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.

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.

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.

Astrocytes and oligodendrocytes undergo subtype-specific transcriptional changes in Alzheimer's disease.

Resolving glial contributions to Alzheimer's disease (AD) is necessary because changes in neuronal function, such as reduced synaptic density, altered electrophysiological properties, and degeneration, are not entirely cell autonomous. To improve understanding of transcriptomic heterogeneity in glia during AD, we used single-nuclei RNA sequencing (snRNA-seq) to characterize astrocytes and oligodendrocytes from apolipoprotein (APOE) Ɛ2/3 human AD and age- and genotype-matched non-symptomatic (NS) brains. We enriched astrocytes before sequencing and characterized pathology from the same location as the sequenced material. We characterized baseline heterogeneity in both astrocytes and oligodendrocytes and identified global and subtype-specific transcriptomic changes between AD and NS astrocytes and oligodendrocytes. We also took advantage of recent human and mouse spatial transcriptomics resources to localize heterogeneous astrocyte subtypes to specific regions in the healthy and inflamed brain. Finally, we integrated our data with published AD snRNA-seq datasets, highlighting the power of combining datasets to resolve previously unidentifiable astrocyte subpopulations.

Neurotoxic reactive astrocytes induce cell death via saturated lipids.

Astrocytes regulate the response of the central nervous system to disease and injury and have been hypothesized to actively kill neurons in neurodegenerative disease1-6. Here we report an approach to isolate one component of the long-sought astrocyte-derived toxic factor5,6. Notably, instead of a protein, saturated lipids contained in APOE and APOJ lipoparticles mediate astrocyte-induced toxicity. Eliminating the formation of long-chain saturated lipids by astrocyte-specific knockout of the saturated lipid synthesis enzyme ELOVL1 mitigates astrocyte-mediated toxicity in vitro as well as in a model of acute axonal injury in vivo. These results suggest a mechanism by which astrocytes kill cells in the central nervous system.

Neuroinflammatory astrocyte subtypes in the mouse brain.

Astrocytes undergo an inflammatory transition after infections, acute injuries and chronic neurodegenerative diseases. How this transition is affected by time and sex, its heterogeneity at the single-cell level and how sub-states are spatially distributed in the brain remains unclear. In this study, we investigated transcriptome changes of mouse cortical astrocytes after an acute inflammatory stimulus using the bacterial cell wall endotoxin lipopolysaccharide. We identified fast transcriptomic changes in astrocytes occurring within hours that drastically change over time. By sequencing ~80,000 astrocytes at single-cell resolution, we show that inflammation causes a widespread response with subtypes of astrocytes undergoing distinct inflammatory transitions with defined transcriptomic profiles. We also attribute key sub-states of inflammation-induced reactive astrocytes to specific brain regions using spatial transcriptomics and in situ hybridization. Together, our datasets provide a powerful resource for profiling astrocyte heterogeneity and will be useful for understanding the biological importance of regionally constrained reactive astrocyte sub-states.

Astrocytes.

Philip Hasel and Shane Liddelow introduce astrocytes - glial cells that help to maintain the homeostasis of the central nervous system during development, normal physiology, and aging.

The Developmental Shift of NMDA Receptor Composition Proceeds Independently of GluN2 Subunit-Specific GluN2 C-Terminal Sequences.

The GluN2 subtype (2A versus 2B) determines biophysical properties and signaling of forebrain NMDA receptors (NMDARs). During development, GluN2A becomes incorporated into previously GluN2B-dominated NMDARs. This "switch" is proposed to be driven by distinct features of GluN2 cytoplasmic C-terminal domains (CTDs), including a unique CaMKII interaction site in GluN2B that drives removal from the synapse. However, these models remain untested in the context of endogenous NMDARs. We show that, although mutating the endogenous GluN2B CaMKII site has secondary effects on GluN2B CTD phosphorylation, the developmental changes in NMDAR composition occur normally and measures of plasticity and synaptogenesis are unaffected. Moreover, the switch proceeds normally in mice that have the GluN2A CTD replaced by that of GluN2B and commences without an observable decline in GluN2B levels but is impaired by GluN2A haploinsufficiency. Thus, GluN2A expression levels, and not GluN2 subtype-specific CTD-driven events, are the overriding factor in the developmental switch in NMDAR composition.

Mixed-species RNA-seq for elucidation of non-cell-autonomous control of gene transcription.

Transcriptomic changes induced in one cell type by another mediate many biological processes in the brain and elsewhere; however, achieving artifact-free physical separation of cell types to study them is challenging and generally allows for analysis of only a single cell type. We describe an approach using a co-culture of distinct cell types from different species that enables physical cell sorting to be replaced by in silico RNA sequencing (RNA-seq) read sorting, which is possible because of evolutionary divergence of messenger RNA (mRNA) sequences. As an exemplary experiment, we describe the co-culture of purified neurons, astrocytes, and microglia from different species (12-14 d). We describe how to use our Python tool, Sargasso, to separate the reads from conventional RNA-seq according to species and to eliminate any artifacts borne of imperfect genome annotation (10 h). We show how this procedure, which requires no special skills beyond those that might normally be expected of wet lab and bioinformatics researchers, enables the simultaneous transcriptomic profiling of different cell types, revealing the distinct influence of microglia on astrocytic and neuronal transcriptomes under inflammatory conditions.

