Neuroinflammatory reactive astrocyte formation correlates with adverse outcomes in perinatal white matter injury.

Perinatal white matter injury (WMI) is the leading cause of long-term neurological morbidity in infants born preterm. Neuroinflammation during a critical window of early brain development plays a key role in WMI disease pathogenesis. The mechanisms linking inflammation with the long-term myelination failure that characterizes WMI, however, remain unknown. Here, we investigate the role of astrocyte reactivity in WMI. In an experimental mouse model of WMI, we demonstrate that WMI disease outcomes are improved in mutant mice lacking secretion of inflammatory molecules TNF-α, IL-1α, and C1q known, in addition to other roles, to induce the formation of a neuroinflammatory reactive astrocyte substate. We show that astrocytes express molecular signatures of the neuroinflammatory reactive astrocyte substate in both our WMI mouse model and human tissue affected by WMI, and that this gene expression pattern is dampened in injured mutant mice. Our data provide evidence that a neuroinflammatory reactive astrocyte substate correlates with adverse WMI disease outcomes, thus highlighting the need for further investigation of these cells as potential causal players in WMI pathology.

Immunotherapy-related cognitive impairment after CAR T cell therapy in mice.

Persistent central nervous system (CNS) immune dysregulation and consequent dysfunction of multiple neural cell types is central to the neurobiological underpinnings of a cognitive impairment syndrome that can occur following traditional cancer therapies or certain infections. Immunotherapies have revolutionized cancer care for many tumor types, but the potential long-term cognitive sequelae are incompletely understood. Here, we demonstrate in mouse models that chimeric antigen receptor (CAR) T cell therapy for both CNS and non-CNS cancers can impair cognitive function and induce a persistent CNS immune response characterized by white matter microglial reactivity and elevated cerebrospinal fluid (CSF) cytokines and chemokines. Consequently, oligodendroglial homeostasis and hippocampal neurogenesis are disrupted. Microglial depletion rescues oligodendroglial deficits and cognitive performance in a behavioral test of attention and short-term memory function. Taken together, these findings illustrate similar mechanisms underlying immunotherapy-related cognitive impairment (IRCI) and cognitive impairment following traditional cancer therapies and other immune challenges.

Reactive Astrocytes and Emerging Roles in Central Nervous System (CNS) Disorders.

In addition to their many functions in the healthy central nervous system (CNS), astrocytes respond to CNS damage and disease through a process called "reactivity." Recent evidence reveals that astrocyte reactivity is a heterogeneous spectrum of potential changes that occur in a context-specific manner. These changes are determined by diverse signaling events and vary not only with the nature and severity of different CNS insults but also with location in the CNS, genetic predispositions, age, and potentially also with "molecular memory" of previous reactivity events. Astrocyte reactivity can be associated with both essential beneficial functions as well as with harmful effects. The available information is rapidly expanding and much has been learned about molecular diversity of astrocyte reactivity. Emerging functional associations point toward central roles for astrocyte reactivity in determining the outcome in CNS disorders.

Proteomic profiling of interferon-responsive reactive astrocytes in rodent and human.

Astrocytes are a heterogeneous population of central nervous system glial cells that respond to pathological insults and injury by undergoing a transformation called "reactivity." Reactive astrocytes exhibit distinct and context-dependent cellular, molecular, and functional state changes that can either support or disturb tissue homeostasis. We recently identified a reactive astrocyte sub-state defined by interferon-responsive genes like Igtp, Ifit3, Mx1, and others, called interferon-responsive reactive astrocytes (IRRAs). To further this transcriptomic definition of IRRAs, we wanted to define the proteomic changes that occur in this reactive sub-state. We induced IRRAs in immunopanned rodent astrocytes and human iPSC-differentiated astrocytes using TNF, IL1α, C1Q, and IFNß and characterized their proteomic profile (both cellular and secreted) using unbiased quantitative proteomics. We identified 2335 unique cellular proteins, including IFIT2/3, IFITM3, OASL1/2, MX1/2/3, and STAT1. We also report that rodent and human IRRAs secrete PAI1, a serine protease inhibitor which may influence reactive states and functions of nearby cells. Finally, we evaluated how IRRAs are distinct from neurotoxic reactive astrocytes (NRAs). While NRAs are described by expression of the complement protein C3, it was not upregulated in IRRAs. Instead, we found ~90 proteins unique to IRRAs not identified in NRAs, including OAS1A, IFIT3, and MX1. Interferon signaling in astrocytes is critical for the antiviral immune response and for regulating synaptic plasticity and glutamate transport mechanisms. How IRRAs contribute to these functions is unknown. This study provides the basis for future experiments to define the functional roles of IRRAs in the context of neurodegenerative disorders.

