Fig. 6: NEMO decreases the number of cells with aSyn aggregates in a p62-dependent manner.

a WT NEMO but neither Q330X NEMO nor D311N NEMO decreases the number of cells with aSyn aggregates. SH-SY5Y cells stably expressing aSyn A53T-GFP were transiently transfected with either WT HA-NEMO, Q330X HA-NEMO, D311N HA-NEMO, or mCherry as a control and treated with aSyn A53T seeds 24 h after transfection. The cells were treated 16 h after seeding with Bafilomycin A1 (BafA, 25 nM). 40 h after seeding, the cells were fixed and analyzed by immunocytochemistry and fluorescence microscopy using anti-HA antibodies. The fraction of HA- or mCherry-positive cells containing aSyn-GFP aggregates was quantified. Data are shown as mean ± SD based on 5 independent experiments. At least 750 cells per condition were quantified. Statistics were applied to the entire dataset. For better comparability, the control is shown for each NEMO construct. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test. ***p ≤ 0,001. b Catalytically active HOIP decreases the number of cells with aSyn aggregates. SH-SY5Y cells stably expressing aSyn A53T-GFP were transiently transfected with either WT HA-HOIP, catalytically inactive C885A HA-HOIP, or mCherry as a control, and treated as described in a. The fraction of HA- or mCherry-positive cells containing aSyn-GFP aggregates was quantified. Data are shown as mean ± SD based on 5 independent experiments. At least 750 cells per condition were quantified. Statistics were applied to the entire dataset. For better comparability, the control is shown for each HOIP construct. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test. ***p ≤ 0,001. c NEMO and HOIP silencing increase the number of cells with aSyn-GFP aggregates. SH-SY5Y cells stably expressing aSyn A53T-GFP were transiently transfected with either NEMO- or HOIP-specific siRNAs, treated with aSyn A53T seeds as indicated, and fixed 48 h after seeding. The fraction of cells containing aSyn aggregates was quantified. Data are shown as mean ± SD from 5 biological replicates. At least 750 cells per condition were quantified. Statistics: One-way ANOVA followed by Bonferroni’s multiple comparison test. *p ≤ 0,05, **p ≤ 0,01. d p62 is present at aSyn aggregates. SH-SY5Y cells stably expressing aSyn A53T-GFP were treated with aSyn A53T seeds, fixed 72 h after seeding, and analyzed by immunocytochemistry and SR-SIM fluorescence microscopy using anti-p62 antibodies. Scale bar, 10 µm (overview) and 5 µm (inset). e p62 co-immunoprecipitates with WT NEMO but not with Q330X NEMO. HEK293T cells were transiently transfected with either WT HA-NEMO, Q330X HA-NEMO, or WT FLAG-NEMO to control for unspecific binding. After cell lysis, HA-tagged proteins were immunoprecipitated using anti-HA-beads. Immunoprecipitated proteins were immunoblotted using antibodies against p62, M1-linked ubiquitin, and NEMO. The input was immunoblotted for p62, NEMO, and β-actin. f NEMO and HOIP reduce the number of aSyn aggregates in a p62-dependent manner, which requires the UBA ___domain of p62. p62 KO MEFs were transiently transfected with aSyn A53T-GFP, HA-NEMO, HA-HOIP or mCherry as a control, and p62 or p62ΔUBA, as indicated. One day after transfection, the cells were treated with aSyn A53T seeds, and fixed 48 h after seeding. The fraction of cells containing aSyn aggregates was quantified as described in a. Data are shown as mean ± SD based on 5 individual experiments. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test. **p ≤ 0,01, ***p ≤ 0,001. g The p62-dependent effect of NEMO on aSyn aggregates is sensitive to lysosomal inhibition. p62 KO MEFs were transiently transfected with aSyn A53T-GFP and HA-NEMO, or HA-NEMO and p62, or mCherry as a control, as indicated. The next day, cells were treated with aSyn A53T seeds, and after 16 h treated with Bafilomycin A1 (BafA, 25 nM). 40 h after seeding, cells were fixed and the fraction of cells containing aSyn aggregates was quantified as described in A. Data are shown as mean ± SD based on 5 individual experiments. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test. **p ≤ 0,01, ***p ≤ 0,001.