Fig 2: The expression of mitochondria fission (Drp1 (a), Fis1 (b), MFF (c)) and fusion (Mic60 (d), OPA1 (e)) genes in five tissues of Tibetan sheep. (1–5) Heart, liver, lung, brain, quadriceps femoris. (TS25) 2500 m altitude Tibetan sheep. (TS35) 3500 m altitude Tibetan sheep. (TS45) 4500 m altitude Tibetan sheep. Date shown on graph are means ± SEM. * p < 0.05, ** p < 0.01.

Fig 3: Effect of NaHS on mitochondrial cleavage and fusion proteins activity in HTR-8/SVneo cells. (A) Representative Western blot showing Drp1, p-Drp1 in cytoplasm and mitochondria in HTR-8/SVneo cells after different treatments. (B–E) Bar charts indicating different proteins between different groups. Results in the cytoplasm were normalized against GAPDH, and results in mitochondria were normalized against COX IV. The data are expressed as mean ± SEM. ***Vs. Control, P < 0.001. ##Vs. H2O2, P < 0.01. ###Vs. H2O2, P < 0.001.

Fig 4: Suppression of Drp1 hyperactivation attenuates NLRP3-dependent inflammasome activation and GSDMD-mediated pyroptotic injury in cell culture and AD mice.(A) Mature OLs were infected with lentivirus encoding Drp1 or control shRNA for 48 hours and maintained in the cell culture medium with puromycin (1 µg/ml), followed by treatment with oligomeric Aß1-42 (5 µM) for 24 hours. Western blot analysis of total protein lysates was carried out with the indicated antibodies. The densities of NLRP3, cleaved caspase-1, and GSDMD relative to actin are shown in the histograms. The released IL-1ß into the medium was measured by ELISA. The data from at least three independent experiments were analyzed. (B) Total lysates of mature OLs expressing GFP control vector (GFP Vec), GFP-Drp1 WT (WT), or GFP-Drp1 K38A (K38A) were subjected to Western blot analysis with anti-GSDMD antibody. The density of GSDMD cleavage relative to actin is shown in the histogram. The data are from four independent experiments. Total brain lysates were harvested from 6-month-old WT, CNP;Drp1fl/+, 5XFADhet;Drp1+/+, and 5XFADhet;CNP;Drp1fl/+ mice. n = 3 to 5 mice per group. (C) Drp1 tetramer was analyzed by Western blot analysis in the absence of ß-ME. The relative density of Drp1 tetramer versus the Drp1 monomer is shown in the histogram. (D) Western blot analysis of mouse corpus callosum lysates was carried out with the indicated antibodies. (E) The densities of Drp1, NLRP3, cleaved caspase-1, and GSDMD (CL) relative to actin are shown in the histograms. All the data are expressed as the mean ± SEM and compared by one-way ANOVA with Tukey’s post hoc test.

Fig 5: Drp1 hyperactivation in mature OLs of AD models.(A) Total protein lysates were harvested from the frozen postmortem cortex of AD patients and control subjects (Table 2). Western blot analysis was performed in the absence of 2-mercaptoethanol (ß-ME) to examine Drp1 oligomers. Histogram: the relative density of Drp1 tetramer versus Drp1 monomer is shown. Student’s t test. (B) Postmortem cortex sections from normal subjects and AD patients (Table 1) were stained with anti–p-Drp1 Ser616 and anti-CC1 antibodies. Scale bar, 50 µm. The number of p-Drp1 Ser616+ CC1+ cells per square millimeter was quantified and is presented in the histogram. Student’s t test. (C) Total protein lysates from the corpus callosum of WT and 5XFAD mouse brains were harvested at the ages of 3 and 6 months. Drp1 tetramer was analyzed by Western blot analysis in the absence of ß-ME. The relative density of Drp1 tetramer versus the Drp1 monomer is shown in the histogram. n = 3 mice per group. Student’s t test. (D) Mature OLs were differentiated from OPCs and exposed to oligomeric Aß1-42 for the indicated periods. Drp1 tetramer was analyzed by Western blot analysis of total protein lysates in the absence of ß-ME. The relative density of Drp1 tetramer versus the Drp1 monomer is shown in the histogram. The data from five independent experiments were analyzed using one-way ANOVA with Tukey’s post hoc test. (E) Mature OLs were treated with oligomeric Aß1-42 for 24 hours and stained with anti–p-Drp1 Ser616 (green) and anti-MBP (red) antibodies. The intensity of p-Drp1Ser616 in the MBP+OLs was quantitated and is presented in the histogram. The data from three independent experiments were compared using the Student’s t test. Scale bar, 20 µm. (F) A linear correlation between the relative Drp1 tetramer density and the cleavage of GSDMD is shown. All data are expressed as the mean ± SEM.