The cholinergic system during normal and pathological aging

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

This program emphasizes comparative studies in mice, monkeys and humans 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. A: Normal and pathological aging of cholinergic projections to, and functions of, the entorhinal cortex in humans and rodents (Ananth et al; 2023 Nature Neurosci Rev; Ananth et al; 2023, 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 integrity of cholinergic circuits that underlie emotional memory (Zhong et al.,2022; and Ananth, Watkins, G; M; Zarrabi, Y (Zhong, contd). Young (3 mo) vs aged (12-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 and 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. 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. Over extended: mitochondrial support of very long cholinergic projections from the basal forebrain to the temporal lobe (Freeman, Hospes). Mitochondrial dysfunction is described as an early pathological event in Alzheimers 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. We find that 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, potentially interfering with vesicle recycling and cholinergic transmission. Studies of 18 mo cholinergic mitochondria are underway D. Transcriptomic & structural changes in cholinergic basal forebrain neurons with age related cognitive decline (Ananth, Bai, Kim, Gibson ) We find significant reductions in the integrity of cholinergic axonal projections- in both humans and rodents - as an early feature of age-related cognitive decline. Different cognitive features (object spatial memory vs. emotional memory) decline at different rates and certain BFCN projections are affected sooner and to a greater extent than others. To address possible differences in genetic profile and/or distinct patterns of changes in gene expression as underlying mechanism(s) of vulnerability of BFCNs, we began snRNAseq and spatial transcriptomic assessments of BFCNs from young vs old (3 vs 18 mo.) mice. Single nucleus RNAseq transcriptomes were determined from >20K cholinergic nuclei. Using unbiased clustering approaches, we identified 25 unique clusters of cholinergic neurons in young mice, indicating far more diversity across the basal forebrain than initially hypothesized. Ongoing analyses investigate the spatial patterns of these clusters across the basal forebrain and assess the selective vulnerability or resilience of these subpopulations with age. E. Additional studies on cholinergic signaling in cognitive decline Transcriptomics: We will extend snRNAseq analysis to human postmortem samples from older adult BFCN tissue (w and w/o prior diagnoses of cognitive impairment). A long-range goal is to expand human work to cell biological analyses using patient derived IPSCs induced to form cholinergic neurons. Longitudinal human PET (collaboration with Parsey/ Delorenzo) focus on 2-6 year follow up VAT imaging with the addition of PET scans for ABeta and hyperphosphorylated Tau. Marmoset PET: Other studies focus on a more ageing-tractable primate marmoset. Studies in marmoset repeatedly sample 18FVAT binding in vivo at different phases of life (2-4 yrs and 6- 10 yrs). A collaboration with the Chemistry and Synthesis Center (CSC) synthesizes and delivers the VAT tracer. Because BFCN regions are small and challenging to identify, a parallel set of studies delineate these regions in samples that have undergone MRI scans (T1w and DTI at 14T) followed by whole brain histology.