Single-nuclei transcriptome of enteric neurons in α-synuclein overexpressing mice

Overexpression of the neuronal protein α-synuclein (αSyn) is a risk factor for Parkinson’s disease (PD), and αSyn aggregation promotes neurodegeneration and movement disorders. αSyn overexpressing mice (Thy1-ASO) reproduce many PD-associated phenotypes such as motor deficits and constipation, which impacts a majority of patients. We sought to gain insight into enteric nervous system function in Thy1-ASO mice by isolating neuronal nuclei from mouse intestines, which makes up less than 1% of cells, followed by transcriptomic analysis. To overcome the challenge of profiling gene expression in rare cell populations within complex tissues, we engineered an adeno-associated virus (AAV) that expresses a fluorescent protein in the nuclear membrane under the control of a constitutive neuronal promoter. We generated a high-quality single-nuclei gene expression dataset that revealed transcriptional heterogeneity reflecting populations of 4 putative excitatory motor neuron subtypes, 2 putative inhibitory motor neuron subtypes, 3 putative interneuron subtypes, and a putative sensory neuron gene cluster. There was little difference in expression of individual genes in neurons between wild-type and Thy1-ASO mice in the colon, under the conditions tested. However, pathway analysis revealed changes in the PI3K pathway in excitatory motor neurons (upregulated in Thy1-ASO), MAPK and androgen pathways in inhibitory motor neurons (upregulated in Thy1-ASO), and WNT (upregulated in Thy1-ASO) and p53 (downregulated in Thy1-ASO) pathways in interneurons. This dataset can be leveraged to understand the physiological basis of gut motility deficits in α-synuclein overexpressing mice.