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. Author manuscript; available in PMC: 2022 Aug 22.
Published in final edited form as: Cell. 2022 Jun 9;185(12):2035–2056.e33. doi: 10.1016/j.cell.2022.05.008

Figure 5. αS interacts with Edc4 in human neurons in a dose-dependent manner and modulates P-body number.

Figure 5.

(A) Strategy to generate human neurons with differential SNCA copy numbers (see methods for details). Below; all-in-one PiggyBac construct to induce cortical neuron differentiation from iPSCs.

(B) αS protein levels from whole cell extracts of induced neuron(iN) triplication series. Vcp and Gapdh; endogenous markers.

(C) A modified all-in-one PiggyBac to induce cortical neuron differentiation and in situ biotinylation of αS.

(D) IF images of differentiated induced neurons with pan-neuronal markers, Tuj1 and Map2. Edc4 marks the P-body puncta. Below: fivefold magnification of the inlets.

(E) Biotinylated αS pulldown from induced neurons. PiggyBac depicted in (C) was used for neuronal differentiation and αS-Avi biotinylation. Experiment is repeated after 3 and 4 weeks of differentiation.

(F) Endogenous αS interacts with Edc4 in a dose-dependent manner in iPSC-derived neurons. Left: Immuno-Proximity ligation assay (PLA) for αS and Edc4 in 3-week-old neurons with differing SNCA copy numbers. Right: Super plots of PLA experiment quantification; 4 independent replicates across 3 neuronal differentiations (biological replicates) are shown. Imaged fields represent the total number of images taken (each image > 20 neurons). Summary statistics; ratiometric paired t-test (n=4, p=0.0008). Each independent replicate is color-coded.

(G) Edc4 puncta numbers decrease in SNCA triplication compared wild-type (2-copy) levels of SNCA. Left: IF for endogenous Edc4 and Tuj1(pan-neuronal marker) in 3-week-old iNs with differing SNCA copy numbers. Merged channel images are shown. Right: Super plots of IF quantifications; 4 independent replicates across 3 separate neuronal differentiations. Summary statistics; ratiometric paired t-test (n=4, p=0.0196).

(H) IF of Edc4 puncta in neurons derived from an familial SNCA mutation (A53T) line and its isogenically corrected control. Super plots of IF quantifications across two biological replicates. Summary statistics; ratiometric paired t-test (n=2, p=0.0567)