Figure 6. Pathologic αS increase disrupts decapping-module composition and mRNA turnover rates in human neurons.

(A) Endogenous Edc4 immunoprecipitation (IP) after dox addition (10 ng/ml) in tetON inducible SNCA and wt HEK293 cells (n=3, mean,sd, two-tailed t-test)

(B) Blue native-PAGE analysis of whole cell extracts from a tetON inducible SNCA HEK293 cells.

(C-D) Superplots of PLA between Dcp1 and Edc4 in SNCA 4-copy and 2-copy neurons. 9 independent replicates across 3 neuronal differentiations, ratiometric paired t-test (n=9, p=0.0004).

(E) Artificial tethering of αS to a nanoluciferase reporter mRNA.

(F) Strategy for ActinomycinD (ActD) pulse experiment to measure decay rate differences in patient derived neurons with different SNCA copy numbers.

(G) Volcano plot for the differential gene expression between SNCA 2 and 4 copy neurons (SNCA ~1.7 fold change in linear scale)

(H) Hypothesis tested in ActD pulse experiment in patient derived triplication neurons. Prediction: Δslope(2copy-4copy) < 0 indicating mRNA stabilization.

(I) Left panel: Slope diagram of transcripts with significant differential expression within the first hour of ActD addition (1024 genes, FDR<0.05, Likelihood ratio test (LRT) of DeSeq2). Right panel: Slopes of transcripts among the right panel that decayed in the 2-copy neurons (n=709, unpaired t-test, p<0.001)

(J-L) Δslope histograms (2copy minus 4copy) for all decaying genes within the first hour of ActD treatment in 2-copy neurons. Above the histogram is the question posed. (n; number of decaying transcripts, p-value: paired one-sided t-test for the hypothesis if Δslope<0, The summary of the test is depicted in the inlet). Histogram binning colors differentiate Δslope=0 boundary.

(M) Model proposed for αS accumulation impact of αS accumulation on decapping module composition and mRNA decay rates.