Astrocytic Fate Research Articles

The Role of GLP-1 Affects Fate of Astrocytes Following Ischemic Stroke in Mice

Stroke is one of the leading causes of physical and intellectual disability and mortality in adults of most of the developed countries. The most of studies in ischemic stroke focus on neuronal pathological mechanisms and neuronal death trying to rescue neurons via stopping the detrimental pathological processes or enhancing neuronal viability. Although astrocytes are the most abundant cells and in mammal central nervous system (CNS), they were ignored for period of time but are return to spotlight in recent years. Astrocyte play multiple roles in maintain normal function of central nervous system and respond to all kinds of CNS injury and disease. Responses of astrocytes to CNS damage lead to both detrimental and beneficial consequences. Previous studies have reported that astrocyte inflammatory responses to ischemic stroke may devastate the ischemic lesion, but other astrocytic responses lead to anti-excitotoxicity, secreting neurotrophic factors, neurogenesis, synaptogenesis, axonal remodeling, and angiogenesis promoting neuroprotective effects. Glucagon-like peptide-1 (GLP-1) have been intensively studied on beneficial effects of neurons, but not many studies work on effects of GLP-1 to astrocytes. Our early study has found that activation of GLP-1 receptors prevented ischemia-induced brain cell death and observed newly neuronal progenitor cells were from astrocyte-lineage in penumbral area of 3-day post-ischemic mouse brains by immunohistochemistry (IHC). RT-PCR, immunoblotting, and immunofluorescent results also clearly indicated the astrocytes expressed GLP-1 receptors. Furthermore, Western blotting results showed administration of GLP-1 analogue, exendin-4 (EX-4), suppressed expression of pro-inflammatory proteins in oxygen-glucose deprivation (OGD) treated mouse primary astrocytes. Interestingly, we also observed that infarct site obtained sever DNA damage and less GFAP positive astrocytes, on contrary, the penumbral area had more GFAP positive astrocytes and less DNA damage in IHC images of stroke brains. The results implied that A1 and A2 astrocytes play certain roles in ischemic brains and recovery. The goal of this study is to determine the mechanisms of GLP-1 involving in astrocytic fate decision and approach on minimizing detrimental effects and amplifying the beneficial effects, which possibly develop innovative therapies for ischemic stroke. The study is supported by 108-2320-B-182A-005-MY3 and 112-2320-B-182A-003- from Taiwan National Science and Technology Council. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heimdall, an alternative protein issued from a ncRNA related to kappa light chain variable region of immunoglobulins from astrocytes: a new player in neural proteome

The dogma “One gene, one protein” is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcript. Alternative open reading frames for individual transcripts give proteins originate from the 5′- and 3′-UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12 h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as “NOTCH1, EPHA3, IPO13” as well as membrane receptor protein including “CHRNA9; TGFBR, EPHB6, and TRAM”. However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.

Interfacing reduced graphene oxide with an adipose-derived extracellular matrix as a regulating milieu for neural tissue engineering.

Enthralling evidence of the potential of graphene-based materials for neural tissue engineering is motivating the development of scaffolds using various structures related to graphene such as graphene oxide (GO) or its reduced form. Here, we investigated a strategy based on reduced graphene oxide (rGO) combined with a decellularized extracellular matrix from adipose tissue (adECM), which is still unexplored for neural repair and regeneration. Scaffolds containing up to 50wt% rGO relative to adECM were prepared by thermally induced phase separation assisted by carbodiimide (EDC) crosslinking. Using partially reduced GO enables fine-tuning of the structural interaction between rGO and adECM. As the concentration of rGO increased, non-covalent bonding gradually prevailed over EDC-induced covalent conjugation with the adECM. Edge-to-edge aggregation of rGO favours adECM to act as a biomolecular physical crosslinker to rGO, leading to the softening of the scaffolds. The unique biochemistry of adECM allows neural stem cells to adhere and grow. Importantly, high rGO concentrations directly control cell fate by inducing the differentiation of both NE-4C cells and embryonic neural progenitor cells into neurons. Furthermore, primary astrocyte fate is also modulated as increasing rGO boosts the expression of reactivity markers while unaltering the expression of scar-forming ones.

