Divergent transcriptional regulation of astrocyte reactivity across disorders.

Astrocytes respond to injury and disease in the central nervous system with reactive changes that influence the outcome of the disorder1-4. These changes include differentially expressed genes (DEGs) whose contextual diversity and regulation are poorly understood. Here we combined biological and informatic analyses, including RNA sequencing, protein detection, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and conditional gene deletion, to predict transcriptional regulators that differentially control more than 12,000 DEGs that are potentially associated with astrocyte reactivity across diverse central nervous system disorders in mice and humans. DEGs associated with astrocyte reactivity exhibited pronounced heterogeneity across disorders. Transcriptional regulators also exhibited disorder-specific differences, but a core group of 61 transcriptional regulators was identified as common across multiple disorders in both species. We show experimentally that DEG diversity is determined by combinatorial, context-specific interactions between transcriptional regulators. Notably, the same reactivity transcriptional regulators can regulate markedly different DEG cohorts in different disorders; changes in the access of transcriptional regulators to DNA-binding motifs differ markedly across disorders; and DEG changes can crucially require multiple reactivity transcriptional regulators. We show that, by modulating reactivity, transcriptional regulators can substantially alter disorder outcome, implicating them as therapeutic targets. We provide searchable resources of disorder-related reactive astrocyte DEGs and their predicted transcriptional regulators. Our findings show that transcriptional changes associated with astrocyte reactivity are highly heterogeneous and are customized from vast numbers of potential DEGs through context-specific combinatorial transcriptional-regulator interactions.

Loss-of-function manipulations to identify roles of diverse glia and stromal cells during CNS scar formation.

Scar formation is the replacement of parenchymal cells by stromal cells and fibrotic extracellular matrix. Until as recently as 25 years ago, little was known about the major functional contributions of different neural and non-neural cell types in the formation of scar tissue and tissue fibrosis in the CNS. Concepts about CNS scar formation are evolving rapidly with the availability of different types of loss-of-function technologies that allow mechanistic probing of cellular and molecular functions in models of CNS disorders in vivo. Such loss-of-function studies are beginning to reveal that scar formation and tissue fibrosis in the CNS involves complex interactions amongst multiple types of CNS glia and non-neural stromal cells. For example, attenuating functions of the CNS resident glial cells, astrocytes or microglia, can disrupt the formation of limitans borders that form around stromal cell scars, which leads to increased spread of inflammation, increased loss of neural tissue, and increased fibrosis. Insights are being gained into specific neuropathological mechanisms whereby specific dysfunctions of different types of CNS glia could cause or contribute to disorder-related tissue pathology and dysfunction. CNS glia, as well as fibrosis-producing stromal cells, are emerging as potential major contributors to diverse CNS disorders either through loss- or gain-of-functions, and are thereby emerging as important potential targets for interventions. In this article, we will review and discuss the effects on CNS scar formation and tissue repair of loss-of-function studies targeted at different specific cell types in various disorder models in vivo.

Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.

Simultaneous generation of neural cells and that of the nutrient-supplying vasculature during brain development is called neurovascular coupling. We report on a transgenic mouse with impaired transforming growth factor ß (TGFß)-signalling in forebrain-derived neural cells using a Foxg1-cre knock-in to drive the conditional knock-out of the Tgfbr2. Although the expression of FOXG1 is assigned to neural progenitors and neurons of the telencephalon, Foxg1(cre/+);Tgfbr2(flox/flox) (Tgfbr2-cKO) mutants displayed intracerebral haemorrhage. Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching. Vascular endothelial growth factor (VEGF) A, insulin-like growth factor (IGF) 1, IGF2, TGFß, inhibitor of DNA binding (ID) 1, thrombospondin (THBS) 2, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 1 were altered in either expression levels or tissue distribution. Accordingly, human umbilical vein endothelial cells (HUVEC) displayed branching defects after stimulation with conditioned medium (CM) that was derived from primary neural cultures of the ventral and dorsal telencephalon of Tgfbr2-cKO. Supplementing CM of Tgfbr2-cKO with VEGFA rescued these defects, but application of TGFß aggravated them. HUVEC showed reduced migration towards CM of mutants compared with controls. Supplementing the CM with growth factors VEGFA, fibroblast growth factor (FGF) 2 and IGF1 partially restored HUVEC migration. In contrast, TGFß supplementation further impaired migration of HUVEC. We observed differences along the dorso-ventral axis of the telencephalon with regard to the impact of these factors on the phenotype. Together these data establish a TGFBR2-dependent molecular crosstalk between neural and endothelial cells during brain vessel development. These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimers disease, cerebral amyloid angiopathy or tumour biology.

