Single-nucleus transcriptomics uncovers region-specific molecular features underlying selective aging vulnerability of cholinergic neurons.

Cholinergic neurons exhibit functional and molecular heterogeneity across the basal forebrain (BF), midbrain (MB), and brainstem (BS), while the aging-associated transcriptional features remain poorly characterized. Here, we performed single nucleus RNA sequencing (snRNA-seq) on 108,062 nuclei from the MS/VDB, HDB/SI, PPTG/LDTG, 7N, and 12N in adult and aged mice. BF cells showed greater age-associated transcriptomic alterations than MB and BS cells, with pronounced region-specific divergence among cholinergic neurons. Intercellular communication analysis revealed BF-specific age-related changes in APP and ApoE signaling. Within this altered milieu, intrinsic transcriptional responses varied across brain regions. In aged BF cholinergic neurons, transcriptional responses were characterized by increased oxidative stress and inflammatory signatures and reduced expression of genes involved in neuronal structural maintenance. In contrast, MB and BS cholinergic neurons maintained relatively stable transcriptional profiles. These findings reveal region-specific associations between extracellular signaling and intrinsic transcriptional stress, highlighting a selective imbalance that underlies the vulnerability of BF cholinergic neurons.