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.

A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences.

Astrocytes, one of the most prevalent cell types in the central nervous system (CNS), are critically involved in neural function. Genetically manipulating astrocytes is an essential tool in understanding and affecting their roles. Adeno-associated viruses (AAVs) enable rapid genetic manipulation; however, astrocyte specificity of AAVs can be limited, with high off-target expression in neurons and sparsely in endothelial cells. Here, we report the development of a cassette of four copies of six miRNA targeting sequences (4x6T) which triggers transgene degradation specifically in neurons and endothelial cells. In combination with the GfaABC1D promoter, 4x6T increases astrocytic specificity of Cre with a viral reporter from <50% to >99% in multiple serotypes in mice, and confers astrocyte specificity in multiple recombinases and reporters. We also present empty vectors to add 4x6T to other cargo, independently and in Cre/Dre-dependent forms. This toolbox of AAVs allows rapid manipulation of astrocytes throughout the CNS, is compatible with different AAV serotypes, and demonstrates the efficacy of using multiplexed miRNA targeting sequences to decrease expression in multiple off-target cell populations simultaneously.

Astrocytes in human central nervous system diseases: a frontier for new therapies.

Astroglia are a broad class of neural parenchymal cells primarily dedicated to homoeostasis and defence of the central nervous system (CNS). Astroglia contribute to the pathophysiology of all neurological and neuropsychiatric disorders in ways that can be either beneficial or detrimental to disorder outcome. Pathophysiological changes in astroglia can be primary or secondary and can result in gain or loss of functions. Astroglia respond to external, non-cell autonomous signals associated with any form of CNS pathology by undergoing complex and variable changes in their structure, molecular expression, and function. In addition, internally driven, cell autonomous changes of astroglial innate properties can lead to CNS pathologies. Astroglial pathophysiology is complex, with different pathophysiological cell states and cell phenotypes that are context-specific and vary with disorder, disorder-stage, comorbidities, age, and sex. Here, we classify astroglial pathophysiology into (i) reactive astrogliosis, (ii) astroglial atrophy with loss of function, (iii) astroglial degeneration and death, and (iv) astrocytopathies characterised by aberrant forms that drive disease. We review astroglial pathophysiology across the spectrum of human CNS diseases and disorders, including neurotrauma, stroke, neuroinfection, autoimmune attack and epilepsy, as well as neurodevelopmental, neurodegenerative, metabolic and neuropsychiatric disorders. Characterising cellular and molecular mechanisms of astroglial pathophysiology represents a new frontier to identify novel therapeutic strategies.

Recovery of walking after paralysis by regenerating characterized neurons to their natural target region.

Axon regeneration can be induced across anatomically complete spinal cord injury (SCI), but robust functional restoration has been elusive. Whether restoring neurological functions requires directed regeneration of axons from specific neuronal subpopulations to their natural target regions remains unclear. To address this question, we applied projection-specific and comparative single-nucleus RNA sequencing to identify neuronal subpopulations that restore walking after incomplete SCI. We show that chemoattracting and guiding the transected axons of these neurons to their natural target region led to substantial recovery of walking after complete SCI in mice, whereas regeneration of axons simply across the lesion had no effect. Thus, reestablishing the natural projections of characterized neurons forms an essential part of axon regeneration strategies aimed at restoring lost neurological functions.

Reactive astrocytes transduce inflammation in a blood-brain barrier model through a TNF-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin.

