CA1 pyramidal neuron gene expression mosaics in the Ts65Dn murine model of Down syndrome and Alzheimer's disease following maternal choline supplementation

Abstract

Although there are changes in gene expression and alterations in neuronal density and afferent inputs in the forebrain of trisomic mouse models of Down syndrome (DS) and Alzheimer's disease (AD), there is a lack of systematic assessments of gene expression and encoded proteins within individual vulnerable cell populations, precluding translational investigations at the molecular and cellular level. Further, no effective treatment exists to combat intellectual disability and basal forebrain cholinergic neurodegeneration seen in DS. To further our understanding of gene expression changes before and following cholinergic degeneration in a well-established mouse model of DS/AD, the Ts65Dn mouse, we assessed RNA expression levels from CA1 pyramidal neurons at two adult ages (∼6 months of age and ∼11 months of age) in both Ts65Dn and their normal disomic (2N) littermates. We further examined a therapeutic intervention, maternal choline supplementation (MCS), which has been previously shown to lessen dysfunction in spatial cognition and attention, and have protective effects on the survival of basal forebrain cholinergic neurons in the Ts65Dn mouse model. Results indicate that MCS normalized expression of several genes in key gene ontology categories, including synaptic plasticity, calcium signaling, and AD-associated neurodegeneration related to amyloid-beta peptide (Aβ) clearance. Specifically, normalized expression levels were found for endothelin converting enzyme-2 (Ece2), insulin degrading enzyme (Ide), Dyrk1a, and calcium/calmodulin-dependent protein kinase II (Camk2a), among other relevant genes. Single population expression profiling of vulnerable CA1 pyramidal neurons indicates that MCS is a viable therapeutic for long-term reprogramming of key transcripts involved in neuronal signaling that are dysregulated in the trisomic mouse brain which have translational potential for DS and AD.

Maternal Choline Supplementation Alters Basal Forebrain Cholinergic Neuron Gene Expression in the Ts65Dn Mouse Model of Down Syndrome

“…Recently, studies have demonstrated that dietary MCS during pregnancy and lactation attenuates cognitive dysfunction and BFCN degeneration in adult Ts65Dn progeny (Moon et al , ; Ash et al , ; Kelley et al , ; ; Strupp et al , ; Powers et al , ). Despite the importance of the cholinergic basal forebrain projection system in cognitive function, little is known about the molecular pathobiology of BFCNs in DS (Chrast et al , ; Alldred et al , ; ; ; Perez et al , ). Here, we present single population neuronal expression data showing alterations to the transcriptional signature of MS/VDB cholinergic neurons obtained from offspring of Ts65Dn dams treated with MCS or a choline‐controlled diet during gestation and nursing.…”

“…Nitrocellulose membranes with embedded probes were used for hybridization of cDNA, as described previously (Alldred et al , ). Membranes were stripped in a weak base (0.4N NaOH) in glass tubes placed in a rotating hybridization oven (45 min at 45°C), rinsed (0.2 M Tris pH 7.2, 0.1% SDS 10% (Gibco), 0.1% SSC 20X (Fisher); 1X 1 min, 2X 10 min, 20°C), and incubated in a prehybridization buffer (formamide, Denhardts solution, 20X SSPE (Ambion), 0.1% SDS, and boiled sheared salmon sperm DNA (Ambion) in DEPC‐treated H 2 O; 4 h, 42°C).…”

“…To address this, the present study examined the molecular signature of MS/VDB neurons, identified immunohistochemically using the cholinergic neuron marker choline acetyltransferase (ChAT) and acquired by laser capture microdissection (LCM), in Ts65Dn (Ts) and disomic (2N) mice (Mufson et al , ; Alldred et al , ). Since the MS/VDB region contains a heterogeneous population of neurons, analyses based on whole region homogenates are ill‐suited for BFCN‐specific profile analysis (Mesulam et al , ; Rye et al , ; Ginsberg et al , ; ).…”

Long‐term effects of maternal choline supplementation on CA1 pyramidal neuron gene expression in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease

“…Additionally, these studies examined admixed neuronal, glial, and vascular cell populations and, in some cases, multiple admixed brain regions. Previous work in our lab has shown that MCS attenuated or reversed a subset of transcripts whose aberrant gene expression was seen in the murine DS phenotype (Ts65Dn mice) compared with 2N controls (51). Because of the large and complex datasets emanating from these single population analyses, global MCS gene expression level changes within CA1 pyramidal neurons stratified by age or genotype have not been performed, which are crucial to understand what MCS does mechanistically to gene expression within a vulnerable hippocampal cell type in an aging paradigm relevant toward understanding the pathophysiology of DS and AD.…”

“…A second independent, mixed-gender cohort of ;6-mo-old mice (n = 9/condition for 2N + , 2N, Ts + , and Ts) was perfused with 0.1 M phosphate buffer, and the CA1 subregion was microdissected tissue as previously described (51,62,63) and RNA extracted for quantitative PCR (qPCR) and NanoString nCounter (NanoString Technologies; Seattle, WA, USA) analyses. Taqman qPCR primers (Thermo Fisher Scientific) were utilized for the following genes: CREB binding protein (Crebbp; Mm01342452_m1), embryonic lethal abnormal vision-like 1 (Elavl1; m01243908_m1), and G-protein, a-z polypeptide (Gnaz; Mm01150269_m1).…”