Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

Single-nucleus expression characterization of non-enhancing region of recurrent high-grade glioma.

Background: Non-enhancing (NE) infiltrating tumor cells beyond the contrast-enhancing (CE) bulk of tumor are potential propagators of recurrence after gross total resection of high-grade glioma. Methods: We leveraged single-nucleus RNA sequencing on 15 specimens from recurrent high-grade gliomas (n = 5) to compare prospectively identified biopsy specimens acquired from CE and NE regions. Additionally, 24 CE and 22 NE biopsies had immunohistochemical staining to validate RNA findings. Results: Tumor cells in NE regions are enriched in neural progenitor cell-like cellular states, while CE regions are enriched in mesenchymal-like states. NE glioma cells have similar proportions of proliferative and putative glioma stem cells relative to CE regions, without significant differences in % Ki-67 staining. Tumor cells in NE regions exhibit upregulation of genes previously associated with lower grade gliomas. Our findings in recurrent GBM paralleled some of the findings in a re-analysis of a dataset from primary GBM. Cell-, gene-, and pathway-level analyses of the tumor microenvironment in the NE region reveal relative downregulation of tumor-mediated neovascularization and cell-mediated immune response, but increased glioma-to-nonpathological cell interactions. Conclusions: This comprehensive analysis illustrates differing tumor and nontumor landscapes of CE and NE regions in high-grade gliomas, highlighting the NE region as an area harboring likely initiators of recurrence in a pro-tumor microenvironment and identifying possible targets for future design of NE-specific adjuvant therapy. These findings also support the aggressive approach to resection of tumor-bearing NE regions.

Dysregulation of extracellular potassium distinguishes healthy ageing from neurodegeneration.

Progressive neuronal loss is a hallmark feature distinguishing neurodegenerative diseases from normal ageing. However, the underlying mechanisms remain unknown. Extracellular K+ homeostasis is a potential mediator of neuronal injury as K+ elevations increase excitatory activity. The dysregulation of extracellular K+ and potassium channel expressions during neurodegeneration could contribute to this distinction. Here we measured the cortical extracellular K+ concentration ([K+]e) in awake wild-type mice as well as murine models of neurodegeneration using K+-sensitive microelectrodes. Unexpectedly, aged wild-type mice exhibited significantly lower cortical [K+]e than young mice. In contrast, cortical [K+]e was consistently elevated in Alzheimer's disease (APP/PS1), amyotrophic lateral sclerosis (ALS) (SOD1G93A) and Huntington's disease (R6/2) models. Cortical resting [K+]e correlated inversely with neuronal density and the [K+]e buffering rate but correlated positively with the predicted neuronal firing rate. Screening of astrocyte-selective genomic datasets revealed a number of potassium channel genes that were downregulated in these disease models but not in normal ageing. In particular, the inwardly rectifying potassium channel Kcnj10 was downregulated in ALS and Huntington's disease models but not in normal ageing, while Fxyd1 and Slc1a3, each of which acts as a negative regulator of potassium uptake, were each upregulated by astrocytes in both Alzheimer's disease and ALS models. Chronic elevation of [K+]e in response to changes in gene expression and the attendant neuronal hyperexcitability may drive the neuronal loss characteristic of these neurodegenerative diseases. These observations suggest that the dysregulation of extracellular K+ homeostasis in a number of neurodegenerative diseases could be due to aberrant astrocytic K+ buffering and as such, highlight a fundamental role for glial dysfunction in neurodegeneration.

Remyelination in the Central Nervous System.

The inability of the mammalian central nervous system (CNS) to undergo spontaneous regeneration has long been regarded as a central tenet of neurobiology. However, while this is largely true of the neuronal elements of the adult mammalian CNS, save for discrete populations of granule neurons, the same is not true of its glial elements. In particular, the loss of oligodendrocytes, which results in demyelination, triggers a spontaneous and often highly efficient regenerative response, remyelination, in which new oligodendrocytes are generated and myelin sheaths are restored to denuded axons. Yet remyelination in humans is not without limitation, and a variety of demyelinating conditions are associated with sustained and disabling myelin loss. In this work, we will (1) review the biology of remyelination, including the cells and signals involved; (2) describe when remyelination occurs and when and why it fails, including the consequences of its failure; and (3) discuss approaches for therapeutically enhancing remyelination in demyelinating diseases of both children and adults, both by stimulating endogenous oligodendrocyte progenitor cells and by transplanting these cells into demyelinated brain.

