Varicose-projection astrocytes: a reactive phenotype associated with neuropathology

Glial cells are fundamental for the pathophysiology of all neurological disorders. Astrocytes, the primary home-ostatic cells of the central nervous system (CNS), exhibit species-specific characteristics, with human astrocytes specifically displaying unique structural and functional features. It is thus essential to investigate human-specific astrocytic responses to neuropathology using human-relevant models. Varicose projection (VP) astrocytes, traditionally considered specific to humans and apes, were suggested to reflect pathological burden, albeit direct evidence linking them to neurological diseases has been lacking. Here, we demonstrate for the first time that VP astrocytes are present in mice and tigers ( Panthera tigris ) and we provide evidence from four distinct human-based models that VP astrocytes are not a distinct physiological astrocyte subtype but rather a novel class of reactive astrocytes associated with neuropathology. Using human induced pluripotent stem cell (hiPSC)-derived astrocytes, mixed neural cultures, and cortical organoids, we showed that VP astrocytes are induced by pro-inflammatory cytokines interleukin-1 β (IL-1 β ) and tumor necrosis factor- α (TNF- α ) or LPS. Notably, cytokine withdrawal reverses the VP phenotype of astrocytes, indicating that it is a transient, inflammation-dependent state. We characterized the distinctive components of varicosities, including markers for extracellular vesicles, mitochondria, Golgi and endoplasmic reticulum components, suggesting roles in cellular stress responses and metabolic dysregulation. We further validated the pathological relevance of VP astrocytes by documenting their significant enrichment in postmortem brain samples from patients with several neurodegenerative diseases including as Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis, as well as in surgical resections from patients with epilepsy due to hippocampal sclerosis or brain tumors, including previously unreported subcortical regions such as basal ganglia. Additionally, we identified a higher number of VP astrocytes also in mouse astrocytes upon treatment with pro-inflammatory cytokines, suggesting that formation of VP astrocytes is an evolutionarily conserved astrocytic response to neuroinflammation. Our findings point to VP astrocytes as a novel reactive astrocyte subtype closely linked to neuropathology, highlighting their potential as biomarkers and therapeutic targets in neurological diseases. This study lays the groundwork for future investigations into the mechanisms driving VP astrocyte formation and their broader implications in neuropathology.