze oligodendroglia Ca2+ imaging data at the population and single-cell levels. We describe a new analytical pipeline containing two free, open source and cross-platform software programs, Occam and post-prOccam, that enable the fully automated analysis of one- and two-photon Ca2+ imaging datasets from oligodendroglia obtained by either ex vivo or in vivo Ca2+ imaging techniques. Easily configurable, our software solution is optimized to obtain unbiased results from large datasets acquired with different imaging techniques. Compared to other recent software, our solution proved to be fast, low memory demanding and faithful in the analysis of oligodendroglial Ca2+ signals in all tested imaging conditions. Our versatile and accessible Ca2+ imaging data analysis tool will facilitate the elucidation of Ca2+-mediated mechanisms in oligodendroglia. Its configurability should also ensure its suitability with new use cases such as other glial cell types or even cells outside the CNS.

HiPSC-derived 3D neural models reveal neurodevelopmental pathomechanisms of the Cockayne Syndrome B.

Cockayne Syndrome B (CSB) is a hereditary multiorgan syndrome which-through largely unknown mechanisms-can affect the brain where it clinically presents with microcephaly, intellectual disability and demyelination. Using human induced pluripotent stem cell (hiPSC)-derived neural 3D models generated from CSB patient-derived and isogenic control lines, we here provide explanations for these three major neuropathological phenotypes. In our models, CSB deficiency is associated with (i) impaired cellular migration due to defective autophagy as an explanation for clinical microcephaly; (ii) altered neuronal network functionality and neurotransmitter GABA levels, which is suggestive of a disturbed GABA switch that likely impairs brain circuit formation and ultimately causes intellectual disability; and (iii) impaired oligodendrocyte maturation as a possible cause of the demyelination observed in children with CSB. Of note, the impaired migration and oligodendrocyte maturation could both be partially rescued by pharmacological HDAC inhibition.

Parkinson's disease and multiple system atrophy patient iPSC-derived oligodendrocytes exhibit alpha-synuclein-induced changes in maturation and immune reactive properties.

SignificanceOur results demonstrate the existence of early cellular pathways and network alterations in oligodendrocytes in the alpha-synucleinopathies Parkinson's disease and multiple system atrophy. They further reveal the involvement of an immune component triggered by alpha-synuclein protein, as well as a connection between (epi)genetic changes and immune reactivity in multiple system atrophy. The knowledge generated in this study could be used to devise novel therapeutic approaches to treat synucleinopathies.