Single-Cell Map Reveals Why Some Cholinergic Neurons Age Faster Than Others

Deep inside the brain, a scattered population of neurons that produce the chemical messenger acetylcholine performs some of the nervous system’s most essential work. These cholinergic neurons sharpen attention, anchor memory formation, regulate sleep and arousal, and fine-tune motor control. Yet they do not all age alike. The cholinergic neurons of the basal forebrain, which supply acetylcholine to the cortex and hippocampus, degenerate prominently in normal aging and are among the earliest casualties of Alzheimer’s disease. Their counterparts in the midbrain and brainstem, by contrast, largely retain their numbers and structure into old age. A new single-nucleus RNA sequencing study published in iScience offers the most systematic molecular explanation yet for this puzzling selectivity, showing that the answer lies in sharply divergent, region-specific transcriptional programs that unfold as these neurons grow old.

A research team led by Qianqian Li, Jiale Chen, and Miao Ren of the Hainan University collaboration with the HUST-Suzhou Institute for Brainsmatics dissected specific subregions of the mouse basal forebrain, midbrain, and brainstem in both adult animals aged three to five months and aged animals aged eighteen to twenty-four months. Using the 10x Genomics Chromium platform, they generated single-nucleus RNA sequencing libraries and, after rigorous quality control, assembled an atlas of 108,062 high-quality cells. Computational annotation based on canonical marker genes identified eleven major cell types, including GABAergic and glutamatergic neurons, cholinergic neurons, oligodendrocytes and their precursors, microglia, astrocytes, endothelial cells, ependymal cells, pericytes, and fibroblasts. Reference-based label transfer with Seurat confirmed the assignments with high concordance, giving the team a reliable cellular census across three anatomically and functionally distinct cholinergic territories.

The first striking finding came from comparing how entire brain regions respond to age at the multicellular level. In the aged basal forebrain, upregulated differentially expressed genes were consistently enriched across multiple cell types in pathways tied to p53-mediated inflammatory responses, oxidative phosphorylation, synaptic translation, and protein synthesis. No comparable coordinated transcriptional activation appeared in the aged midbrain or brainstem. Meanwhile, genes downregulated with age in all three regions clustered around axon guidance and neuronal maintenance. In other words, the basal forebrain undergoes a robust, brain-wide transcriptional remodeling characterized by heightened stress and metabolic programs alongside erosion of the pathways that keep neurons structurally intact, a pattern conspicuously absent from its more resilient neighbors.

To probe how cells talk to one another, the researchers applied CellChat, a computational framework that infers intercellular communication from ligand-receptor expression. The analysis revealed a striking regional specificity. The ApoE signaling pathway, a major genetic risk factor for Alzheimer’s disease, was active exclusively in the basal forebrain, where microglia acted as the principal receivers of ApoE signals emitted by cholinergic neurons and other cell types. The aged brainstem instead showed specific activation of insulin-like growth factor signaling, a change the authors interpret as a potentially protective or compensatory mechanism supporting metabolic homeostasis and survival. The aged midbrain displayed enhanced protocadherin signaling, associated with neuronal identity and connectivity. These distinct signaling landscapes suggest that each cholinergic system ages within its own extracellular milieu, shaped by different glial and metabolic partners.

One pathway, however, spanned all three regions: amyloid precursor protein, or APP, signaling, with cholinergic neurons identified as the putative receiver cells. Here the regional differences proved decisive. In the basal forebrain and midbrain, the importance of APP signaling to cholinergic neurons rose substantially with age, driven largely by the App-Tnfrsf21 ligand-receptor pair, whereas the brainstem showed no such age-related increase. Given that APP processing lies at the heart of amyloid-beta generation in Alzheimer’s disease, the selective amplification of APP reception in aged basal forebrain cholinergic neurons hints at an extracellular signaling context that may prime these cells for degeneration. The finding dovetails with recent work showing that aging selectively increases the vulnerability of the cholinergic system to amyloid pathology.

