Maternal choline supplementation protects against age-associated cholinergic and GABAergic basal forebrain neuron degeneration in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease.

Down syndrome (DS) is a genetic disorder caused by triplication of human chromosome 21. In addition to intellectual disability, DS is defined by a premature aging phenotype and Alzheimer's disease (AD) neuropathology, including septohippocampal circuit vulnerability and degeneration of basal forebrain cholinergic neurons (BFCNs). The Ts65Dn mouse model recapitulates key aspects of DS/AD pathology, namely age-associated atrophy of BFCNs and cognitive decline in septohippocampal-dependent behavioral tasks. We investigated whether maternal choline supplementation (MCS), a well-tolerated treatment modality, protects vulnerable BFCNs from age- and genotype-associated degeneration in trisomic offspring. We also examined the effect of trisomy, and MCS, on GABAergic basal forebrain parvalbumin neurons (BFPNs), an unexplored neuronal population in this DS model. Unbiased stereological analyses of choline acetyltransferase (ChAT)-immunoreactive BFCNs and parvalbumin-immunoreactive BFPNs were conducted using confocal z-stacks of the medial septal nucleus and the vertical limb of the diagonal band (MSN/VDB) in Ts65Dn mice and disomic (2N) littermates at 3-4 and 10-12 months of age. MCS trisomic offspring displayed significant increases in ChAT-immunoreactive neuron number and density compared to unsupplemented counterparts, as well as increases in the area of the MSN/VDB occupied by ChAT-immunoreactive neuropil. MCS also rescued BFPN number and density in Ts65Dn offspring, a novel rescue of a non-cholinergic cell population. Furthermore, MCS prevented age-associated loss of BFCNs and MSN/VDB regional area in 2N offspring, indicating genotype-independent neuroprotective benefits. These findings demonstrate MCS provides neuroprotection of vulnerable BFCNs and non-cholinergic septohippocampal BFPNs, indicating this modality has translational value as an early life therapy for DS, as well as extending benefits to the aging population at large.

A method for quantification of vesicular compartments within cells using 3D reconstructed confocal z-stacks: Comparison of ImageJ and Imaris to count early endosomes within basal forebrain cholinergic neurons.

BACKGROUND: Phenotypic changes in vesicular compartments are an early pathological hallmark of many peripheral and central diseases. For example, accurate assessment of early endosome pathology is crucial to the study of Down syndrome (DS) and Alzheimer's disease (AD), as well as other neurological disorders with endosomal-lysosomal pathology. NEW METHOD: We describe a method for quantification of immunolabeled early endosomes within transmitter-identified basal forebrain cholinergic neurons (BFCNs) using 3-dimensional (3D) reconstructed confocal z-stacks employing Imaris software. RESULTS: Quantification of 3D reconstructed z-stacks was performed using two different image analysis programs: ImageJ and Imaris. We found ImageJ consistently overcounted the number of early endosomes present within individual BFCNs. Difficulty separating densely packed early endosomes within defined BFCNs was observed in ImageJ compared to Imaris. COMPARISON WITH EXISTING METHODS: Previous methods quantifying endosomal-lysosomal pathology relied on confocal microscopy images taken in a single plane of focus. Since early endosomes are distributed throughout the soma and neuronal processes of BFCNs, critical insight into the abnormal early endosome phenotype may be lost as a result of analyzing only a single image of the perikaryon. Rather than relying on a representative sampling, this protocol enables precise, direct quantification of all immunolabeled vesicles within a defined cell of interest. CONCLUSIONS: Imaris is an ideal program for accurately counting punctate vesicles in the context of dual label confocal microscopy. Superior image resolution and detailed algorithms offered by Imaris make precise and rigorous quantification of individual early endosomes dispersed throughout a BFCN in 3D space readily achievable.