Fig. 1: P56S-VAPB leads to defective autophagy in ALS8.

a Representative H&E stained section from ALS8 index patient muscle biopsy showing typical neurogenic atrophy; presence of angular, flatten and group fibre atrophy. Scale bar: 60 µm. b Prominent immunoreactivity of globular VAPB aggregates (arrowheads, right panels) in grouped angular or flattened, atrophic, denervated muscle fibres of ALS8 compared to the normal control (left panel) showing diffuse pattern of sarcomeric VAPB immunolabelling in normal-sized fibres. Scale bars: 70 µm. c Immunolabeling of human fibroblasts using VAPB antibody showing small but enormous punctate P56S-VAPB aggregates in ALS8 fibroblast (right panel, white arrowheads), compared to control (left panel). Note the increased formation of globular P56S-VAPB aggregates (arrowheads) in ALS8 fibroblasts, after treatment with proteasome inhibitor MG132 (2 µM) and Bafilomycin A (200 nM) for 4 h. Scale bars: 10 µm. d DAB immunohistochemistry performed on ALS8 muscle biopsy showing increased accumulation of autophagy marker p62 and LC3 in atrophic and partially atrophic fibres. Note the numerous large autophagic vacuoles (white arrows) present in hypertrophic fibres. Scale bars: 50 µm. e Double immunofluorescence labelling of ALS8 muscle biopsy using antibodies against VAPB and for the autophagy marker p62 and LC3 showing co-localisation of the aggregated p62 and LC3 together with the mutant VAPB aggregates. Scale bars: 25 µm. f Punctate P56S-VAPB aggregates co-localised with LC3 and p62 accumulations in ALS8 fibroblasts. Scale bars: 10 µm. g Immunoblot analysis in NSC-34 cells transiently transfected either with empty GFP vector, wt-VAPB and P56S-VAPB. Note the increased levels of ER stress, autophagy markers as well as presence of gel top smear aggregate of P56S-VAPB (black arrowhead) and aggregated p62 (red arrowhead) in P56S-VAPB expressing cells. Corresponding densitometric data are shown at the bottom; representing the relative band intensity of LC3-II/LC3-I, normalised with tubulin levels. Graph pad prism, unpaired Student’s t test for comparison between two sample groups. Values were expressed as mean ± standard error of mean (SEM) from three independent experiments. The asterisks denote significant differences (*p < 0.05). h, i Co-labelling of wt-VAPB and P56S-VAPB with LC3 (h) and with p62 (i). Note that the globular accumulations of both LC3 and p62 and their co-localisation with P56S aggregates. Scale bar: 10 µm. j, k Increased accumulation of autophagosomes (white arrowheads) in the stable autophagy reporter cell line NIH3T3-GFP transfected with P56S-VAPB. Green: GFP-LC3; red: VAPB; scale bar: 10 µm (j). Immunoblot analysis showing increased levels of both p62 and LC3 in cells transfected with P56S-VAPB (k). Unpaired Student’s t test for comparison between two sample groups. Values were expressed as mean ± standard error of mean (SEM) from three independent experiments. The asterisks denote significant differences (*p < 0.05). l Immunoblot analysis of A431 cells transfected with EGFP, wt-VAPB and P56S-VAPB showing the increased levels of the autophagy markers EGFR, p62 and LC3 in P56S-VAPB expressing cells. Unpaired Student’s t-test for comparison between two sample groups. Values were expressed as mean ± standard error of mean (SEM) from three independent experiments. The asterisks denote significant differences (*p < 0.05). m A431 cells were transfected as described above. 24 h later, transfected cells were processed for the EGFR degradation assay (see Methods) and analysed by immunoblotting with the EGFR antibody. Note the delayed EGFR degradation in P56S-VAPB expressing cells. Right panel: quantification of immunoblots analysis. Graph pad prism, unpaired Student’s t test for comparison between two sample groups. Values were expressed as mean ± standard error of mean (SEM) from three independent experiments. The asterisks denote significant differences (*p < 0.05), while # denotes absence of a significant difference. n NSC-34 cells overexpressing wt-VAPB and P56S-VAPB were fixed with 2.5% buffered glutaraldehyde and processed for EM. Several membrane-bound vacuolar structures (white arrows) often containing cargoes and probably derived from the ER in a representative P56S-VAPB-expressing cell compared to cells expressing wt-VAPB control. Higher magnification showing the ER membrane proliferation into tubular structures (black arrow; lower left panel), and large autophagic vacuoles often filled with cargoes (white arrow; lower panel) in representative P56S-VAPB expressing cells. Scale bars: 1 µm (upper panel), 0.5 µm (lower panel).