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

Excitotoxic glutamate levels drive spinal cord ependymal stem cell proliferation and fate specification through CP-AMPAR signaling.

The adult spinal cord contains a population of ependymal-derived neural stem/progenitor cells (epNSPCs) that are normally quiescent, but are activated to proliferate, differentiate, and migrate after spinal cord injury. The mechanisms that regulate their response to injury cues, however, remain unknown. Here, we demonstrate that excitotoxic levels of glutamate promote the proliferation and astrocytic fate specification of adult spinal cord epNSPCs. We show that glutamate-mediated calcium influx through calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (CP-AMPARs) in concert with Notch signaling increases the proliferation of epNSPCs via pCREB, and induces astrocytic differentiation through Hes1 upregulation. Furthermore, the invivo targeting of this pathway via positive modulation of AMPARs after spinal cord injury enhances epNSPC proliferation, astrogliogenesis, neurotrophic factor production and increases neuronal survival. Our study uncovers an important mechanism by which CP-AMPARs regulate the growth and phenotype of epNSPCs, which can be targeted therapeutically to harness the regenerative potential of these cells after injury.

Injury primes mutation-bearing astrocytes for dedifferentiation in later life

Despite their latent neurogenic potential, most normal parenchymal astrocytes fail to dedifferentiate to neural stem cells in response to injury. In contrast, aberrant lineage plasticity is a hallmark of gliomas, and this suggests that tumor suppressors may constrain astrocyte dedifferentiation. Here, we show that p53, one of the most commonly inactivated tumor suppressors in glioma, is a gatekeeper of astrocyte fate. In the context of stab-wound injury, p53 loss destabilized the identity of astrocytes, priming them to dedifferentiate in later life. This resulted from persistent and age-exacerbated neuroinflammation at the injury site and EGFR activation in periwound astrocytes. Mechanistically, dedifferentiation was driven by the synergistic upregulation of mTOR signaling downstream of p53 loss and EGFR, which reinstates stemness programs via increased translation of neurodevelopmental transcription factors. Thus, our findings suggest that first-hit mutations remove the barriers to injury-induced dedifferentiation by sensitizing somatic cells to inflammatory signals, with implications for tumorigenesis.

Профили астроцитарной дифференцировки переднего мозга человека на внутриутробном этапе онтогенеза

Introduction. Brain functioning is kept by both neuronal cell activity and macroglia, i.e., astrocytes and oligodendrocytes. The current data on the prenatal development of the human brain are scarce, and gliogenesis is less studied than cortical neurogenesis. Normal limits and variations and spatiotemporal patterns of glial differentiation in human brain development remain poorly studied. Materials and methods. We used human fetal autopsy samples from the Collection of the Laboratory of Nervous System Development of Avtsyn Research Institute of Human Morphology. For immune and morphological analysis, samples of 38 fetal cerebral hemispheres at stages from 8 postconceptional weeks to birth were chosen. Results. We provided the results of the pilot comparative immune and morphological study with the panel of markers (GFAP, ALDH1L1, FABP-7) of the fetal human telencephalon in prenatal ontogenesis. Specific differentiation and maturation of the astrocyte population on the telencephalon start before early fetal period (12–13 gestational weeks). GFAP+ and ALDH1L1+ astrocyte populations in early human telencephalon are still to be studied for their homology. Analysis of GFAP+ and ALDH1L1+ glioblast distribution proposes dorsal proliferative zone as a source for fibrous cortical astrocytes. Comparative immune and morphological analysis of FABP-7+ neuroblasts in the fetal telencephalon questions whether FABP-7 cells belong to astrocyte population at early prenatal human ontogenesis. Conclusion. In the telencephalon, temporal and/or spatiotemporal translational profiles of these three antigens differ, which indicates that the astrocyte population is heterogeneous in early ontogenesis. Keywords: human brain development, telencephalon, glial differentiation, astrocytes, astrocyte fate lineage, GFAP, ALDH1L1, FABP-7

Glial progenitor cells of the adult human white and grey matter are contextually distinct.