Lymphatic endothelial-like cells promote glioblastoma stem cell growth through cytokine-driven cholesterol metabolism.

Glioblastoma is the most lethal primary brain tumor with glioblastoma stem cells (GSCs) atop a cellular hierarchy. GSCs often reside in a perivascular niche, where they receive maintenance cues from endothelial cells, but the role of heterogeneous endothelial cell populations remains unresolved. Here, we show that lymphatic endothelial-like cells (LECs), while previously unrecognized in brain parenchyma, are present in glioblastomas and promote growth of CCR7-positive GSCs through CCL21 secretion. Disruption of CCL21-CCR7 paracrine communication between LECs and GSCs inhibited GSC proliferation and growth. LEC-derived CCL21 induced KAT5-mediated acetylation of HMGCS1 on K273 in GSCs to enhance HMGCS1 protein stability. HMGCS1 promoted cholesterol synthesis in GSCs, favorable for tumor growth. Expression of the CCL21-CCR7 axis correlated with KAT5 expression and HMGCS1K273 acetylation in glioblastoma specimens, informing patient outcome. Collectively, glioblastomas contain previously unrecognized LECs that promote the molecular crosstalk between endothelial and tumor cells, offering potentially alternative therapeutic strategies.

Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema.

Cerebral oedema is associated with morbidity and mortality after traumatic brain injury (TBI)1. Noradrenaline levels are increased after TBI2-4, and the amplitude of the increase in noradrenaline predicts both the extent of injury5 and the likelihood of mortality6. Glymphatic impairment is both a feature of and a contributor to brain injury7,8, but its relationship with the injury-associated surge in noradrenaline is unclear. Here we report that acute post-traumatic oedema results from a suppression of glymphatic and lymphatic fluid flow that occurs in response to excessive systemic release of noradrenaline. This post-TBI adrenergic storm was associated with reduced contractility of cervical lymphatic vessels, consistent with diminished return of glymphatic and lymphatic fluid to the systemic circulation. Accordingly, pan-adrenergic receptor inhibition normalized central venous pressure and partly restored glymphatic and cervical lymphatic flow in a mouse model of TBI, and these actions led to substantially reduced brain oedema and improved functional outcomes. Furthermore, post-traumatic inhibition of adrenergic signalling boosted lymphatic export of cellular debris from the traumatic lesion, substantially reducing secondary inflammation and accumulation of phosphorylated tau. These observations suggest that targeting the noradrenergic control of central glymphatic flow may offer a therapeutic approach for treating acute TBI.

CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance.

The naked mole rat (NMR) is the longest-lived rodent, resistant to multiple age-related diseases including neurodegeneration. However, the mechanisms underlying the NMR's resistance to neurodegenerative diseases remain elusive. Here, we isolated oligodendrocyte progenitor cells (OPCs) from NMRs and compared their transcriptome with that of other mammals. Extracellular matrix (ECM) genes best distinguish OPCs of long- and short-lived species. Notably, expression levels of CD44, an ECM-binding protein that has been suggested to contribute to NMR longevity by mediating the effect of hyaluronan (HA), are not only high in OPCs of long-lived species but also positively correlate with longevity in multiple cell types/tissues. We found that CD44 localizes to the endoplasmic reticulum (ER) and enhances basal ATF6 activity. CD44 modifies proteome and membrane properties of the ER and enhances ER stress resistance in a manner dependent on unfolded protein response regulators without the requirement of HA. HA-independent role of CD44 in proteostasis regulation may contribute to mammalian longevity.

Young glial progenitor cells competitively replace aged and diseased human glia in the adult chimeric mouse brain.

