Single-Nucleus RNA Sequencing Reveals the Spatiotemporal Dynamics of Disease-Associated Microglia in Amyotrophic Lateral Sclerosis

Abstract

Disease-associated microglia (DAM) are observed in neurodegenerative diseases, demyelinating disorders, and aging. However, the spatiotemporal dynamics and evolutionary trajectory of DAM during the progression of amyotrophic lateral sclerosis (ALS) remain unclear. Using a mouse model of ALS that expresses a human SOD1 gene mutation, we found that the microglia subtype DAM begins to appear following motor neuron degeneration, primarily in the brain stem and spinal cord. Using reverse transcription quantitative polymerase chain reaction, RNAscope in situ hybridization, and flow cytometry, we found that DAM increased in number as the disease progressed, reaching their peak in the late disease stage. DAM responded to disease progression in both SOD1G93A mice and sporadic ALS and C9orf72-mutated patients. Motor neuron loss in SOD1G93A mice exhibited 2 accelerated phases: P90 to P110 (early stage) and P130 to P150 (late stage). Some markers were synchronized with the accelerated phase of motor neuron loss, suggesting that these proteins may be particularly responsive to disease progression. Through pseudotime trajectory analysis, we tracked the dynamic transition of homeostatic microglia into DAM and cluster 6 microglia. Interestingly, we used the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia in SOD1G93A mice and observed that DAM survival is independent of CSF1R. An in vitro phagocytosis assay directly confirmed that DAM could phagocytose more beads than other microglia subtypes. These findings reveal that the induction of the DAM phenotype is a shared cross-species and cross-subtype characteristic in ALS. Inducing the DAM phenotype and enhancing its function during the early phase of disease progression, or the time window between P130 and P150 where motor neuron loss slows, could serve as a neuroprotective strategy for ALS.