Fig. 7
Kaempferol enhances IP3R and VDAC1 association promoting mitochondrial Ca2+ uptake. a Representative images of proximity ligation assay (PLA) between IP3R and VDAC1 in C9-500 + saline and C9-500 + KMP treated mice, showing a notable increase in the number of puncta in C9-500 + KMP treated animals (One- way ANOVA: F = 100.0, P < 0.0001***, Sidak’s multiple comparison test: WT + saline vs C9-500 + saline, t = 5.303, ***; C9-500 + saline vs C9-500 + KMP, t = 16.31, ***). Scale bar = 10 μm, n = 3–4 mice per genotype/treatment. b Baseline and stimulated mitochondrial Ca2+ uptake traces in WT and C9-500 cortical neurons (dotted lines) and after KMP treatment (bold lines). KMP treatment significantly improves Ca2+ uptake in the mitochondria of C9-500 cortical neurons (number of neurons WT: 22, C9-500: 24, WT + KMP:21, C9-500 + KMP: 21; multiple t-test 100 s C9-500 mean = 0.967, C9-500 + KMP mean = 2.285, P < 0.0001). c Representative confocal images of spinal cord immunolabeled for GRP75 shows a significant increase in C9-500 and WT MNs treated with KMP (One-way ANOVA F = 102.8, P < 0.0001***, Sidak’s multiple comparison test: WT saline vs C9-500 saline t = 6.203, ***; C9-500 saline vs C9-500 + KMP t = 16.42, ***). Scale bars: 30 μm. d List of proteins found to specifically interact with GRP75 in C9-500 ventral spinal cord but not in WT after mass spectroscopy (MS) analysis of immunoprecipitation for GRP75. Specifically, in red are highlighted GRP75 and VDAC1 as an internal control of the experiment (present in both WT and C9-500 samples), and in green is highlighted ATP5J (ATP synthase factor 6 of the mitochondria), whose interaction with GRP75 was detected in C9-500 ventral spinal cord lysates, which are enriched for MNs. e PLA between ATP5J and GRP75 in WT and C9-500 at P125 and P200, showing notable increase in number of puncta in C9-500 at P125 when MNs express high levels of GRP75 (Unpaired t-test P125 WT vs C9-500, t = 25.28, ***; P200 WT vs C9-500, t = 3.58, ***). f Representative confocal images of WT and C9-500 MNs stained for GRP75 and ATP5J. MNs expressing high levels of GRP75 also express high levels of ATP5J. Right: Q.A of ATP5J expression across different disease stages: One-way ANOVA F = 95.75, P < 0.0001***; Sidak’s multiple comparison test: P30 WT n = 26 vs P30 C9-500 n = 46, t = 0.4823, n.s.; P125 WT n = 42 vs P125 C9-500 n = 59, t = 16.29***, P200 WT n = 27 vs P200 C9-500 n = 27, t = 3.795***. Scale bars: 30 μm. g Representative images of ATP5J staining depicting the increased intensity of ATP5J expression in WT and C9-500 treated with KMP (One-way ANOVA: F = 90.48, P < 0.0001***, Sidak’s multiple comparison test: WT saline vs WT + KMP t = 4.928, ***; C9-500 saline vs C9-500 + KMP t = 15.55, ***). Scale bars: 10 μm