The cholinergic system during normal and pathological aging

National Institute Of Neurological Disorders And Stroke

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Abstract

This program emphasizes comparative studies in mice, monkeys and humans (by collaboration) to examine BFCNs known to be affected in neurodegenerative diseases that involve cognitive decline. Our goal is to identify vulnerable vs resilient populations of BFCNs by combinations of molecular, structural and functional profiling. An additional goal is to examine the se A: Normal and pathological aging of cholinergic projections to, and functions of, the entorhinal cortex in humans and rodents (Ananth et al; 2023 Ananth et al; 2024, in revision) We conducted a human/rodent comparison of cognitive performance and cholinergic innervation of the lateral entorhinal cortex (LEC) the area in humans that has been identified as the earliest site of vulnerability in cognitive decline. The relationship between cholinergic health and EC function in humans was assayed using PET imaging with a novel probe, 18FVAT, which labels cholinergic presynaptic sites. We compared the integrity of cholinergic terminal fields in the EC of healthy, older vs. age-matched participants with mild cognitive impairment. In mice, we also assayed the cholinergic terminal field density in the EC, comparing mice with and without a genetically induced aging pathology. Cognitive tasks in mice also focused on displaced object recognition to specifically assess EC function. Use of mouse models allowed us to test mechanisms underlying EC dysfunction, using genetically encoded activity and functional probes to quantify the extent of engagement of EC- and the EC projecting cholinergic neurons with concurrent assays of behavioral performance. Human participants with MCI had significantly lower levels of VAT binding in the EC compared to healthy, cognitively intact counterparts. The cross correlation of performance and VAT binding density in humans emphasized the link between behavior and cholinergic integrity. We compared these findings with those in a novel genetic mouse model that results in spontaneous increases in phospho-tau and overt cognitive deficits by 3 months of age (vs 12 mo in WT). In mice, lower EC cholinergic projection density corresponded with lower EC function. The loss of cholinergic terminal fields in EC preceded loss of cholinergic neurons in EC projecting nuclei. Genetically modified mice also had fewer learning-activated neurons in the EC and fewer activated cholinergic neurons in the basal forebrain than young animals performing an EC-engaging task. Finally, chemogenetic inhibition of EC projecting cholinergic neurons in WT mice recapitulated the performance deficits and activity profile of mutant mice. These studies provide the first detailed comparison of the cholinergic integrity of EC based cognitive performance in humans and rodents. The findings also highlight the differential vulnerability of distinct BFCN populations and their target fields. Parallel projects examine the consequence of age on emotional memory, assessing the integrity of cholinergic signaling in BLA and ventral (anterior in human) hippocampus. B: Age-related changes in the integrity of cholinergic circuits that underlie emotional memory. Young (3 mo) vs aged (12-18-mo) XY and XX WT mice were studied in a cue-associated threat task (see ZIA NS009416). In young adults, performance requires activation of cholinergic neurons within the NBM, consequent ACh release and signaling in the BLA during memory recall. Aged animals had blunted recall, with a correspondingly lower activation of neurons in the anterior BLA Ananth & Muir et al, 2026). Given the importance of cholinergic signaling in the BLA to cued threat performance (Jiang et al.,2016, Rajebhosale et al; 2023) and age-related deterioration of cholinergic terminal fields in EC with decline in DOR performance (A; above), we next compared young vs old cholinergic axonal terminals in BLA. Reductions in cholinergic terminal density in the BLA correspond with altered expression of fear learning behavior and impaired BLA activation in aged animals. The axonal changes in cholinergic terminal fields within the BLA precede those in the cholinergic cell body regions, reminiscent of findings in entorhinal cortex of human and mouse. C: Further comparative analysis of BLA-projecting cholinergic neurons in NBM/SI were conducted by comparison with macaque (Luo & Li, 2025). Thanks to the NHPCC (a consortium of investigators from 5 different neuro ICs that work together to maximize utilization of macaque tissue; see IRP collaborators) we have been able to extend our analysis to a rigorous comparison of the morphoelectric profiles of mouse vs. macaque BLA-projecting, NBM/ SI, cholinergic neurons. This work used a combination of retrograde labeling from BLA with fluorospheres and individual cell loading with biocytin. In initial studies, we examined the feasibility of a cholinergic enhancer virus (Fishell, Harvard U.) for in vivo identification, however comparison with post hoc ChAT staining revealed that the reliability of the probe (SE972E) was only ~60%. As such, we identified all cholinergic neurons by post hoc staining with ChAT. Following relocation with biocytin+ChAT IHC we conducted proximal arbor reconstruction of >100 mouse vs macaque, BLA projecting, cholinergic NBM neurons. These ex vivo studies of functionally analogous populations revealed striking differences in the 18 electrophysiological and 13 morphological features studied. Because of the profound effect of experience on the electrophysiology of NBM/SI neurons in mouse, we plan additional assays in animals of more comparable age/sex and experience. D. Over extended: mitochondrial support of very long cholinergic projections from the basal forebrain to the temporal lobe (Freeman, Hospes in prep). Mitochondrial dysfunction is described as an early pathological event in Alzheimer's disease. Cholinergic neurons extend extremely long and highly branched axonal projections to the entire temporal lobe, but there is no work examining the mitochondria in cholinergic axonal projections to cortex or hippocampus. To address this gap in knowledge, we use super-resolution SDCM and intersectional genetics to quantify changes in mitochondria in cholinergic terminals fields from mice aged 1-18 mo. At 1 mo., cholinergic mitochondria have a median volume of 0.05 um3 - similar in size to other non-cholinergic populations of axonal mitochondria in young mice. Mitochondria are densely packed along axons and cluster near release sites, marked by VAT. By 6 mo. of age, the volume of cholinergic mitochondria is reduced, but without changes in cholinergic axonal density. At 12 mo., the terminal field density of cholinergic mitochondria was dramatically reduced: they appear hyper-fragmented with a median volume of 0.02 um3 and are not associated with VAT clusters, likely related to altered vesicle recycling and release. The latter is being tested. By 18 mo most cholinergic mitochondria are in aggregates being engulfed by numerous microglia. E. Transcriptomic & structural changes in cholinergic basal forebrain neurons with age-related cognitive decline. A key feature of age-related cognitive decline is the ordered reduction in the integrity of cholinergic axonal projections, followed by death of specific subpopulations. To address possible differences in genetic profile and/or distinct patterns of changes in gene expression as underlying mechanisms (s) of vulnerability of BFCNs, we began with snRNAseq and spatial transcriptomic assessments of BFCNs from young vs old (3 vs 18 mo.) mice. We find more than 20 subgroups of ChAT+, basal forebrain cholinergic neurons by unsupervised sorting methods. There are important differences in specific subgroups as a function of age, including dIfferences in NRG expression that may be predictive of relative resilience vs vulnerability of BFCNs.