A CellProfiler Pipeline for Quantification of p-SNCA in Mouse Striatal Cholinergic and Medium Spiny Neurons v2

This protocol describes an automated image analysis workflow using CellProfiler (1) to quantify Ser129 phosphorylated α-synuclein (pSNCA) within Cholinergic and Medium Spiny Neurons (MSNs) in the striatum of Synuclein G51D mutant mice (2). Brain sections are immunostained for Ser129 phospho-α-synuclein, DARPP-32 to identify MSNs, choline acetyltransferase (ChAT) to label cholinergic neurons, and DAPI to identify nuclei. Confocal Z-stacks are processed into consecutive three-slice maximum-intensity projections (MIPs) using a custom FIJI/ImageJ macro prior to segmentation and analysis. Quantitative outputs include integrated pSNCA intensity and neuronal mask area, which are normalised using a custom R script to yield cell type specific pSNCA burden per unit area. This workflow enables cell type resolved quantification of α-synuclein pathology from fluorescence imaging datasets. (1) Stirling DR, Swain-Bowden MJ, Lucas AM, Carpenter AE, Cimini BA, Goodman A (2021). CellProfiler 4:improvements in speed, utility and usability. BMC Bioinformatics, 22 (1), 433.. PMID: 34507520 PMCID: PMC8431850. (2) Kim Y, McInnes J, Kim J, Liang YHW, Veeraragavan S, Garza AR, Belfort BDW, Arenkiel B, Samaco R, Zoghbi HY. Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice. Proc Natl Acad Sci U S A. 2024 Sep 24;121(39):e2406479121. doi: 10.1073/pnas.2406479121.