Momoka Hikosaka

Sporadic amyotrophic lateral sclerosis (ALS) is characterized by selective motor neuron vulnerability accompanied by prominent glial responses, yet their molecular heterogeneity and spatial organization in the human central nervous system and spinal cord remain incompletely defined. Here, we delineate ALS-associated glial states in situ by spatially resolved, single-cell proteomic profiling of postmortem human sporadic ALS and non-neurological control tissues across the motor cortex, spinal cord, and cerebellum. We identify subpopulations of astrocytes and microglia co-expressing both canonical ALS markers together with newly implicated ALS-associated proteins, including phosphorylated TDP-43, UBQLN2, TREM2, IL6ST, and glutaminase (GLS). These glial states are consistent with transcriptomic signatures characterized by TP53 and GLS upregulation, indicating convergence on senescence stress- and glutamine metabolism-related pathways. Notably, p53–GLS-associated profiles are selectively enriched in anatomically vulnerable regions in ALS, supporting region-specific glial metabolic alterations in human disease. Together, these findings provide insight into the metabolic alterations in ALS-affected human tissue and highlight the relevance of spatial context in shaping glial responses to neurodegeneration.

The functional alteration of microglia arises in brains exposed to external stress during early development. Pathophysiological findings of neurodevelopmental disorders such as schizophrenia and autism spectrum disorder suggest cerebellar functional deficits. However, the link between stress-induced microglia reactivity and cerebellar dysfunction is missing. Here, we investigate the developmental immune environment in translational mouse models that combine two risk factors: maternal infection and repeated social defeat stress (2HIT). We find the synergy of inflammatory stress insults, leading to microglial increase specifically in the cerebellum of both sexes. Microglial turnover correlates with the Purkinje neuron loss in 2HIT mice. Highly multiplexed imaging-mass-cytometry identifies a cell transition to TREM2(+) stress-associated microglia in the cerebellum. Single-cell-proteomic clustering reveals IL-6- and TGFβ-signaling association with microglial cell transitions. Reduced excitability of remaining Purkinje cells, cerebellum-involved brain-wide functional dysconnectivity, and behavioral abnormalities indicate cerebellar cognitive dysfunctions in 2HIT animals, which are ameliorated by both systemic and cerebellum-specific microglia replacement.

The functional alteration of microglia emerges in the brains exposed to extrinsic stress insults during early development. Pathophysiological findings of psychiatric disorders such as schizophrenia and autism implied a localized deficit of the cerebellum. However, the link between microglia reactivity and cerebellar dysfunction is missing. Here, we investigated the developmental immune environment of translational mouse models that combine two risk factors: maternal infection and repeated social defeat stress (i.e., 2HIT). We found synergy of inflammatory stress insults produced reactive microglia, specifically in the cerebellum in both sexes. The microglial proliferation correlated with the Purkinje neuron loss in 2HIT mice. Highly multiplexed imaging-mass-cytometry demonstrated a TREM2-positive population of stress-associated microglia in the cerebellum. Single-cell-proteomic clustering revealed IL-6- and TGFβ-signaling association with microglial cell transition. Purkinje cells reduced excitability, cerebellum-involved brain-wide functional dysconnectivity, and behavioral anomalies underpin the cerebellar cognitive dysfunctions in 2HIT animals, ameliorated by cerebellum-specific microglia replacement.