Genomic analyses have revealed heterogeneity among glial progenitor cells (GPCs), but the compartment selectivity of human GPCs (hGPCs) is unclear. Here, we asked if GPCs of human grey and white brain matter are distinct in their architecture and associated gene expression. RNA profiling of NG2-defined hGPCs derived from adult human neocortex and white matter differed in their expression of genes involved in Wnt, NOTCH, BMP and TGFβ signaling, suggesting compartment-selective biases in fate and self-renewal. White matter hGPCs over-expressed the BMP antagonists BAMBI and CHRDL1, suggesting their tonic suppression of astrocytic fate relative to cortical hGPCs, whose relative enrichment of cytoskeletal genes presaged their greater morphological complexity. In human glial chimeric mice, cortical hGPCs assumed larger and more complex morphologies than white matter hGPCs, and both were more complex than their mouse counterparts. These findings suggest that human grey and white matter GPCs comprise context-specific pools with distinct functional biases.

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.

Reliable generation of glial enriched progenitors from human fibroblast-derived iPSCs

White matter stroke (WMS) occurs as small infarcts in deep penetrating blood vessels in the brain and affects the regions of the brain that carry connections, termed the subcortical white matter. WMS progresses over years and has devastating clinical consequences. Unlike large grey matter strokes, WMS disrupts the axonal architecture of the brain and depletes astrocytes, oligodendrocyte lineage cells, axons and myelinating cells, resulting in abnormalities of gait and executive function. An astrocytic cell-based therapy is positioned as a strong therapeutic candidate after WMS. In this study we report, the reliable generation of a novel stem cell-based therapeutic product, glial enriched progenitors (GEPs) derived from human induced pluripotent stem cells (hiPSCs). By transient treatment of hiPSC derived neural progenitors (hiPSC-NPCs) with the small molecule deferoxamine, a prolyl hydroxylase inhibitor, for three days hiPSC-NPCs become permanently biased towards an astrocytic fate, producing hiPSC-GEPs. In preparation for clinical application, we have developed qualification assays to ensure identity, safety, purity, and viability of the cells prior to manufacture. Using tailored q-RT-PCR-based assays, we have demonstrated the lack of pluripotency in our final therapeutic candidate cells (hiPSC-GEPs) and we have identified the unique genetic profile of hiPSC-GEPs that is clearly distinct from the parent lines, hiPSCs and iPSC-NPCs. After completion of the viability assay, we have stablished the therapeutic window of use for hiPSC-GEPs in future clinical applications (7 h). Lastly, we were able to reliably and consistently produce a safe therapeutic final product negative for contamination by any human or murine viral pathogens, selected bacteria, common laboratory mycoplasmas, growth of any aerobes, anaerobes, yeast, or fungi and 100 times less endotoxin levels than the maximum acceptable value. This study demonstrates the reliable and safe generation of patient derived hiPSC-GEPs that are clinically ready as a cell-based therapeutic approach for WMS.

Reliable Generation of Glial Enriched Progenitors from Human Fibroblast-Derived iPSCs