Competition among adult brain cells has not been extensively researched. To investigate whether healthy glia can outcompete diseased human glia in the adult forebrain, we engrafted wild-type (WT) human glial progenitor cells (hGPCs) produced from human embryonic stem cells into the striata of adult mice that had been neonatally chimerized with mutant Huntingtin (mHTT)-expressing hGPCs. The WT hGPCs outcompeted and ultimately eliminated their human Huntington's disease (HD) counterparts, repopulating the host striata with healthy glia. Single-cell RNA sequencing revealed that WT hGPCs acquired a YAP1/MYC/E2F-defined dominant competitor phenotype upon interaction with the host HD glia. WT hGPCs also outcompeted older resident isogenic WT cells that had been transplanted neonatally, suggesting that competitive success depended primarily on the relative ages of competing populations, rather than on the presence of mHTT. These data indicate that aged and diseased human glia may be broadly replaced in adult brain by younger healthy hGPCs, suggesting a therapeutic strategy for the replacement of aged and diseased human glia.

Astrocytic engagement of the corticostriatal synaptic cleft is disrupted in a mouse model of Huntington's disease.

Astroglial dysfunction contributes to the pathogenesis of Huntington's disease (HD), and glial replacement can ameliorate the disease course. To establish the topographic relationship of diseased astrocytes to medium spiny neuron (MSN) synapses in HD, we used 2-photon imaging to map the relationship of turboRFP-tagged striatal astrocytes and rabies-traced, EGFP-tagged coupled neuronal pairs in R6/2 HD and wild-type (WT) mice. The tagged, prospectively identified corticostriatal synapses were then studied by correlated light electron microscopy followed by serial block-face scanning EM, allowing nanometer-scale assessment of synaptic structure in 3D. By this means, we compared the astrocytic engagement of single striatal synapses in HD and WT brains. R6/2 HD astrocytes exhibited constricted domains, with significantly less coverage of mature dendritic spines than WT astrocytes, despite enhanced engagement of immature, thin spines. These data suggest that disease-dependent changes in the astroglial engagement and sequestration of MSN synapses enable the high synaptic and extrasynaptic levels of glutamate and K+ that underlie striatal hyperexcitability in HD. As such, these data suggest that astrocytic structural pathology may causally contribute to the synaptic dysfunction and disease phenotype of those neurodegenerative disorders characterized by network overexcitation.

Astrocytes as master modulators of neural networks: Synaptic functions and disease-associated dysfunction of astrocytes.

Astrocytes are the most abundant glial cell type in the central nervous system and are essential to the development, plasticity, and maintenance of neural circuits. Astrocytes are heterogeneous, with their diversity rooted in developmental programs modulated by the local brain environment. Astrocytes play integral roles in regulating and coordinating neural activity extending far beyond their metabolic support of neurons and other brain cell phenotypes. Both gray and white matter astrocytes occupy critical functional niches capable of modulating brain physiology on time scales slower than synaptic activity but faster than those adaptive responses requiring a structural change or adaptive myelination. Given their many associations and functional roles, it is not surprising that astrocytic dysfunction has been causally implicated in a broad set of neurodegenerative and neuropsychiatric disorders. In this review, we focus on recent discoveries concerning the contributions of astrocytes to the function of neural networks, with a dual focus on the contribution of astrocytes to synaptic development and maturation, and on their role in supporting myelin integrity, and hence conduction and its regulation. We then address the emerging roles of astrocytic dysfunction in disease pathogenesis and on potential strategies for targeting these cells for therapeutic purposes.

Astrocytic contributions to Huntington's disease pathophysiology.

Huntington's disease (HD) is a fatal, monogenic, autosomal dominant neurodegenerative disease caused by a polyglutamine-encoding CAG expansion in the huntingtin (HTT) gene that results in mutant huntingtin proteins (mHTT) in cells throughout the body. Although large parts of the central nervous system (CNS) are affected, the striatum is especially vulnerable and undergoes marked atrophy. Astrocytes are abundant within the striatum and contain mHTT in HD, as well as in mouse models of the disease. We focus on striatal astrocytes and summarize how they participate in, and contribute to, molecular pathophysiology and disease-related phenotypes in HD model mice. Where possible, reference is made to pertinent astrocyte alterations in human HD. Astrocytic dysfunctions related to cellular morphology, extracellular ion and neurotransmitter homeostasis, and metabolic support all accompany the development and progression of HD, in both transgenic mouse and human cellular and chimeric models of HD. These findings reveal the potential for the therapeutic targeting of astrocytes so as to restore synaptic as well as tissue homeostasis in HD. Elucidation of the mechanisms by which astrocytes contribute to HD pathogenesis may inform a broader understanding of the role of glial pathology in neurodegenerative disorders and, by so doing, enable new strategies of glial-directed therapeutics.

