Fig 1: Mitochondrial dynamics and function change upon PGAM5 deletion.a Mitochondrial morphology outlined by Tom20 antibodies in control and PGAM5−/− ARPE-19 cells. Scale bar = 20 µm. n = 5. b Western blots showing upregulation of phosphor-Drp1(S637) but not total Drp1 in PGAM5−/− ARPE-19 cells. α-Tubulin was used as loading control. n = 4. c Western blots showing PGAM5 cleavage and Drp1(S637) dephosphorylation in ARPE-19 cells by CCCP treatment. n = 3. d Co-immunoprecipitation experiment using Axin1 antibody, showing that Axin1 interacts with both Drp1 and cleaved PGAM5 in ARPE-19 cells. n = 3. e Western blots showing upregulation of mitochondrial proteins (Tom20, CYTC, CYPD) and downregulation of PGC1α in PGAM5−/− ARPE-19 cells. α-Tubulin was used as loading control. n = 4. f Increased mitochondrial DNA in PGAM5−/− ARPE-19 cells. n = 5. *p = 0.0111, two-tailed unpaired t-tests; error bars, mean ± s.e.m. g Increased mitochondrial protein Cypd in the RPE/choroid of Pgam5−/− mice. α-Tubulin was used as loading control. n = 3. h Decreased mitochondrial turnover in PGAM5−/− ARPE-19 by MitoTimer transfection and labeling. Scale Bar equals to 20 µm. n = 3. i Mitochondrial membrane potential change as labeled by JC-1 in WT and PGAM5−/− ARPE-19 cells. Scale bar = 20 µm. n = 5. j ATP level change as measured in short-term (1 week) and long-term (8 weeks) culture of WT and PGAM5−/− ARPE-19 cells. n = 3, ****p < 0.0001, two-way ANOVA Tukey’s multiple comparisons test; error bars, mean ± s.e.m. k ROS change as measured in short-term (1 week) and long-term (8 weeks) culture of WT and PGAM5−/− ARPE-19 cells. n = 3, *p < 0.05; ****p < 0.0001, two-way ANOVA Tukey’s multiple comparisons test. n.s. represents no significance. Error bars, mean ± s.e.m.; for assays in the figure, n represents the number of biologically independent experiments. Images were captured under same settings, and representative images were shown. Source data are available as a Source Data file.
Fig 2: Sevoflurane anesthesia reduces binding of CypD and ANT. (A) CypD mainly locates in isolated mitochondria, but not cytosol, and its level can be increased in the isolated mitochondria of WT NPCs following the sevoflurane anesthesia. (B) RT-PCR shows that sevoflurane anesthesia does not significantly increase the mRNA levels of CypD as compared to the control condition in WT NPCs (P = 0.802, Student’s t test, N = 3). (C) Co-immunoprecipitation studies show that sevoflurane anesthesia reduces the binding of CypD with ANT as compared to the control condition in H4 human neuroglioma cells. (D) Immunocytochemistry images show that there are both CypD bound with ANT (yellow arrow) and CypD separated with ANT (white arrow) in the H4 human neuroglioma cells following sevoflurane anesthesia. Specifically, the green dots demonstrated CypD (D, top left panel), and the red dots showed ANT (D, top right panel). The white arrows in the bottom two panels of D indicated the CypD that did not bind to ANT, and the yellow arrows in the bottom panels of D revealed the CypD that bonded to ANT.
Fig 3: Hypothesized pathway of CypD associated sevoflurane-induced cognitive impairment in young mice. Sevoflurane increases levels of CypD via reducing the binding of CypD with ANT. The increased CypD then causes mitochondrial dysfunctions and impairment of neurogenesis, eventually leading to cognitive impairment in young mice.
Fig 4: CypD derived fusion constructs exhibit mitochondrial localization. (A) Schematic drawing of the three peptide sequences inserted upstream of the genetically encoded Ca2+ indicator jRCaMP1b. (B–D) CypD-jRCaMP1b (shortened as CypD-jR), CypDS42A-jRCaMP1b (shortened as S42A-jR) and CypDN30-jRCaMP1b (shortened as N30-jR) all exhibited predominant localization inside the mitochondria of C2C12 cells. Immunostaining of TOM20 (green) highlights the outer mitochondrial membrane. Boxes highlight enlarged area. Grey strips in the enlarged image highlight areas for relative intensity (F − Fmin)/(Fmax − Fmin) profiling on the right. Scale bars: 5 µm, 1 µm.
Fig 5: Observation of mitoflash associated mitochondrial Ca2+ efflux in the perinuclear region of myofibers. (A) Representative time-lapse recording of mouse FDB myofibers comparing CypD-jRCaMP1b (red hot pseudo-color) and mt-cpYFP (rainbow RGB pseudo-color) signals in the perinuclear mitochondria. X-Y view of the myofiber is collapsed along the time axis (T) and X-T view is collapsed along the Y axis. Relative intensity changes (ΔF/F0) are measured at the perinuclear area (white arrows). Black arrows denote drop of jRCaMP1b intensity accompanying mitoflash events. (B) Representative recording of mouse FDB myofibers comparing CypDS42A-jRCaMP1b and mt-cpYFP signals in perinuclear mitochondria. Black arrows denote drop of jRCaMP1b intensity accompanying mitoflash events. (C) Representative recording of mouse FDB myofibers comparing CypDN30-jRCaMP1b and mt-cpYFP in perinuclear mitochondria. Scale bars, 10 µm.
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