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The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases. The human body relies on a molecule called ATP as an energy source and as a messenger. When cells die, for example if they are damaged or because of inflammation, they release large amounts of ATP into their environment. Their neighbors can detect the outpouring of ATP through specific receptors, the proteins that sit at the cell’s surface and can bind external agents. Scientists believe that one of these ATP-binding receptors, P2X7, responds to high levels of ATP by triggering a cascade of reactions that results in inflammation and cell death. P2X7 also seems to play a role in several brain diseases such as epilepsia and Alzheimer’s, but the exact mechanisms are not known. In particular, how this receptor is involved in the death of neurons is unclear, and researchers still debate whether P2X7 is present in neurons and in other types of brain cells. To answer this, Kaczmarek-Hájek, Zhang, Kopp et al. created genetically modified mice in which the P2X7 receptors carry a fluorescent dye. Powerful microscopes can pick up the light signal from the dye and help to reveal which cells have the receptors. These experiments show that neurons do not carry the protein; instead, P2X7 is present in certain brain cells that keep the neurons healthy. For example, it is found in the immune cells that ‘clean up’ the organ, and the cells that support and insulate neurons. Kaczmarek-Hájek et al. further provide preliminary data suggesting that, under certain conditions, if too many P2X7 receptors are present in these cells neuronal damage might be increased. It is therefore possible that the brain cells that carry P2X7 indirectly contribute to the death of neurons when large amounts of ATP are released. The genetically engineered mouse designed for the experiments could be used in further studies to dissect the role that P2X7 plays in diseases of the nervous system. In particular, this mouse model might help to understand whether the receptor could become a drug target for neurodegenerative conditions.

Astrocyte heterogeneity has been well explored, but our understanding of white matter (WM) astrocytes and their distinctions from gray matter (GM) astrocytes remains limited. Here, we compared astrocytes from cortical GM and WM/corpus callosum (WM/CC) using single-cell RNA sequencing and spatial transcriptomics of the murine forebrain. The comparison revealed similarities but also significant differences between WM and GM astrocytes, including cytoskeletal and metabolic hallmarks specific to WM astrocytes with molecular properties also shared with human WM astrocytes. When we compared murine astrocytes from two different WM regions, the cortex and cerebellum, we found that they exhibited distinct, region-specific molecular properties, with the cerebellum lacking, for example, a specific cluster of WM astrocytes expressing progenitor and proliferation genes. Functional experiments confirmed astrocyte proliferation in the WM/CC, but not in the cerebellar WM, suggesting that the WM/CC may be a source of continued astrogenesis. White matter (WM) astrocytes differ significantly from gray matter astrocytes, with WM astrocytes in the forebrain exhibiting unique proliferation capacity, which is absent in cerebellar WM, suggesting region-specific astrocyte generation.

Traumatic brain injury leads to a highly orchestrated immune- and glial cell response partially responsible for long-lasting disability and the development of secondary neurodegenerative diseases. A holistic understanding of the mechanisms controlling the responses of specific cell types and their crosstalk is required to develop an efficient strategy for better regeneration. Here, we combine spatial and single-cell transcriptomics to chart the transcriptomic signature of the injured male murine cerebral cortex, and identify specific states of different glial cells contributing to this signature. Interestingly, distinct glial cells share a large fraction of injury-regulated genes, including inflammatory programs downstream of the innate immune-associated pathways Cxcr3 and Tlr1/2. Systemic manipulation of these pathways decreases the reactivity state of glial cells associated with poor regeneration. The functional relevance of the discovered shared signature of glial cells highlights the importance of our resource enabling comprehensive analysis of early events after brain injury. Glial cells and their crosstalk after injury are crucial for brain regeneration. Here, the authors show the spatial, temporal, and single-cell responses of glial cells after injury and identify shared pathways controlling glial reactivity.

Decreasing the activation of pathology-activated microglia is crucial to prevent chronic inflammation and tissue scarring. In this study, we used a stab wound injury model in zebrafish and identified an injury-induced microglial state characterized by the accumulation of lipid droplets and TAR DNA-binding protein of 43 kDa (TDP-43) + condensates. Granulin-mediated clearance of both lipid droplets and TDP-43 + condensates was necessary and sufficient to promote the return of microglia back to the basal state and achieve scarless regeneration. Moreover, in postmortem cortical brain tissues from patients with traumatic brain injury, the extent of microglial activation correlated with the accumulation of lipid droplets and TDP-43 + condensates. Together, our results reveal a mechanism required for restoring microglia to a nonactivated state after injury, which has potential for new therapeutic applications in humans. Zambusi, Novoselc et al. show that granulin-mediated clearance of cytoplasmic TDP-43 + condensates and lipid droplets in injury-activated microglia is required for their return to the homeostatic state and successful brain regeneration.

Glial cells influence brain function and disease progression. This study identifies signals that elicit hemorrhage-specific glia plasticity, including proliferation and the acquisition of neural stem cell properties. It thereby sets a foundation for aligning glia reactivity with disease progression and for attempting to use this endogenous stem cell pool for brain repair.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters—preferably in vivo—plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions. Good–bad binary classifications fail to describe reactive astrocytes in CNS disorders. Here, 81 researchers reach consensus on widespread misconceptions and provide definitions and recommendations for future research on reactive astrocytes.

The glial environment influences neurological disease progression, yet much of our knowledge still relies on preclinical animal studies, especially regarding astrocyte heterogeneity. In murine models of traumatic brain injury, beneficial functions of proliferating reactive astrocytes on disease outcome have been unraveled, but little is known regarding if and when they are present in human brain pathology. Here we examined a broad spectrum of pathologies with and without intracerebral hemorrhage and found a striking correlation between lesions involving blood–brain barrier rupture and astrocyte proliferation that was further corroborated in an assay probing for neural stem cell potential. Most importantly, proteomic analysis unraveled a crucial signaling pathway regulating this astrocyte plasticity with GALECTIN3 as a novel marker for proliferating astrocytes and the GALECTIN3-binding protein LGALS3BP as a functional hub mediating astrocyte proliferation and neurosphere formation. Taken together, this work identifies a therapeutically relevant astrocyte response and their molecular regulators in different pathologies affecting the human cerebral cortex. Intracerebral hemorrhage triggers astrocyte proliferation and mediates neural stem cell potential via Galectin3 signaling.