Fig. 3: Dissociation of ATP synthase F₁ from F_O occurs under glutamate excitotoxic conditions.

A, C Immunoblot analysis of mitochondria isolated from hippocampal neurons before and after glutamate treatment in the absence or presence of CsA (n = 3 independent cultures, **P < 0.0017, ***P < 0.0003, ****P < 0.0001) or the absence or presence of a proteasome inhibitor (PI) (n = 3 independent cultures, ****P < 0.0001), respectively, one-way ANOVA Dunnett’s multiple comparisons test. These data confirm that F₁ separates from c-subunit, then F₁ components are proteolyzed. B, D Group data for cell death (lactate dehydrogenase (LDH) release) of hippocampal neurons under the indicated conditions. CsA, but not proteasome inhibitor (PI), inhibits cell death by preventing F₁/F_O dissociation. For CsA data: n = 3 independent cultures, ***P = 0.0007 for Con vs. Glu; *P = 0.0377 for Con vs. Glu+CsA; *P = 0.0434 for Glu vs. Glu+CsA, **P = 0.0053 for Glu vs. CsA. For proteasome inhibitor (PI) group data: n = 3 independent cultures, *P = 0.0376 for Con vs. Glu; *P = 0.0135 for Con vs. Glu+PI; *P = 0.0376 for Glu+PI vs. PI. Statistics for group data were calculated using one-way ANOVA Dunnett’s multiple comparisons test. Error bars refer to SEM. Antibodies for different ATP synthase subunits were used as indicated. E, F Images of propidium iodide (PID) staining of cultured hippocampal neurons before and after glutamate treatment (cells were treated with 20 μM glutamate at DIV 12–14 after transfection and stained with propidium iodide 18 h after glutamate stimulation). Primary hippocampal neurons express GFP, GFP plus c-subunit shRNA or GFP plus human c-subunit. Green: GFP; Red: PID. G Group data of propidium iodide staining of cultured hippocampal neurons, expressing the indicated constructs with or without glutamate treatment. 65–106 micrographs were used, n = 3–5 independent cultures, ***P = 0.0004, ****P < 0.0001, one-way ANOVA. Glutamate produces substantial cell death, which is increased by c-subunit overexpression and inhibited by knockdown of c-subunit with shRNA. The histogram represents the death of only GFP-expressing cells. H, I Images and group data of cell death measured with propidium iodide for HEK 293 cells transfected with constructs expressing GFP or GFP plus human c-subunit constructs, measured at 24 h after a 30 min. exposure to 1 mM H₂O₂ (10–23 wells under each condition, ***p < 0.0001, unpaired t-test). Green: GFP; Red: PID. c-subunit overexpression substantially increases cell death compared with H₂O₂ treatment alone. The histogram represents cell death of only GFP-expressing cells. J Immunoblot analysis after non-denaturing Blue Native Page (BNP) of mitochondria isolated from HEK 293 cells overexpressing Myc-tagged human c-subunit. Antibody for Myc-tag was used. c-subunit is only partially assembled in ATP synthase, detected as a band ~720 kDa position, while the band at ~242 kDa represents the higher-order c-ring oligomer uncomplexed with F₁. The gel is representative of 3 gels. K Schematic representation of non-reversible dissociation of ATP synthase F₁ from F_O during glutamate-induced excitotoxic conditions. ATP synthase subunits are drawn as ribbon representations (modified PDB ID code: 6J5I) [14].