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

In vitro heterogeneity of human oligodendroglia

Oligodendrocytes in the central nervous system (CNS) wrap their plasma membrane extensions around large calibre neuronal axons formulating the multi-layered myelin sheath. Myelin is essential since it allows fast conduction of action potentials and it also provides metabolic support to the environmentally isolated neuronal axon. Even though oligodendrocytes were considered to be a relatively homogeneous population, they demonstrate morphological heterogeneity and behavioural differences, associated with their developmental source and region of presence as well as the expression of distinct molecular markers depending on their differentiation stage. Recent transcriptomics studies have also shown oligodendrocyte heterogeneity both in mouse and in human, with each sub-population of myelinating oligodendrocytes potentially associated with a different specific oligodendrocyte function. For example, some oligodendrocytes might be better at providing metabolic support for axons, while others might be better at myelination, thus displaying a promising remyelinating capacity. Furthermore, numerous studies have shown differences between rodent and human oligodendroglia, with the possibility that remyelination may also be mediated by mature surviving oligodendrocytes in humans, while remyelination in animal models is mostly mediated by newly generated oligodendrocytes, although surviving oligodendrocytes also contribute. Hence, it is important to study human oligodendrocyte biology in health and disease, especially in the context of demyelinating disorders like multiple sclerosis (MS), where animal models do not accurately recapitulate all the aspects of the disease simultaneously (inflammation, neurodegeneration, age). Here, I used the recently developed single-cell RNA sequencing (scRNAseq) technology to phenotype human embryonic stem cell (hES)-derived oligodendrocytes exposed to three different culture/model systems using multiple gene transcripts as a readout of their function : a) a monolayer (2D) oligodendroglia model, where oligodendrocytes express myelin associated proteins but are unable to form myelin sheaths, b) a cortical brain organoid (3D) model where oligodendrocytes express myelin associated proteins and form some myelin sheaths around axons and c) a chimeric model of transplantation of hES-derived monolayer oligodendroglia into immunocompromised Shiverer mice, where they can extensively myelinate the corpus callosum (cc). Transcriptional analysis of scRNAseq data at three different timepoints showed that hES-derived oligodendroglia grown in a monolayer were a heterogeneous population which can be divided into distinct clusters of oligodendroglia. Gene expression analysis demonstrated the presence of three different oligodendrocyte fine clusters in addition to oligodendrocyte progenitor cells (OPCs), oligodendrocyte and astrocyte progenitor cells (OAPC), primitive oligodendrocyte progenitors (pri_OPC), cycling progenitors (CyP) and committed oligodendrocyte progenitor cells (COP). Canonical correlation analysis (CCA) to map hES-derived oligodendroglia transcriptional data onto human adult post-mortem oligodendroglia single-nuclei RNA sequencing (snRNAseq) data (previously generated in the lab) showed that hES-derived monolayer oligodendroglia are immature when compared to their adult counterparts and in fact resemble foetal oligodendroglia, as suggested by integration analysis with our 20weeks old human foetal snRNAseq data. Transcriptional analysis of scRNAseq data at four different timepoints acquired from cortical brain organoids revealed limited heterogeneity with similar oligodendroglia populations. Integration analysis revealed three distinct populations of oligodendrocytes, in addition to OPCs, COPs, OAPCs and pri_OPCs, similarly to hES-derived monolayer oligodendroglia. The classical developmental differentiation from OPCs to the most mature oligodendroglia was seen and there was increased transcriptional similarity with human adult post-mortem oligodendroglia compared to hES-derived monolayer oligodendrocytes, although still remaining quite immature. Transcriptional analysis of scRNAseq data from hES-derived oligodendroglia transplanted into immunocompromised Shiverer mice isolated at 10 weeks from the cc of chimeric mice demonstrated heterogeneity by the presence of oligodendrocyte populations with distinct transcriptional expression profiles. Four different immature and mature oligodendrocyte populations were identified among OPCs, COPs and CyPs. Transcriptional comparison of hES-derived chimeric oligodendroglia with human adult post-mortem oligodendroglia exhibited increased overlap between the two populations both in the progenitor and mature cells, suggesting that these oligodendroglia achieved increased levels of maturation. This transcriptional heterogeneity was also validated using in situ hybridization (ISH) and immunofluorescence (IF) for unique or enriched RNA markers of individual clusters within the oligodendrocyte lineage, as identified in a post-mortem human brain dataset by Jäkel & Agirre et al. Thus, the chimeric model can more accurately recapitulate adult human oligodendroglia and this may help understand human oligodendrocyte function in myelination and remyelination. To test this, I used two repurposed drugs thought to enhance myelination and/or remyelination, either directly (clemastine) or indirectly by adult OPC rejuvenation (metformin), to assess their effectiveness in promoting hES-derived oligodendroglia differentiation and myelination as well as examine whether they could generate a shift towards a specific oligodendroglia population, for example one that has a transcriptomic signature of better myelinating/remyelinating capacity. Metformin, a licenced drug for type II diabetes mellitus (T2DM), was able to increase oligodendrocyte differentiation in all three hES-derived oligodendroglial systems. Electron microscopy (EM) analysis also demonstrated a significant increase in the percentage of compact myelinated axons in the metformin-treated chimeric model. Additionally, transcriptional analysis of treated and control chimeras revealed oligodendroglia clusters selectively present in the treated animals, while differential gene expression analysis suggested a drug mechanism of action through mitochondrial function and mostly through mitochondrial respiratory chain (MRC) Complex I. Clemastine, a widely used antihistamine, was also able to significantly increase mature oligodendrocytes in all hES-derived models, as well as increase the percentage of axons with compact myelin in hES-derived chimeras as demonstrated by IF and EM analysis respectively. However, transcriptional analysis of treated and control animals did not reveal any shifts in oligodendroglial populations, suggesting that clemastine may act on oligodendroglia post-transcriptionally. Overall, these results demonstrate that hES-derived oligodendroglia are a heterogeneous population which can accurately recapitulate adult human oligodendroglia in models with increasing environmental complexity and that metformin can increase hES-derived oligodendroglia differentiation and shift them towards a state associated with increased myelination, possibly through increased mitochondrial energy supply mediated by MRC Complex I gene upregulation

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

Abstract 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