White matter stroke (WMS) occurs as small infarcts in deep penetrating blood vessels in the brain and affects the regions of the brain that carry connections, termed the subcortical white matter. WMS progresses over years and has devastating clinical consequences. Unlike large grey matter strokes, WMS disrupts the axonal architecture of the brain and depletes astrocytes, oligodendrocyte lineage cells, axons and myelinating cells, resulting in abnormalities of gait and executive function. An astrocytic cell-based therapy is positioned as a strong therapeutic candidate after WMS. In this study we report, the reliable generation of a novel stem cell-based therapeutic product, glial enriched progenitors (GEPs) derived from human induced pluripotent stem cells (hiPSCs). By transient treatment of hiPSC derived neural progenitors (hiPSC-NPCs) with the small molecule deferoxamine, a prolyl hydroxylase inhibitor, for three days hiPSC-NPCs become permanently biased towards an astrocytic fate, producing hiPSC-GEPs. In preparation for clinical application, we have developed qualification assays to ensure identity, safety, purity, and viability of the cells prior to manufacture. Using tailored q-RT-PCR-based assays, we have demonstrated the lack of pluripotency in our final therapeutic candidate cells (hiPSC-GEPs) and we have identified the unique genetic profile of hiPSC-GEPs that is clearly distinct from the parent lines, hiPSCs and iPSC-NPCs. After completion of the viability assay, we have stablished the therapeutic window of use for hiPSC-GEPs in future clinical applications (7 hours). Lastly, we were able to reliably and consistently produce a safe therapeutic final product negative for contamination by any human or murine viral pathogens, selected bacteria, common laboratory mycoplasmas, growth of any aerobes, anaerobes, yeast, or fungi and 100 times less endotoxin levels than the maximum acceptable value. This study demonstrates the reliable and safe generation of patient derived hiPSC-GEPs that are clinically ready as a cell-based therapeutic approach for WMS.

Serglycin activates pro-tumorigenic signaling and controls glioblastoma cell stemness, differentiation and invasive potential

Despite the functional role of serglycin as an intracellular proteoglycan, a variety of malignant cells depends on its expression and constitutive secretion to advance their aggressive behavior. Serglycin arose to be a biomarker for glioblastoma, which is the deadliest and most treatment-resistant form of brain tumor, but its role in this disease is not fully elucidated. In our study we suppressed the endogenous levels of serglycin in LN-18 glioblastoma cells to decipher its involvement in their malignant phenotype. Serglycin suppressed LN-18 (LN-18shSRGN) glioblastoma cells underwent astrocytic differentiation characterized by induced expression of GFAP, SPARCL-1 and SNAIL, with simultaneous loss of their stemness capacity. In particular, LN-18shSRGN cells presented decreased expression of glioma stem cell-related genes and ALDH1 activity, accompanied by reduced colony formation ability. Moreover, the suppression of serglycin in LN-18shSRGN cells retarded the proliferative and migratory rate, the invasive potential in vitro and the tumor burden in vivo. The lack of serglycin in LN-18shSRGN cells was followed by G2 arrest, with subsequent reduction of the expression of cell-cycle regulators. LN-18shSRGN cells also exhibited impaired expression and activity of proteolytic enzymes such as MMPs, TIMPs and uPA, both in vitro and in vivo. Moreover, suppression of serglycin in LN-18shSRGN cells eliminated the activation of pro-tumorigenic signal transduction. Of note, LN-18shSRGN cells displayed lower expression and secretion levels of IL-6, IL-8 and CXCR-2. Concomitant, serglycin suppressed LN-18shSRGN cells demonstrated repressed phosphorylation of ERK1/2, p38, SRC and STAT-3, which together with PI3K/AKT and IL-8/CXCR-2 signaling control LN-18 glioblastoma cell aggressiveness. Collectively, the absence of serglycin favors an astrocytic fate switch and a less aggressive phenotype, characterized by loss of pluripotency, block of the cell cycle, reduced ability for ECM proteolysis and pro-tumorigenic signaling attenuation.

Development of glial restricted human neural stem cells for oligodendrocyte differentiation in vitro and in vivo

