Benefits of Maternal choline supplementation on aged basal forebrain cholinergic neurons in a mouse model of Down syndrome and Alzheimer's disease
Down syndrome (DS) stemming from triplication of chromosome 21 causes intellectual disability, with early onset of Alzheimer's disease (AD) pathology in mid-life.  Early intervention studies to reduce cognitive impairments and onset of pathology are lacking.  One such treatment, maternal choline supplementation (MCS), has shown beneficial effects on behavior and gene expression in neurodegenerative and neurodevelopmental diseases in rodent models including DS mouse models. Basal forebrain cholinergic neuron (BFCN) degeneration and enlarged endosomal phenotype is reduced due to maternal choline supplementation (MCS) in a mouse model of Down Syndrome (Ts65Dn) with concomitant beneficial behavioral effects. However, the underlying mechanisms and gene expression alterations during aging and BFCN degeneration are understudied.  We postulate cellular mechanisms driving these alterations will be elucidated with novel therapeutic targets identified by examining the modulation in MCS treated aged DS mice.   Immunohistochemical ChAT staining in the medial septal/ventral diagonal band area of the basal forebrain in ~11 MO DS and control (2N) mice with and without MCS was performed. Approximately 500 cholinergic positive neurons per animal were microisolated for RNA-sequencing. Bioinformatic analysis elucidated gene expression alterations in the aged DS mouse model compared to controls which were attenuated by MCS treatment in the DS and control animals, including cognitive impairment and NAD signaling pathway.  In conclusion, MCS treatment shows moderate effects on gene expression in aged BFCNs, indicating that MCS prolongs the healthspan in this DS mouse model, but does not eliminate disease onset. Overall design: Ts65Dn dams were given choline normal (1.1 g/kg; control diet) or choline supplemented (5.0 g/kg; treatment diet) chow during gestation and weaning to investigate the lifelong effects of maternal choline supplementation (MCS) in a mouse model of Down syndrome.We aged the pups to ~11MO and performed a transcardial perfusion with phosphate buffer to clear the brain of peripheral contaminants.  Brains were collected from control (2N mice) and from DS mice (Ts65Dn), flash frozen and stored at -80*C.  The brains were sectioned and mounted on LCM PEN slides and a rapid immunohistochemistry protocol was used to identify cholinergic positive excitatory neurons, while ensuring preservation of RNA integrity.  Excitatory neurons from the medial septal/ventral diagonal band region of the basal forebrain were isolated by laser capture and RNA-seq analysis was performed on the purified RNA to determine lifelong gene expression changes in the DS and control animals due to MCS treatment. (N=10-12 per genotype per dietary condition)