PI3K-p110-alpha-subtype signalling mediates survival, proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2.

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFß) and insulin-like growth factor 1 (IGF1) have a central role. The TGFß- and IGF1-pathways cross-talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down-stream molecules of TGFß and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5-derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5-derived cells. TGFß has little effect at E13.5. It does not activate the PI3K- and mTOR-signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5-derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K-p110-alpha signalling. PI3K-p110-beta signalling activates mTORC2 in E16.5-derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms. Within developing cortical cells TGFß- and IGF-signalling activities are timely separated. TGFß dominates in E16.5-derived cells and drives neuronal differentiation. IGF influences survival, proliferation and neuronal differentiation in E13.5-derived cells. mTORC2-signalling in E16.5-derived cells influences survival, proliferation and differentiation, activated through PI3K-p110-alpha. PI3K-p110-beta-signalling activates a different mTORC2. Both PI3K/mTORC2-signalling pathways are required but not directly activated in TGFß-mediated neuronal differentiation.

Regulation of cell distancing in peri-plaque glial nets by Plexin-B1 affects glial activation and amyloid compaction in Alzheimer's disease.

Communication between glial cells has a profound impact on the pathophysiology of Alzheimer's disease (AD). We reveal here that reactive astrocytes control cell distancing in peri-plaque glial nets, which restricts microglial access to amyloid deposits. This process is governed by guidance receptor Plexin-B1 (PLXNB1), a network hub gene in individuals with late-onset AD that is upregulated in plaque-associated astrocytes. Plexin-B1 deletion in a mouse AD model led to reduced number of reactive astrocytes and microglia in peri-plaque glial nets, but higher coverage of plaques by glial processes, along with transcriptional changes signifying reduced neuroinflammation. Additionally, a reduced footprint of glial nets was associated with overall lower plaque burden, a shift toward dense-core-type plaques and reduced neuritic dystrophy. Altogether, our study demonstrates that Plexin-B1 regulates peri-plaque glial net activation in AD. Relaxing glial spacing by targeting guidance receptors may present an alternative strategy to increase plaque compaction and reduce neuroinflammation in AD.

Diversified transcriptional responses of myeloid and glial cells in spinal cord injury shaped by HDAC3 activity.

The innate immune response influences neural repair after spinal cord injury (SCI). Here, we combined myeloid-specific transcriptomics and single-cell RNA sequencing to uncover not only a common core but also temporally distinct gene programs in injury-activated microglia and macrophages (IAM). Intriguingly, we detected a wide range of microglial cell states even in healthy spinal cord. Upon injury, IAM progressively acquired overall reparative, yet diversified transcriptional profiles, each comprising four transcriptional subtypes with specialized tasks. Notably, IAM have both distinct and common gene signatures as compared to neurodegeneration-associated microglia, both engaging phagocytosis, autophagy, and TyroBP pathways. We also identified an immediate response microglia subtype serving as a source population for microglial transformation and a proliferative subtype controlled by the epigenetic regulator histone deacetylase 3 (HDAC3). Together, our data unveil diversification of myeloid and glial subtypes in SCI and an extensive influence of HDAC3, which may be exploited to enhance functional recovery.

RNA Profiling of Mouse Ependymal Cells after Spinal Cord Injury Identifies the Oncostatin Pathway as a Potential Key Regulator of Spinal Cord Stem Cell Fate.