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 TGFß 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.

Pathway-based approach reveals differential sensitivity to E2F1 inhibition in glioblastoma.

Analysis of tumor gene expression is an important approach for the classification and identification of therapeutic vulnerabilities. However, targeting glioblastoma (GBM) based on molecular subtyping has not yet translated into successful therapies. Here, we present an integrative approach based on molecular pathways to expose new potentially actionable targets. We used gene set enrichment analysis (GSEA) to conduct an unsupervised clustering analysis to condense the gene expression data from bulk patient samples and patient-derived gliomasphere lines into new gene signatures. We identified key targets that are predicted to be differentially activated between tumors and were functionally validated in a library of gliomasphere cultures. Resultant cluster-specific gene signatures associated not only with hallmarks of cell cycle and stemness gene expression, but also with cell-type specific markers and different cellular states of GBM. Several upstream regulators, such as PIK3R1 and EBF1 were differentially enriched in cells bearing stem cell like signatures and bear further investigation. We identified the transcription factor E2F1 as a key regulator of tumor cell proliferation and self-renewal in only a subset of gliomasphere cultures predicted to be E2F1 signaling dependent. Our in vivo work also validated the functional significance of E2F1 in tumor formation capacity in the predicted samples. E2F1 inhibition also differentially sensitized E2F1-dependent gliomasphere cultures to radiation treatment. Our findings indicate that this novel approach exploring cancer pathways highlights key therapeutic vulnerabilities for targeting GBM.

A molecular interactome of the glioblastoma perivascular niche reveals integrin binding sialoprotein as a mediator of tumor cell migration.

Glioblastoma (GBM) is characterized by extensive microvascular hyperproliferation. In addition to supplying blood to the tumor, GBM vessels also provide trophic support to glioma cells and serve as conduits for migration into the surrounding brain, promoting recurrence. Here, we enrich CD31-expressing glioma vascular cells (GVCs) and A2B5-expressing glioma tumor cells (GTCs) from primary GBM and use RNA sequencing to create a comprehensive molecular interaction map of the secreted and extracellular factors elaborated by GVCs that can interact with receptors and membrane molecules on GTCs. To validate our findings, we utilize functional assays, including a hydrogel-based migration assay and in vivo mouse models to demonstrate that one identified factor, the little-studied integrin binding sialoprotein (IBSP), enhances tumor growth and promotes the migration of GTCs along the vasculature. This perivascular niche interactome will serve as a resource to the research community in defining the potential functions of the GBM vasculature.

P300 promotes tumor recurrence by regulating radiation-induced conversion of glioma stem cells to vascular-like cells.

Glioma stem cells (GSC) exhibit plasticity in response to environmental and therapeutic stress leading to tumor recurrence, but the underlying mechanisms remain largely unknown. Here, we employ single-cell and whole transcriptomic analyses to uncover that radiation induces a dynamic shift in functional states of glioma cells allowing for acquisition of vascular endothelial-like and pericyte-like cell phenotypes. These vascular-like cells provide trophic support to promote proliferation of tumor cells, and their selective depletion results in reduced tumor growth post-treatment in vivo. Mechanistically, the acquisition of vascular-like phenotype is driven by increased chromatin accessibility and H3K27 acetylation in specific vascular genes allowing for their increased expression post-treatment. Blocking P300 histone acetyltransferase activity reverses the epigenetic changes induced by radiation and inhibits the adaptive conversion of GSC into vascular-like cells and tumor growth. Our findings highlight a role for P300 in radiation-induced stress response, suggesting a therapeutic approach to prevent glioma recurrence.

Liver-secreted fluorescent blood plasma markers enable chronic imaging of the microcirculation.