Author Correction: Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism.

This corrects the article DOI: 10.1038/ncomms15132.

Corrigendum: Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system.

This corrects the article DOI: 10.1038/ncomms7761.

Neurons and neuronal activity control gene expression in astrocytes to regulate their development and metabolism.

The influence that neurons exert on astrocytic function is poorly understood. To investigate this, we first developed a system combining cortical neurons and astrocytes from closely related species, followed by RNA-seq and in silico species separation. This approach uncovers a wide programme of neuron-induced astrocytic gene expression, involving Notch signalling, which drives and maintains astrocytic maturity and neurotransmitter uptake function, is conserved in human development, and is disrupted by neurodegeneration. Separately, hundreds of astrocytic genes are acutely regulated by synaptic activity via mechanisms involving cAMP/PKA-dependent CREB activation. This includes the coordinated activity-dependent upregulation of major astrocytic components of the astrocyte-neuron lactate shuttle, leading to a CREB-dependent increase in astrocytic glucose metabolism and elevated lactate export. Moreover, the groups of astrocytic genes induced by neurons or neuronal activity both show age-dependent decline in humans. Thus, neurons and neuronal activity regulate the astrocytic transcriptome with the potential to shape astrocyte-neuron metabolic cooperation.

Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.

Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.

Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals.

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

Neuronal development is promoted by weakened intrinsic antioxidant defences due to epigenetic repression of Nrf2.

Forebrain neurons have weak intrinsic antioxidant defences compared with astrocytes, but the molecular basis and purpose of this is poorly understood. We show that early in mouse cortical neuronal development in vitro and in vivo, expression of the master-regulator of antioxidant genes, transcription factor NF-E2-related-factor-2 (Nrf2), is repressed by epigenetic inactivation of its promoter. Consequently, in contrast to astrocytes or young neurons, maturing neurons possess negligible Nrf2-dependent antioxidant defences, and exhibit no transcriptional responses to Nrf2 activators, or to ablation of Nrf2's inhibitor Keap1. Neuronal Nrf2 inactivation seems to be required for proper development: in maturing neurons, ectopic Nrf2 expression inhibits neurite outgrowth and aborization, and electrophysiological maturation, including synaptogenesis. These defects arise because Nrf2 activity buffers neuronal redox status, inhibiting maturation processes dependent on redox-sensitive JNK and Wnt pathways. Thus, developmental epigenetic Nrf2 repression weakens neuronal antioxidant defences but is necessary to create an environment that supports neuronal development.

Synaptic NMDA receptor activity is coupled to the transcriptional control of the glutathione system.

How the brain's antioxidant defenses adapt to changing demand is incompletely understood. Here we show that synaptic activity is coupled, via the NMDA receptor (NMDAR), to control of the glutathione antioxidant system. This tunes antioxidant capacity to reflect the elevated needs of an active neuron, guards against future increased demand and maintains redox balance in the brain. This control is mediated via a programme of gene expression changes that boosts the synthesis, recycling and utilization of glutathione, facilitating ROS detoxification and preventing Puma-dependent neuronal apoptosis. Of particular importance to the developing brain is the direct NMDAR-dependent transcriptional control of glutathione biosynthesis, disruption of which can lead to degeneration. Notably, these activity-dependent cell-autonomous mechanisms were found to cooperate with non-cell-autonomous Nrf2-driven support from astrocytes to maintain neuronal GSH levels in the face of oxidative insults. Thus, developmental NMDAR hypofunction and glutathione system deficits, separately implicated in several neurodevelopmental disorders, are mechanistically linked.

Selective dendritic susceptibility to bioenergetic, excitotoxic and redox perturbations in cortical neurons.

Neurodegenerative and neurological disorders are often characterised by pathological changes to dendrites, in advance of neuronal death. Oxidative stress, energy deficits and excitotoxicity are implicated in many such disorders, suggesting a potential vulnerability of dendrites to these situations. Here we have studied dendritic vs. somatic responses of primary cortical neurons to these types of challenges in real-time. Using a genetically encoded indicator of intracellular redox potential (Grx1-roGFP2) we found that, compared to the soma, dendritic regions exhibited more dramatic fluctuations in redox potential in response to sub-lethal ROS exposure, and existed in a basally more oxidised state. We also studied the responses of dendritic and somatic regions to excitotoxic NMDA receptor activity. Both dendritic and somatic regions experienced similar increases in cytoplasmic Ca²âº. Interestingly, while mitochondrial Ca²âº uptake and initial mitochondrial depolarisation were similar in both regions, secondary delayed mitochondrial depolarisation was far weaker in dendrites, potentially as a result of less NADH depletion. Despite this, ATP levels were found to fall faster in dendritic regions. Finally we studied the responses of dendritic and somatic regions to energetically demanding action potential burst activity. Burst activity triggered PDH dephosphorylation, increases in oxygen consumption and cellular NADH:NAD ratio. Compared to somatic regions, dendritic regions exhibited a smaller degree of mitochondrial Ca²âº uptake, lower fold-induction of NADH and larger reduction in ATP levels. Collectively, these data reveal that dendritic regions of primary neurons are vulnerable to greater energetic and redox fluctuations than the cell body, which may contribute to disease-associated dendritic damage. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.