Anna Lasne

Investigating the consequences of astrocyte loss in the intact brain is both important and challenging. As integral components of the neuro-glia-vascular unit, astrocytes are involved in a variety of brain processes including water homeostasis, metabolic supply, regulation of cerebral blood flow, and coordination of neuronal circuit activity. Astrocyte impairment has been associated with numerous neurological disorders. However, experimental models combining focal astrocyte ablation with longitudinal in vivo imaging in the intact adult brain have been lacking, limiting efforts to define the causal contribution of astrocyte loss to central nervous system (CNS) pathology and repair.Here, we present an in vivo model of antibody-mediated astrocyte ablation that enables longitudinal imaging and detailed investigation of ensuing cellular responses. It integrates focal induction of aquaporin-4 antibody-mediated astrocyte loss, chronic in vivo two-photon imaging, genetically encoded sensors, and reporter mouse lines. This advancement allows visualization and quantification of cellular and subcellular events in living organisms during lesion progression and recovery. It overcomes many longstanding limitations of previous models that are either constrained by non-specific hypoxic or mechanical tissue damage or require sacrificing animals at discrete time points, hindering the ability to monitor dynamic biological processes over time. In contrast, the selective targeting of astrocytes prevents the formation of the glial border, enabling the investigation of CNS response in a scar-free environment.Overall, this new approach represents a significant technical advancement, enabling comprehensive longitudinal studies of CNS responses to astrocyte loss, thus opening new avenues for understanding astrocytopathy-driven pathology, evaluating therapeutic interventions, and promoting translational research.

Investigating the consequences of astrocyte loss in the intact brain is both important and challenging. As integral components of the neuro-glia-vascular unit, astrocytes are involved in a variety of brain processes including water homeostasis, metabolic supply, regulation of cerebral blood flow, and coordination of neuronal circuit activity. Astrocyte impairment has been associated with numerous neurological disorders. However, experimental models combining focal astrocyte ablation with longitudinal in vivo imaging in the intact adult brain have been lacking, limiting efforts to define the causal contribution of astrocyte loss to central nervous system (CNS) pathology and repair. Here, we present an in vivo model of antibody-mediated astrocyte ablation that enables longitudinal imaging and detailed investigation of ensuing cellular responses. It integrates focal induction of aquaporin-4 antibody-mediated astrocyte loss, chronic in vivo two-photon imaging, genetically encoded sensors, and reporter mouse lines. This advancement allows visualization and quantification of cellular and subcellular events in living organisms during lesion progression and recovery. It overcomes many longstanding limitations of previous models that are either constrained by non-specific hypoxic or mechanical tissue damage or require sacrificing animals at discrete time points, hindering the ability to monitor dynamic biological processes over time. In contrast, the selective targeting of astrocytes prevents the formation of the glial border, enabling the investigation of CNS response in a scar-free environment. Overall, this new approach represents a significant technical advancement, enabling comprehensive longitudinal studies of CNS responses to astrocyte loss, thus opening new avenues for understanding astrocytopathy-driven pathology, evaluating therapeutic interventions, and promoting translational research. Significance: Astrocyte loss has been implicated in various neurological disorders, either as a primary cause or as a contributing factor throughout disease progression. However, there remains a critical lack of experimental models capable of directly assessing the impact of astrocyte loss on CNS integrity. Here, we introduce an in vivo model of focal astrocyte ablation that enables multimodal, longitudinal investigation of astrocyte regeneration at both the population and single-cell levels, as well as of dynamic interactions between astrocytes and other CNS cell types. Applications of this model can substantially advance our understanding of the consequences of astrocyte loss in the adult brain, opening new opportunities to investigate its implications in neurological disorders.

Astrocyte loss occurs in various neurological conditions and can disrupt local tissue homeostasis. While astrocytes surrounding border-forming lesions adopt reactive states without restoring astrocyte networks, how astrocytes respond to spatially confined astrocyte loss remains poorly understood. Here we used longitudinal in vivo two-photon microscopy, combined with spatiotemporal transcriptional profiling, to examine astrocyte responses following focal aquaporin-4 antibody-mediated ablation in the somatosensory cortex of adult mouse brain, a model of astrocytopathy relevant to neuromyelitis optica spectrum disorder. Here we show that perilesional astrocytes undergo pronounced structural remodeling during lesion repopulation, characterized by cell proliferation, prolonged multinucleated astrocyte states, polarized process extension into the depleted area and gradual displacement of nuclei into previously unoccupied astrocyte territories. Spatial transcriptomics reveal an injury-associated molecular response that resolves as the astrocyte network is restored. Together, our findings delineate the spatiotemporal dynamics of astrocyte regeneration after astrocyte loss, extending current understanding of astroglial plasticity in the adult brain.

Summary: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by increasing disability. The cellular and molecular drivers of clinical transition towards progressive disease are poorly understood. Here, we combine single-cell profiling technologies with genetic and pharmacological perturbations across the course of murine CNS inflammation to dissect the role of the local immune landscape in disease progression. We uncover a chronic monocyte-to-phagocyte transition as a hallmark of progressive disease, characterized by the emergence of maladaptive, lipid-associated macrophages (LAMs) marked by lysosomal activation and fibrotic features. Spatial transcriptomics and multiplexed imaging revealed that these LAMs localized to the leptomeninges in close proximity to parenchymal colony-stimulating factor (CSF)-1 producing disease-associated microglia (DAMs) and meningeal granulocyte-macrophage (GM)-CSF-expressing T helper cells that license their differentiation. Interference with this local cytokine network revealed a protective role for resident microglia and implicated monocyte-derived phagocytes as key drivers of progressive neuroinflammation. Notably, LAM-like macrophages could also be identified in the meninges of people with MS, indicating a homology to human disease. By elucidating their ontogeny, spatial niche, and regulatory cytokine milieu, we provide a mechanistic framework for targeting harmful myeloid states while preserving reparative CNS immunity in progressive MS.