Ependymal cells reside in the adult spinal cord and display stem cell properties in vitro. They proliferate after spinal cord injury and produce neurons in lower vertebrates but predominantly astrocytes in mammals. The mechanisms underlying this glial-biased differentiation remain ill-defined. We addressed this issue by generating a molecular resource through RNA profiling of ependymal cells before and after injury. We found that these cells activate STAT3 and ERK/MAPK signaling post injury and downregulate cilia-associated genes and FOXJ1, a central transcription factor in ciliogenesis. Conversely, they upregulate 510 genes, seven of them more than 20-fold, namely Crym, Ecm1, Ifi202b, Nupr1, Rbp1, Thbs2 and Osmr-the receptor for oncostatin, a microglia-specific cytokine which too is strongly upregulated after injury. We studied the regulation and role of Osmr using neurospheres derived from the adult spinal cord. We found that oncostatin induced strong Osmr and p-STAT3 expression in these cells which is associated with reduction of proliferation and promotion of astrocytic versus oligodendrocytic differentiation. Microglial cells are apposed to ependymal cells in vivo and co-culture experiments showed that these cells upregulate Osmr in neurosphere cultures. Collectively, these results support the notion that microglial cells and Osmr/Oncostatin pathway may regulate the astrocytic fate of ependymal cells in spinal cord injury.

Microglia and macrophages promote corralling, wound compaction and recovery after spinal cord injury via Plexin-B2.

Tissue repair after spinal cord injury requires the mobilization of immune and glial cells to form a protective barrier that seals the wound and facilitates debris clearing, inflammatory containment and matrix compaction. This process involves corralling, wherein phagocytic immune cells become confined to the necrotic core, which is surrounded by an astrocytic border. Here we elucidate a temporally distinct gene signature in injury-activated microglia and macrophages (IAMs) that engages axon guidance pathways. Plexin-B2 is upregulated in IAMs and is required for motor sensory recovery after spinal cord injury. Plexin-B2 deletion in myeloid cells impairs corralling, leading to diffuse tissue damage, inflammatory spillover and hampered axon regeneration. Corralling begins early and requires Plexin-B2 in both microglia and macrophages. Mechanistically, Plexin-B2 promotes microglia motility, steers IAMs away from colliding cells and facilitates matrix compaction. Our data therefore establish Plexin-B2 as an important link that integrates biochemical cues and physical interactions of IAMs with the injury microenvironment during wound healing.

Epigenetic Regulation Of Axon Regeneration and Glial Activation in Injury Responses.

Injury to the nervous system triggers a multicellular response in which epigenetic mechanisms play an important role in regulating cell type-specific transcriptional changes. Here, we summarize recent progress in characterizing neuronal intrinsic and extrinsic chromatin reconfigurations and epigenetic changes triggered by axonal injury that shape neuroplasticity and glial functions. We specifically discuss regeneration-associated transcriptional modules comprised of transcription factors and epigenetic regulators that control axon growth competence. We also review epigenetic regulation of neuroinflammation and astroglial responses that impact neural repair. These advances provide a framework for developing epigenetic strategies to maximize adaptive alterations while minimizing maladaptive stress responses in order to enhance axon regeneration and achieve functional recovery after injury.

Author Correction: HDAC3 inhibition ameliorates spinal cord injury by immunomodulation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

HDAC3 inhibition ameliorates spinal cord injury by immunomodulation.

Following spinal cord injury (SCI), the innate immune response of microglia and infiltrating macrophages clears up cellular debris and promotes tissue repair, but it also inflicts secondary injury from inflammatory responses. Immunomodulation aimed at maximizing the beneficial effects while minimizing the detrimental roles of the innate immunity may aid functional recovery after SCI. However, intracellular drivers of global reprogramming of the inflammatory gene networks in the innate immune cells are poorly understood. Here we show that SCI resulted in an upregulation of histone deacetylase 3 (HDAC3) in the innate immune cells at the injury site. Remarkably, blocking HDAC3 with a selective small molecule inhibitor shifted microglia/macrophage responses towards inflammatory suppression, resulting in neuroprotective phenotypes and improved functional recovery in SCI model. Mechanistically, HDAC3 activity is largely responsible for histone deacetylation and inflammatory responses of primary microglia to classic inflammatory stimuli. Our results reveal a novel function of HDAC3 inhibitor in promoting functional recovery after SCI by dampening inflammatory cytokines, thus pointing towards a new direction of immunomodulation for SCI repair.