In this study, we have developed highly expandable neural stem cells (NSCs) from HESCs and iPSCs that artificially express the oligodendrocyte (OL) specific transcription factor gene Zfp488. This is enough to restrict them to an exclusive oligodendrocyte progenitor cell (OPC) fate during differentiation in vitro and in vivo. During CNS development, Zfp488 is induced during the early stages of OL generation, and then again during terminal differentiation of OLs. Interestingly, the human ortholog Znf488, crucial for OL development in human, has been recently identified to function as a dorsoventral pattering regulator in the ventral spinal cord for the generation of P1, P2/pMN, and P2 neural progenitor domains. Forced expression of Zfp488 gene in human NSCs led to the robust generation of OLs and suppression of neuronal and astrocyte fate in vitro and in vivo. Zfp488 expressing NSC derived oligodendrocytes are functional and can myelinate rat dorsal root ganglion neurons in vitro, and form myelin in Shiverer mice brain in vivo. After transplantation near a site of demyelination, Zfp488 expressing hNSCs migrated to the lesion and differentiated into premyelinating OLs. A certain fraction also homed in the subventricular zone (SVZ). Zfp488-ZsGreen1-hNSC derived OLs formed compact myelin in Shiverer mice brain seen under the electron microscope. Transplanted human neural stem cells (NSC) that have the potential to differentiate into functional oligodendrocytes in response to remyelinating signals can be a powerful therapeutic intervention for disorders where oligodendrocyte (OL) replacement is beneficial.

Notch signaling in astrocytes mediates their morphological response to an inflammatory challenge

In the nervous system, Notch pathway has a prominent role in the control of neuronal morphology and in the determination of the astrocyte fate. However, the role of Notch in morphological astrocyte plasticity is unknown. Here, we have explored the role of Notch activity on the morphological reactivity of primary astrocytes in response to LPS, an inflammatory stimulus. We found that LPS induces reactive astrocyte morphology by the inhibition of Notch signaling via NFκB activation and Jagged upregulation. In contrast, IGF-1, an anti-inflammatory molecule, inhibits LPS-induced reactive astrocyte morphological phenotype by enhancing Notch signaling through the inhibition of NFκB and the activation of MAPK. Therefore, Notch signaling pathway emerges as a mediator of the regulation of astrocyte morphology by inflammatory and anti-inflammatory stimuli.

Stage-Specific Transcription Factors Drive Astrogliogenesis by Remodeling Gene Regulatory Landscapes

SummaryA broad molecular framework of how neural stem cells are specified toward astrocyte fate during brain development has proven elusive. Here we perform comprehensive and integrated transcriptomic and epigenomic analyses to delineate gene regulatory programs that drive the developmental trajectory from mouse embryonic stem cells to astrocytes. We report molecularly distinct phases of astrogliogenesis that exhibit stage- and lineage-specific transcriptomic and epigenetic signatures with unique primed and active chromatin regions, thereby revealing regulatory elements and transcriptional programs underlying astrocyte generation and maturation. By searching for transcription factors that function at these elements, we identified NFIA and ATF3 as drivers of astrocyte differentiation from neural precursor cells while RUNX2 promotes astrocyte maturation. These transcription factors facilitate stage-specific gene expression programs by switching the chromatin state of their target regulatory elements from primed to active. Altogether, these findings provide integrated insights into the genetic and epigenetic mechanisms steering the trajectory of astrogliogenesis.

Snail regulates BMP and TGF\u03b2 pathways to control the differentiation status of glioma-initiating cells

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor β (TGFβ) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFβ1 signaling activity. Exogenous TGFβ1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFβ pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.

Age-Dependent Decline in Fate Switch from NG2 Cells to Astrocytes After Olig2 Deletion.

