Basal forebrain cholinergic neurons are vulnerable in a mouse model of Down syndrome and their molecular fingerprint is rescued by maternal choline supplementation

Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Down syndrome (DS) and Alzheimer's disease (AD). Current therapeutics in these disorders have been unsuccessful in slowing disease progression, likely due to poorly understood complex pathological interactions and dysregulated pathways. The Ts65Dn trisomic mouse model recapitulates both cognitive and morphological deficits of DS and AD, including BFCN degeneration and has shown lifelong behavioral changes due to maternal choline supplementation (MCS). To test the impact of MCS on trisomic BFCN, we performed laser capture microdissection to individually isolate choline acetyltransferase-immunopositive neurons in Ts65Dn and disomic littermates, in conjunction with MCS at the onset of BFCN degeneration. We utilized single population RNA sequencing (RNA-seq) to interrogate transcriptomic changes within medial septal nucleus (MSN) BFCN. Leveraging multiple bioinformatic analysis programs on differentially expressed genes (DEGs) by genotype and diet, we identified key canonical pathways and altered physiological functions within Ts65Dn MSN BFCN, which were attenuated by MCS in trisomic offspring, including the cholinergic, glutamatergic and GABAergic pathways. We linked differential gene expression bioinformatically to multiple neurological functions, including motor dysfunction/movement disorder, early onset neurological disease, ataxia and cognitive impairment via Ingenuity Pathway Analysis. DEGs within these identified pathways may underlie aberrant behavior in the DS mice, with MCS attenuating the underlying gene expression changes. We propose MCS ameliorates aberrant BFCN gene expression within the septohippocampal circuit of trisomic mice through normalization of principally the cholinergic, glutamatergic, and GABAergic signaling pathways, resulting in attenuation of underlying neurological disease functions. Pups born of Ts65Dn dams were given choline normal (1.1 g/kg; control diet) or choline supplemented (5.0 g/kg; treatment diet) chow during gestation through weaning to investigate the lifelong gene expression changes in basal forbrain cholinergic neurons due to maternal choline supplementation (MCS) in a mouse model of Down syndrome. After weaning, pups were aged on control diet to ~6MO for analysis. Fresh frozen brains were isolated, sectioned and immonhistochemical anlaysis was done to identify basal forebrain cholinergic neurons (BFCNs), with a rapid detection method to preserve RNA. BFCNs were isolated by laser capture and RNA-seq analysis was performed on single population BFCNs (~500 neurons per sample) to determine lifelong gene expression changes in the DS and control animals due to MCS treatment. Cohort were n=6 male mice per genotype and treatment (24 total). Comparisons for RNA-seq analysis was done based on genotype and treatment.