Zooming in on the cholinergic neurons themselves, unbiased subclustering revealed that they segregate cleanly by anatomical origin, forming the most regionally distinctive transcriptional profile of any cell type in the atlas. Basal forebrain cholinergic neurons highly expressed Lhx8, Ngfr, and Ntrk1, the latter two encoding receptors for nerve growth factor, a trophic signal long known to sustain these cells. Midbrain neurons were enriched for Tgfbr2, Egflam, and Nos1, while brainstem neurons expressed Sv2b, Ryr3, and Nfia. RNAscope in situ hybridization validated the star of this list: Ngfr was expressed in nearly all basal forebrain cholinergic neurons and virtually absent from the midbrain and brainstem populations. Functional enrichment underscored the divide, with basal forebrain genes tied to neurodegenerative disease pathways and synaptic signaling, midbrain genes to nitric oxide and Wnt signaling, and brainstem genes to longevity-regulating and PI3K-Akt pathways.

High-dimensional weighted gene co-expression network analysis then exposed how aging rewrites these programs differently in each region. In the basal forebrain, an age-correlated module was enriched in neurodegenerative disease pathways, oxidative phosphorylation, ATP biosynthesis, and apoptotic signaling, and contained the aging-linked genes Malat1 and Ube3a. The midbrain’s age module pointed instead toward RNA processing, nuclear transport, and chromatin remodeling, while the brainstem’s declining module involved neuronal migration, axon guidance, and neurotransmitter transport. Gene set variation analysis confirmed the asymmetry: stress and apoptosis pathways climbed in aged basal forebrain cholinergic neurons, whereas metabolic homeostasis and growth signaling pathways, including insulin receptor recycling and IGF1 signaling, rose in the midbrain and brainstem while falling in the basal forebrain.

Differential expression between adult and aged cholinergic neurons within each region revealed remarkably little overlap, with 186 genes uniquely upregulated and 92 uniquely downregulated in the aged basal forebrain, and hundreds of region-specific changes in the brainstem but none in the midbrain. The basal forebrain losses were especially telling, encompassing axonal and cytosolic transport, neurotrophic signaling, lipid membrane metabolism, and the somatic and synaptic architecture that keeps a neuron connected. The brainstem, by contrast, upregulated gene sets for ion homeostasis, energy metabolism, cytoskeletal maintenance, and cell survival, an adaptive profile consistent with resilience. Two genes with opposite regional trajectories stood out. Oxr1, a conserved antioxidant protein that protects mitochondria and guards against oxidative damage, rose with age in basal forebrain cholinergic neurons and fell in the brainstem. Ptprs, a receptor tyrosine phosphatase governing axon growth, adhesion, and synapse formation, dropped in the basal forebrain and trended upward in the brainstem.

Using the machine-learning framework scTenifoldKnk, the team simulated virtual knockouts of these two candidates in basal forebrain cholinergic neurons. Perturbing Oxr1 disrupted 81 downstream genes, including 22 that were both age-related and hub genes of the oxidative stress module, with enrichment in the ERK1/2 cascade, synapse assembly, and neuron projection extension. Virtual knockout of Ptprs perturbed 69 genes, among them neurodevelopmental regulators such as Nfib and Reln, converging on axon guidance, synaptic organization, and neurotransmitter cycling. Both perturbations thus struck the same target: the gene networks that maintain neuronal structure and synaptic function. The authors are careful to note the caveats. Tissues from multiple mice were pooled per library, limiting replicate-level statistics; both sexes were combined, precluding assessment of sex-specific effects; and the findings remain transcriptomic predictions awaiting experimental validation in living models. Many brainstem cholinergic neurons are also motor neurons with fundamentally different physiological demands, which may partly explain their apparent resilience. Even so, the study delivers a compelling molecular framework for one of neuroscience’s enduring questions: why the neurons that sustain our memory and attention are precisely the ones that time attacks first, and where, in their shifting gene networks, the seeds of that vulnerability lie.

Subject of Research: Region-specific molecular mechanisms underlying selective aging vulnerability of cholinergic neurons in the mouse basal forebrain, midbrain, and brainstem

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

Article References: Li, Q., Chen, J., Liu, X., Gong, H., Li, X., & Ren, M. (2026). Single-nucleus transcriptomics uncovers region-specific molecular features underlying selective aging vulnerability of cholinergic neurons. iScience, 29(11), Article 117778. https://doi.org/10.1016/j.isci.2026.117778

Image Credits: AI Generated

DOI: 10.1016/j.isci.2026.117778

Keywords: cholinergic neurons, basal forebrain, single-nucleus RNA sequencing, brain aging, Alzheimer's disease, ApoE signaling, APP signaling, Oxr1, Ptprs, neurodegeneration, CellChat, transcriptomics