Fig. 4

ERp57 preserves neuromuscular junction connectivity of ALS mice. a Neuromuscular junction (NMJ) staining of lumbrical muscle at post-natal day 90. Anti-SV2 and anti-NF-M staining correspond to pre-synaptic component (pseudocolored red). α-Bungarotoxin coupled to Alexa 488 (pseudocolored green) corresponds to post-synaptic endplate. Representative confocal optical sections of three animals per genotype are shown. Scale bar: 50 μm. b Analysis of NMJ integrity of lumbrical muscle at post-natal day 90. Confocal optical sections of pre- and post-synaptic components were binarized and automatically subtracted to quantify endplate area without pre-synaptic component (unoccupied NMJ). Representative confocal optical sections of non-Tg and SOD1^G93A mice are shown. Scale bar: 50 μm. c Distribution of NMJ occupied area (as percentage of endplate area) in lumbrical muscle at post-natal day 90. NMJ overlap histograms with Gaussian fits are shown. Statistical analysis was performed using non-linear fit followed by extra sum-of-squares F test comparing different genotype curve fits to non-Tg fit. p values: n.s., p > 0.05; ***, p ≤ 0.001 (n = 4 animals per genotype with 65–201 NMJ per animal quantified). d Analysis of c showing NMJ occupied area as average percentage of total endplate areas. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. Mean ± S.E. is shown; p values: *, p ≤ 0.05; ***, p ≤ 0.001 (n = 4 animals per genotype with 65–201 NMJ per animal quantified). e NSC-34 cell lines stably expressing wild-type SOD1 (SOD1^WT) or mutant SOD1 (SOD1^G93A) were transfected with constructs to express wild-type human ERp57 coupled to V5 tag (ERp57^WT-V5) or YFP (Control, pseudocolored green). Cells were differentiated by serum deprivation for 24 h. Immunofluorescence staining against V5 tag was performed (pseudocolored green) along with Hoechst 33,342 staining (pseudocolored blue). Scale bar: 20 μm. f Analysis of neurite sprout of cells described in e. –FBS: serum deprivation. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. Mean ± S.E. is shown; p values: n.s., p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001 (n = 3 independent experiments). g Volcano plots of proteomic analysis of lumbar spinal cord at post-natal day 90. Each panel shows a different comparison between genotypes. Statistical analysis was performed using multiple t-test with two-stage step-up method using Benjamini, Krieger and Yekutieli approach with a False Discovery Rate of 5%. Hits with q-value ≤ 0.05 and p value ≤ 0.05 are highlighted on each plot (grey dots and black border). Selected hits with q-value ≤ 0.05 and p value ≤ 0.05 that are contributions from each genotype are highlighted on each plot (ERp57^WT: solid green, SOD1^G93A: solid red, SOD1^G93A/ERp57^WT: solid blue) (n = 3–4 animals per genotype). h Venn diagram of proteomic hits from genotype pair comparisons. Hits with q-value ≤ 0.05 and p value ≤ 0.05 were considered for analysis. i Schematic representation of ERp57 involvement in molecular and cellular pathways of ALS pathophysiology