Temporal and spatial analysis of astrocytes following stroke identifies novel drivers of reactivity.

Astrocytes undergo robust gene expression changes in response to a variety of perturbations, including ischemic injury. How these transitions are affected by time, and how heterogeneous and spatially distinct various reactive astrocyte populations are, remain unclear. To address these questions, we performed spatial transcriptomics as well as single nucleus RNAseq of ~138,000 mouse forebrain astrocytes at 1, 3, and 14 days after ischemic injury. We observed a widespread and temporally diverse response across many astrocyte subtypes. We identified astrocyte clusters unique in injury, including a transiently proliferative substate that may be BRCA1-dependent. We also found an interferon-responsive population that rapidly expands to the perilesion cortex at 1 day and persists up to 14 days post stroke. These lowly abundant, spatially restricted populations are likely functionally important in post-injury stabilization and resolution. These datasets offer valuable insights into injury-induced reactive astrocyte heterogeneity and can be used to guide functional interrogation of biologically meaningful reactive astrocyte substates to understand their pro- and anti-reparative functions following acute injuries such as stroke.

Sex Chromosomes and Gonads Shape the Sex-Biased Transcriptomic Landscape in Tlr7-Mediated Demyelination During Aging.

Demyelination occurs in aging and associated diseases, including Alzheimer's disease. Several of these diseases exhibit sex differences in prevalence and severity. Biological sex primarily stems from sex chromosomes and gonads releasing sex hormones. To dissect mechanisms underlying sex differences in demyelination of aging brains, we constructed a transcriptomic atlas of cell type-specific responses to illustrate how sex chromosomes, gonads, and their interaction shape responses to demyelination. We found that sex-biased oligodendrocyte and microglial responses are driven by interaction of sex chromosomes and gonads prior to myelin loss. Post demyelination, sex chromosomes mainly guide microglial responses, while gonadal composition influences oligodendrocyte signaling. Significantly, ablation of the X-linked gene Toll-like receptor 7 (Tlr7), which exhibited sex-biased expression during demyelination, abolished the sex-biased responses and protected against demyelination.

Blocking Formation of Neurotoxic Reactive Astrocytes is Beneficial Following Stroke.

Microglia and astrocytes play an important role in the neuroinflammatory response and contribute to both the destruction of neighboring tissue as well as the resolution of inflammation following stroke. These reactive glial cells are highly heterogeneous at both the transcriptomic and functional level. Depending upon the stimulus, microglia and astrocytes mount a complex, and specific response composed of distinct microglial and astrocyte substates. These substates ultimately drive the landscape of the initiation and recovery from the adverse stimulus. In one state, inflammation- and damage-induced microglia release tumor necrosis factor (TNF), interleukin 1α (IL1α), and complement component 1q (C1q), together 'TIC'. This cocktail of cytokines drives astrocytes into a neurotoxic reactive astrocyte (nRA) substate. This nRA substate is associated with loss of many physiological astrocyte functions (e.g., synapse formation and maturation, phagocytosis, among others), as well as a gain-of-function release of neurotoxic long-chain fatty acids which kill neighboring cells. Here we report that transgenic removal of TIC led to reduction of gliosis, infarct expansion, and worsened functional deficits in the acute and delayed stages following stroke. Our results suggest that TIC cytokines, and likely nRAs play an important role that may maintain neuroinflammation and inhibit functional motor recovery after ischemic stroke. This is the first report that this paradigm is relevant in stroke and that therapies against nRAs may be a novel means to treat patients. Since nRAs are evolutionarily conserved from rodents to humans and present in multiple neurodegenerative diseases and injuries, further identification of mechanistic role of nRAs will lead to a better understanding of the neuroinflammatory response and the development of new therapies.

Determinants of motor neuron functional subtypes important for locomotor speed.

Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss of prdm16 or mecom causes fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.

Longitudinal scRNA-seq analysis in mouse and human informs optimization of rapid mouse astrocyte differentiation protocols.

Macroglia (astrocytes and oligodendrocytes) are required for normal development and function of the central nervous system, yet many questions remain about their emergence during the development of the brain and spinal cord. Here we used single-cell/single-nucleus RNA sequencing (scRNA-seq/snRNA-seq) to analyze over 298,000 cells and nuclei during macroglia differentiation from mouse embryonic and human-induced pluripotent stem cells. We computationally identify candidate genes involved in the fate specification of glia in both species and report heterogeneous expression of astrocyte surface markers across differentiating cells. We then used our transcriptomic data to optimize a previous mouse astrocyte differentiation protocol, decreasing the overall protocol length and complexity. Finally, we used multi-omic, dual single-nuclei (sn)RNA-seq/snATAC-seq analysis to uncover potential genomic regulatory sites mediating glial differentiation. These datasets will enable future optimization of glial differentiation protocols and provide insight into human glial differentiation.

Functional roles of reactive astrocytes in neuroinflammation and neurodegeneration.

Despite advances in uncovering the mechanisms that underlie neuroinflammation and neurodegenerative disease, therapies that prevent neuronal loss remain elusive. Targeting of disease-defining markers in conditions such as Alzheimer disease (amyloid-ß and tau) or Parkinson disease (α-synuclein) has been met with limited success, suggesting that these proteins do not act in isolation but form part of a pathological network. This network could involve phenotypic alteration of multiple cell types in the CNS, including astrocytes, which have a major neurosupportive, homeostatic role in the healthy CNS but adopt reactive states under acute or chronic adverse conditions. Transcriptomic studies in human patients and disease models have revealed the co-existence of many putative reactive sub-states of astrocytes. Inter-disease and even intra-disease heterogeneity of reactive astrocytic sub-states are well established, but the extent to which specific sub-states are shared across different diseases is unclear. In this Review, we highlight how single-cell and single-nuclei RNA sequencing and other 'omics' technologies can enable the functional characterization of defined reactive astrocyte states in various pathological scenarios. We provide an integrated perspective, advocating cross-modal validation of key findings to define functionally important sub-states of astrocytes and their triggers as tractable therapeutic targets with cross-disease relevance.

Defining the molecular identity and morphology of glia limitans superficialis astrocytes in mouse and human.

Astrocytes are a highly abundant glial cell type that perform critical homeostatic functions in the central nervous system. Like neurons, astrocytes have many discrete heterogenous subtypes. The subtype identity and functions are, at least in part, associated with their anatomical location and can be highly restricted to strategically important anatomical domains. Here, we report that astrocytes forming the glia limitans superficialis, the outermost border of brain and spinal cord, are a highly specialized astrocyte subtype and can be identified by a single marker: Myocilin (Myoc). We show that Myoc+ astrocytes cover the entire brain and spinal cord surface, exhibit an atypical morphology, and are evolutionarily conserved from rodents to humans. Identification of this highly specialized astrocyte subtype will advance our understanding of CNS homeostasis and potentially be targeted for therapeutic intervention to combat peripheral inflammatory effects on the CNS.

Molecular and metabolic heterogeneity of astrocytes and microglia.

Astrocytes and microglia are central players in a myriad of processes in the healthy and diseased brain, ranging from metabolism to immunity. The crosstalk between these two cell types contributes to pathology in many if not all neuroinflammatory and neurodegenerative diseases. Recent advancements in integrative multimodal sequencing techniques have begun to highlight how heterogeneous both cell types are and the importance of metabolism to their regulation. We discuss here the transcriptomic, metabolic, and functional heterogeneity of astrocytes and microglia and highlight their interaction in health and disease.

APOE and immunity: Research highlights.

INTRODUCTION: At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias. METHODS: The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry. RESULTS: During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα]). DISCUSSION: This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development.

Microglial INPP5D limits plaque formation and glial reactivity in the PSAPP mouse model of Alzheimer's disease.