NG2 cells are a resident glial progenitor cell population that is uniformly distributed throughout the developing and mature mammalian CNS. Those in the postnatal CNS generate exclusively myelinating and non-myelinating oligodendrocytes and are thus equated with oligodendrocyte precursor cells. Prenatally, NG2 cells in the ventral gray matter of the forebrain generate protoplasmic astrocytes as well as oligodendrocytes. The fate conversion from NG2 cells into protoplasmic astrocytes is dependent on downregulation of the key oligodendrocyte transcription factor Olig2. We showed previously that constitutive deletion of Olig2 in NG2 cells converts NG2 cells in the neocortex into protoplasmic astrocytes at the expense of oligodendrocytes. In this study, we show that postnatal deletion of Olig2 caused NG2 cells in the neocortex but not in other gray matter regions to become protoplasmic astrocytes. However, NG2 cells in the neocortex became more resistant to astrocyte fate switch over the first 3 postnatal weeks. Fewer NG2 cells differentiated into astrocytes and did so with longer latency after Olig2 deletion at postnatal day 18 (P18) compared with deletion at P2. The high-mobility group transcription factor Sox10 was not downregulated for at least 1 month after Olig2 deletion at P18 despite an early transient upregulation of the astrocyte transcription factor NFIA. Furthermore, inhibiting cell proliferation in slice culture reduced astrocyte differentiation from Olig2-deleted perinatal NG2 cells, suggesting that cell division might facilitate nuclear reorganization needed for astrocyte transformation.SIGNIFICANCE STATEMENT NG2 cells are glial progenitor cells that retain a certain degree of lineage plasticity. In the normal postnatal neocortex, they generate mostly oligodendrocyte lineage cells. When the oligodendrocyte transcription factor Olig2 is deleted in NG2 cells in the neocortex, they switch their fate to protoplasmic astrocytes. However, the efficiency of the fate switch decreases with age over the first 3 postnatal weeks and is reduced when cell proliferation is inhibited. As the neocortex matures, sustained expression of the oligodendrocyte lineage-specific key transcription factor Sox10 becomes less dependent on Olig2. Together, our findings suggest a gradual stabilization of the oligodendrocyte lineage genes and loss of lineage plasticity during the first 3 weeks after birth, possibly due to nuclear reorganization.

Fibrinogen Activates BMP Signaling in Oligodendrocyte Progenitor Cells and Inhibits Remyelination after Vascular Damage

Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate invitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination invivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure.

Interaction of reactive astrocytes with type I collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury.

Central nervous system (CNS) injury transforms naive astrocytes into reactive astrocytes, which eventually become scar-forming astrocytes that can impair axonal regeneration and functional recovery. This sequential phenotypic change, known as reactive astrogliosis, has long been considered unidirectional and irreversible. However, we report here that reactive astrocytes isolated from injured spinal cord reverted in retrograde to naive astrocytes when transplanted into a naive spinal cord, whereas they formed astrocytic scars when transplanted into injured spinal cord, indicating the environment-dependent plasticity of reactive astrogliosis. We also found that type I collagen was highly expressed in the spinal cord during the scar-forming phase and induced astrocytic scar formation via the integrin-N-cadherin pathway. In a mouse model of spinal cord injury, pharmacological blockade of reactive astrocyte-type I collagen interaction prevented astrocytic scar formation, thereby leading to improved axonal regrowth and better functional outcomes. Our findings reveal environmental cues regulating astrocytic fate decisions, thereby providing a potential therapeutic target for CNS injury.

Nogo receptor blockade overcomes remyelination failure after white matter stroke and stimulates functional recovery in aged mice

White matter stroke is a distinct stroke subtype, accounting for up to 25% of stroke and constituting the second leading cause of dementia. The biology of possible tissue repair after white matter stroke has not been determined. In a mouse stroke model, white matter ischemia causes focal damage and adjacent areas of axonal myelin disruption and gliosis. In these areas of only partial damage, local white matter progenitors respond to injury, as oligodendrocyte progenitors (OPCs) proliferate. However, OPCs fail to mature into oligodendrocytes (OLs) even in regions of demyelination with intact axons and instead divert into an astrocytic fate. Local axonal sprouting occurs, producing an increase in unmyelinated fibers in the corpus callosum. The OPC maturation block after white matter stroke is in part mediated via Nogo receptor 1 (NgR1) signaling. In both aged and young adult mice, stroke induces NgR1 ligands and down-regulates NgR1 inhibitors during the peak OPC maturation block. Nogo ligands are also induced adjacent to human white matter stroke in humans. A Nogo signaling blockade with an NgR1 antagonist administered after stroke reduces the OPC astrocytic transformation and improves poststroke oligodendrogenesis in mice. Notably, increased white matter repair in aged mice is translated into significant poststroke motor recovery, even when NgR1 blockade is provided during the chronic time points of injury. These data provide a perspective on the role of NgR1 ligand function in OPC fate in the context of a specific and common type of stroke and show that it is amenable to systemic intervention to promote recovery.