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function. Pathological transformation of astrocytes to reactive states can be protective or harmful to BBB function. Here, using a human induced pluripotent stem cell (iPSC)-derived BBB co-culture model, we show that tumor necrosis factor (TNF) transitions astrocytes to an inflammatory reactive state that causes BBB dysfunction through activation of STAT3 and increased expression of SERPINA3, which encodes alpha 1-antichymotrypsin (α1ACT). To contextualize these findings, we correlated astrocytic STAT3 activation to vascular inflammation in postmortem human tissue. Further, in murine brain organotypic cultures, astrocyte-specific silencing of Serpina3n reduced vascular inflammation after TNF challenge. Last, treatment with recombinant Serpina3n in both ex vivo explant cultures and in vivo was sufficient to induce BBB dysfunction-related molecular changes. Overall, our results define the TNF-STAT3-α1ACT signaling axis as a driver of an inflammatory reactive astrocyte signature that contributes to BBB dysfunction.

CRISPRi screens in human iPSC-derived astrocytes elucidate regulators of distinct inflammatory reactive states.

Astrocytes become reactive in response to insults to the central nervous system by adopting context-specific cellular signatures and outputs, but a systematic understanding of the underlying molecular mechanisms is lacking. In this study, we developed CRISPR interference screening in human induced pluripotent stem cell-derived astrocytes coupled to single-cell transcriptomics to systematically interrogate cytokine-induced inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-κB activation drove two distinct inflammatory reactive signatures, one promoted by STAT3 and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in human brains in Alzheimer's disease and hypoxic-ischemic encephalopathy. Furthermore, we validated that markers of these signatures were regulated by STAT3 in vivo using a mouse model of neuroinflammation. These results and the platform that we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

Transcription factor network analysis identifies REST/NRSF as an intrinsic regulator of CNS regeneration in mice.

The inability of neurons to regenerate long axons within the CNS is a major impediment to improving outcome after spinal cord injury, stroke, and other CNS insults. Recent advances have uncovered an intrinsic program that involves coordinate regulation by multiple transcription factors that can be manipulated to enhance growth in the peripheral nervous system. Here, we use a systems genomics approach to characterize regulatory relationships of regeneration-associated transcription factors, identifying RE1-Silencing Transcription Factor (REST; Neuron-Restrictive Silencer Factor, NRSF) as a predicted upstream suppressor of a pro-regenerative gene program associated with axon regeneration in the CNS. We validate our predictions using multiple paradigms, showing that mature mice bearing cell type-specific deletions of REST or expressing dominant-negative mutant REST show improved regeneration of the corticospinal tract and optic nerve after spinal cord injury and optic nerve crush, which is accompanied by upregulation of regeneration-associated genes in cortical motor neurons and retinal ganglion cells, respectively. These analyses identify a role for REST as an upstream suppressor of the intrinsic regenerative program in the CNS and demonstrate the utility of a systems biology approach involving integrative genomics and bio-informatics to prioritize hypotheses relevant to CNS repair.

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.

Astrocyte plasticity in mice ensures continued endfoot coverage of cerebral blood vessels following injury and declines with age.

Astrocytes extend endfeet that enwrap the vasculature, and disruptions to this association which may occur in disease coincide with breaches in blood-brain barrier (BBB) integrity. Here we investigate if focal ablation of astrocytes is sufficient to disrupt the BBB in mice. Targeted two-photon chemical apoptotic ablation of astrocytes induced a plasticity response whereby surrounding astrocytes extended processes to cover vascular vacancies. In young animals, replacement processes occur in advance of endfoot retraction, but this is delayed in aged animals. Stimulation of replacement astrocytes results in constriction of pre-capillary arterioles, suggesting that replacement astrocytes are functional. Pharmacological inhibition of pSTAT3, as well as astrocyte specific deletion of pSTAT3, reduces astrocyte replacement post-ablation, without perturbations to BBB integrity. Similar endfoot replacement occurs following astrocyte cell death due to reperfusion in a stroke model. Together, these studies uncover the ability of astrocytes to maintain cerebrovascular coverage via substitution from nearby cells.

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.

Engineering spinal cord repair.