Studying blood microcirculation is vital for gaining insights into vascular diseases. Blood flow imaging in deep tissue is currently achieved by acute administration of fluorescent dyes in the blood plasma. This is an invasive process, and the plasma fluorescence decreases within an hour of administration. Here, we report an approach for the longitudinal study of vasculature. Using a single intraperitoneal or intravenous administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to chronically label the blood circulation in mice. This approach allows for longitudinal observation of circulation from 2 weeks to over 4 months after vector administration. We demonstrate the chronic assessment of vascular functions including functional hyperemia and vascular plasticity in micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease animal models.

A TCF7L2-responsive suppression of both homeostatic and compensatory remyelination in Huntington disease mice.

Huntington's disease (HD) is characterized by defective oligodendroglial differentiation and white matter disease. Here, we investigate the role of oligodendrocyte progenitor cell (OPC) dysfunction in adult myelin maintenance in HD. We first note a progressive, age-related loss of myelin in both R6/2 and zQ175 HD mice compared with wild-type controls. Adult R6/2 mice then manifest a significant delay in remyelination following cuprizone demyelination. RNA-sequencing and proteomic analysis of callosal white matter and OPCs isolated from both R6/2 and zQ175 mice reveals a systematic downregulation of genes associated with oligodendrocyte differentiation and myelinogenesis. Gene co-expression and network analysis predicts repressed Tcf7l2 signaling as a major driver of this expression pattern. In vivo Tcf7l2 overexpression restores both myelin gene expression and remyelination in demyelinated R6/2 mice. These data causally link impaired TCF7L2-dependent transcription to the poor development and homeostatic retention of myelin in HD and provide a mechanism for its therapeutic restoration.

Translating cell therapies for neurodegenerative diseases: Huntington's disease as a model disorder.

There has been substantial progress in the development of regenerative medicine strategies for CNS disorders over the last decade, with progression to early clinical studies for some conditions. However, there are multiple challenges along the translational pipeline, many of which are common across diseases and pertinent to multiple donor cell types. These include defining the point at which the preclinical data are sufficiently compelling to permit progression to the first clinical studies; scaling-up, characterization, quality control and validation of the cell product; design, validation and approval of the surgical device; and operative procedures for safe and effective delivery of cell product to the brain. Furthermore, clinical trials that incorporate principles of efficient design and disease-specific outcomes are urgently needed (particularly for those undertaken in rare diseases, where relatively small cohorts are an additional limiting factor), and all processes must be adaptable in a dynamic regulatory environment. Here we set out the challenges associated with the clinical translation of cell therapy, using Huntington's disease as a specific example, and suggest potential strategies to address these challenges. Huntington's disease presents a clear unmet need, but, importantly, it is an autosomal dominant condition with a readily available gene test, full genetic penetrance and a wide range of associated animal models, which together mean that it is a powerful condition in which to develop principles and test experimental therapeutics. We propose that solving these challenges in Huntington's disease would provide a road map for many other neurological conditions. This white paper represents a consensus opinion emerging from a series of meetings of the international translational platforms Stem Cells for Huntington's Disease and the European Huntington's Disease Network Advanced Therapies Working Group, established to identify the challenges of cell therapy, share experience, develop guidance and highlight future directions, with the aim to expedite progress towards therapies for clinical benefit in Huntington's disease.

Cell-intrinsic glial pathology is conserved across human and murine models of Huntington's disease.

Glial pathology is a causal contributor to the striatal neuronal dysfunction of Huntington's disease (HD). We investigate mutant HTT-associated changes in gene expression by mouse and human striatal astrocytes, as well as in mouse microglia, to identify commonalities in glial pathobiology across species and models. Mouse striatal astrocytes are fluorescence-activated cell sorted (FACS) from R6/2 and zQ175 mice, which respectively express exon1-only or full-length mHTT, and human astrocytes are generated either from human embryonic stem cells (hESCs) expressing full-length mHTT or from fetal striatal astrocytes transduced with exon1-only mHTT. Comparison of differential gene expression across these conditions, all with respect to normal HTT controls, reveals cell-type-specific changes in transcription common to both species, yet with differences that distinguish glia expressing truncated mHTT versus full-length mHTT. These data indicate that the differential gene expression of glia expressing truncated mHTT may differ from that of cells expressing full-length mHTT, while identifying a conserved set of dysregulated pathways in HD glia.

Conservation and divergence of vulnerability and responses to stressors between human and mouse astrocytes.

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans.