Fig. 4: Neuron-specific regional differences in cellular signaling and cytokine levels.

a Cartoon showing standard and adapted Luminex methods to measure total and biotinylated proteins. b Cartoon representation of standard and adapted Luminex approaches to measure cytokines, MAPK and Akt/mTOR phospho-proteins in brain lysates from Rosa26^TurboID/wt AAV and transgenic cohorts. c–e Fluorescence values for cytokines, MAPK and Akt/mTOR phospho-proteins measured using standard and adapted assays, from Rosa26^TurboID/wt/AAV9-hSyn-Cre mice (n = 3/group). Predominantly neuron-derived cytokines or phospho-proteins have similar fluorescence values in both standard and adapted assays, while predominantly non-neuronal cytokines or phospho-proteins have a markedly lower adapted assay readout as compared to the standard assay, determined by pairwise comparison between control and labeled mice (also see Supplementary Data 17). Data shown as mean ± SEM. f PCA of adapted Luminex assays performed on brain lysates from the Rosa26^TurboID/wt transgenic cohort, showing regional differences (CTX, ST, PM, and CB) based on cytokines and MAPK and Akt/mTOR phospho-proteins. Fluorescence values from adapted assays from labeled (Rosa26^TurboID/wt/Camk2a-Cre^Ert2) and non-labeled controls (Camk2a-Cre^Ert2) brain lysates were normalized to TurboID abundance from MS studies after which signal from adapted assays from control mice was subtracted. g, h Heat map summarizing HCA of normalized data from adapted Luminex assays. Analyses of cytokines and phospho-proteins from MAPK and Akt/mTOR pathways are shown, revealing region specific signatures. Overall, neuron-derived cytokines had higher levels in the cerebellum and relatively lower levels in the cortex while phospho-proteins, particularly from the Akt/mTOR pathway had higher levels in the cortex and lower levels in the cerebellum. Related raw and normalized Luminex data from all animals from AAV and transgenic cohorts are included in Supplementary Data 19–21. i An integrated tSNE of core regional proteomic signatures and Luminex data showed clustering of Akt/mTOR signaling with cortex-specific proteins while elevated cytokines clustered with the cerebellar proteomic signature and MAPK clustered with striatal/thalamic proteomic signature. (see Supplementary Data 18 for statistics). The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. Source data are provided as a Source Data file.