Neurological damage caused by spinal cord injury in humans has been observed for over three thousand years and impacts the lives of several hundred thousand people worldwide. Despite this prevalence and its associated consequences, there is no treatment to repair the injured spinal cord. Evidence gathered over the last several decades has provided mechanistic information on the complex cascade of events following traumatic spinal cord injury and this is paving the way towards mechanism based repair strategies. In this review, we summarize state-of-the-art biological and engineering repair strategies and posit that complete repair will be dependent on cataloguing the molecular signatures and growth requirements of the different neuron subpopulations in the brain and spinal cord.

Molecular and functional properties of cortical astrocytes during peripherally induced neuroinflammation.

Astrocytic contributions to neuroinflammation are widely implicated in disease, but they remain incompletely explored. We assess medial prefrontal cortex (PFC) and visual cortex (VCX) astrocyte and whole-tissue gene expression changes in mice following peripherally induced neuroinflammation triggered by a systemic bacterial endotoxin, lipopolysaccharide, which produces sickness-related behaviors, including anhedonia. Neuroinflammation-mediated behavioral changes and astrocyte-specific gene expression alterations peak when anhedonia is greatest and then reverse to normal. Notably, region-specific molecular identities of PFC and VCX astrocytes are largely maintained during reactivity changes. Gene pathway analyses reveal alterations of diverse cell signaling pathways, including changes in cell-cell interactions of multiple cell types that may underlie the central effects of neuroinflammation. Certain astrocyte molecular signatures accompanying neuroinflammation are shared with changes reported in Alzheimer's disease and mouse models. However, we find no evidence of altered neuronal survival or function in the PFC even when neuroinflammation-induced astrocyte reactivity and behavioral changes are significant.

HepaCAM shapes astrocyte territories, stabilizes gap-junction coupling, and influences neuronal excitability.

How astrocytes form non-overlapping territories within synaptic neuropil is not understood. In this issue of Neuron, Baldwin et al. (2021) report that the cell adhesion molecule hepaCAM shapes murine astrocyte territories and that hepaCAM loss impairs gap-junction cell coupling and the balance between synaptic excitation and inhibition.

Foreign body responses in mouse central nervous system mimic natural wound responses and alter biomaterial functions.

Biomaterials hold promise for therapeutic applications in the central nervous system (CNS). Little is known about molecular factors that determine CNS foreign body responses (FBRs) in vivo, or about how such responses influence biomaterial function. Here, we probed these factors in mice using a platform of injectable hydrogels readily modified to present interfaces with different physiochemical properties to host cells. We found that biomaterial FBRs mimic specialized multicellular CNS wound responses not present in peripheral tissues, which serve to isolate damaged neural tissue and restore barrier functions. We show that the nature and intensity of CNS FBRs are determined by definable properties that significantly influence hydrogel functions, including resorption and molecular delivery when injected into healthy brain or stroke injuries. Cationic interfaces elicit stromal cell infiltration, peripherally derived inflammation, neural damage and amyloid production. Nonionic and anionic formulations show minimal levels of these responses, which contributes to superior bioactive molecular delivery. Our results identify specific molecular mechanisms that drive FBRs in the CNS and have important implications for developing effective biomaterials for CNS applications.

Astrocyte Reactivity: Subtypes, States, and Functions in CNS Innate Immunity.

Astrocytes are neural parenchymal cells that ubiquitously tile the central nervous system (CNS). In addition to playing essential roles in healthy tissue, astrocytes exhibit an evolutionarily ancient response to all CNS insults, referred to as astrocyte reactivity. Long regarded as passive and homogeneous, astrocyte reactivity is being revealed as a heterogeneous and functionally powerful component of mammalian CNS innate immunity. Nevertheless, concepts about what astrocyte reactivity comprises and what it does are incomplete and sometimes controversial. This review discusses the goal of differentiating reactive astrocyte subtypes and states based on composite pictures of molecular expression, cell morphology, cellular interactions, proliferative state, normal functions, and disease-induced dysfunctions. A working model and conceptual framework is presented for characterizing the diversity of astrocyte reactivity.