Figure 1.

Alpha-synuclein A53T mutation and triplication induce nuclear envelope abnormalities and sensitivity to stressors. (A-H) Immunocytochemistry for Lamin B1 in A53T and SNCA-Tri hiPSC-derived MD NPCs and their isogenic corrected controls in regular conditions and upon heat-shock. (A, B) A53T and (C, D) SNCA-Tri hiPSC-derived NPCs showed a loss in the nuclear envelope circularity compared with their isogenic controls. (E, F) A53T and (G-H) SNCA-Tri hiPSC-derived MD NPCs showed a higher susceptibility to heat-shock demonstrated by greater loss in the nuclear envelope circularity compared with their isogenic controls. Quantification of the nuclear envelope circularity in the (I) A53T and (J) SNCA-Tri hiPSC-derived MD NPCs demonstrated loss in circularity in the mutated lines compared with the isogenic corrected line and increased susceptibility to heat-shock in the mutated lines. The data are plotted as frequency distributions for 200 cells. n = 2 independent differentiation protocols. (K-R) Immunocytochemistry for Lamin A/C in A53T and SNCA-Tri hiPSC-derived MD NPCs and their isogenic corrected controls in regular conditions and upon heat-shock. Percentages indicate the proportion of cells with folded/blebbed nuclear morphology, white arrows denote abnormal nuclei. (K, L) A53T and (M, N) SNCA-Tri hiPSC-derived MD NPCs showed higher percentage of abnormal nuclei compared with their isogenic controls. (O, P) A53T and (Q, R) SNCA-Tri hiPSC-derived MD NPCs showed a higher susceptibility to heat-shock demonstrated by higher percentage of abnormal nuclei compared with their isogenic controls. (U-V) Absorption₂₆₀ curve for supernatant from nuclei isolated from (U) A53T hiPSC-derived MD NPCs compared with the corresponding isogenic corrected line, and (V) SNCA-Tri hiPSC-derived MD NPCs compared with the corresponding isogenic corrected line. A53T and SNCA-Tri showed higher susceptibility to osmotic stress compared with their corresponding isogenic corrected lines (n = 3). (I, J) ^****P < 0.0001, Kolmogorov–Smirnov test; (S-V) ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, Student’s t-test. Scale bars: 25 μm.