INTRODUCTION: The inositol polyphosphate-5-phosphatase D (INPP5D) gene encodes a dual-specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins. Single nucleotide polymorphisms in INPP5D impact risk for developing late onset sporadic Alzheimer's disease (LOAD). METHODS: To assess the consequences of inducible Inpp5d knockdown in microglia of APPKM670/671NL /PSEN1Δexon9 (PSAPP) mice, we injected 3-month-old Inpp5dfl/fl /Cx3cr1CreER/+ and PSAPP/Inpp5dfl/fl /Cx3cr1CreER/+ mice with either tamoxifen (TAM) or corn oil (CO) to induce recombination. RESULTS: At age 6 months, we found that the percent area of 6E10+ deposits and plaque-associated microglia in Inpp5d knockdown mice were increased compared to controls. Spatial transcriptomics identified a plaque-specific expression profile that was extensively altered by Inpp5d knockdown. DISCUSSION: These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques. Spatial transcriptomics highlighted an extended gene expression signature associated with plaques and identified CST7 (cystatin F) as a novel marker of plaques. HIGHLIGHTS: Inpp5d knockdown increases plaque burden and plaque-associated microglia number. Spatial transcriptomics identifies an expanded plaque-specific gene expression profile. Plaque-induced gene expression is altered by Inpp5d knockdown in microglia. Our plaque-associated gene signature overlaps with human Alzheimer's disease gene networks.

The choroid plexus: a missing link in our understanding of brain development and function.

Studies of the choroid plexus lag behind those of the more widely known blood-brain barrier, despite a much longer history. This review has two overall aims. The first is to outline long-standing areas of research where there are unanswered questions, such as control of cerebrospinal fluid (CSF) secretion and blood flow. The second aim is to review research over the past 10 years where the focus has shifted to the idea that there are choroid plexuses located in each of the brain's ventricles that make specific contributions to brain development and function through molecules they generate for delivery via the CSF. These factors appear to be particularly important for aspects of normal brain growth. Most research carried out during the twentieth century dealt with the choroid plexus, a brain barrier interface making critical contributions to the composition and stability of the brain's internal environment throughout life. More recent research in the twenty-first century has shown the importance of choroid plexus-generated CSF in neurogenesis, influence of sex and other hormones on choroid plexus function, and choroid plexus involvement in circadian rhythms and sleep. The advancement of technologies to facilitate delivery of brain-specific therapies via the CSF to treat neurological disorders is a rapidly growing area of research. Conversely, understanding the basic mechanisms and implications of how maternal drug exposure during pregnancy impacts the developing brain represents another key area of research.

Blocking of microglia-astrocyte proinflammatory signaling is beneficial following stroke.

Microglia and astrocytes play an important role in the neuroinflammatory response and contribute to both the destruction of neighboring tissue as well as the resolution of inflammation following stroke. These reactive glial cells are highly heterogeneous at both the transcriptomic and functional level. Depending upon the stimulus, microglia and astrocytes mount a complex, and specific response composed of distinct microglial and astrocyte substates. These substates ultimately drive the landscape of the initiation and recovery from the adverse stimulus. In one state, inflammation- and damage-induced microglia release tumor necrosis factor (TNF), interleukin 1α (IL1α), and complement component 1q (C1q), together "TIC." This cocktail of cytokines drives astrocytes into a neurotoxic reactive astrocyte (nRA) substate. This nRA substate is associated with loss of many physiological astrocyte functions (e.g., synapse formation and maturation, phagocytosis, among others), as well as a gain-of-function release of neurotoxic long-chain fatty acids which kill neighboring cells. Here we report that transgenic removal of TIC led to reduction of gliosis, infarct expansion, and worsened functional deficits in the acute and delayed stages following stroke. Our results suggest that TIC cytokines, and likely nRAs play an important role that may maintain neuroinflammation and inhibit functional motor recovery after ischemic stroke. This is the first report that this paradigm is relevant in stroke and that therapies against nRAs may be a novel means to treat patients. Since nRAs are evolutionarily conserved from rodents to humans and present in multiple neurodegenerative diseases and injuries, further identification of mechanistic role of nRAs will lead to a better understanding of the neuroinflammatory response and the development of new therapies.

Repurposing the cardiac glycoside digoxin to stimulate myelin regeneration in chemically-induced and immune-mediated mouse models of multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na+ /K+ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG35-55 (PLG-MOG35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.