Fig. 6

Pathogenic mutations prolong α-syn, TDP-43 and tau half-life. a-f Recombinant, full-length WT or A53T α-syn (a, b), WT or Q331K TDP-43 (c, d), and WT or N279K tau (e, f) was incubated with 50 μg of human lysosome extract for 30 min. The reaction was subjected to Western blot and results quantified. g-l, Differentiated SH-SY5Y cells expressing inducible FLAG-tagged WT or mutant α-syn (g, h), TDP-43 (i, j) or tau (k, l) were treated with doxycycline for 24 h to induce protein expression. Doxycycline was removed (t = 0 days), MG132 (100 nm) was added to inhibit proteasomal degradation and lysates were collected at each time point as indicated. Samples then underwent SDS-PAGE and anti-FLAG Western blotting (n = 3 for all cell-lines tested). Samples were normalized for quantification using GAPDH as a loading control. Representative images showing the gradual clearance of of WT or mutant protein over time are shown in g, I and k with quantification of three independent replicates shown in (h, j) and (i). m-q Control or mutant iNeurons were generated as indicated, and then exposed to MG132 treatment (100 nm) for 5 days. Lysates were collected at day 0 and day 5 and underwent SDS-PAGE and anti- α-syn, anti-TDP-43, or anti-tau Western blotting (n = 3 for all cell-lines tested). Samples were normalized for quantification using GAPDH as a loading control. Representative images demonstrating WT or mutant protein over time are shown in k with quantification of three independent replicates shown in (l). r Proposed model for how mutations in α-syn, TDP-43 and tau can gradually increase steady state levels of protein over decades to predispose to impaired proteostasis, protein aggregation and neurodegeneration. *p < 0.05, **p < 0.01, or ***p < 0.001