Fig. 3: Effects of cardiolipin (CL) alteration on neuronal death.

A, B Cultured spinal cord neurons were treated with the designated concentrations of oxidized CL (OXCL) and non-oxidized CL for 24 h. Neuronal cell death was measured by LDH, a stable cytoplasmic enzyme that is present in all cells but only released when the plasma membrane is damaged. MTT assay was used to determine the cell viability and mitochondrial activity, since tetrazolium is reduced to formazan by mitochondrial dehydrogenase activity. The oxidized CL induced spinal cord neuronal death (A) as measured by LDH release and mitochondrial dysfunction (B) as measured by MTT in a dose-dependent manner. **P < 0.01 (Two-way ANOVA, Tukey’s multiple comparisons test). Data represent the mean ± s.e.m. from 3 independent experiments. C–H The spinal neuronal cultures were exposed to rotenone (125 nM) or H₂O₂ (50 μM) either in the absence or presence of 10 μM XJB-5-131 (XJB) for 24 h. XJB was added 30 min before rotenone or H₂O₂ administration, and the culture medium was removed for LDH at 24 h after oxidative stress. XJB-5-131 reversed rotenone or H₂O₂-induced CL loss (C, F), mitochondrial dysfunction (D, G), and neuronal death (E, H). *P < 0.05, **P < 0.01 (One-way ANOVA, Tukey post hoc test, n = 6/group). Data represent the mean ± s.e.m. from 3 independent experiments. I–P Effects of CL synthase (CLS) siRNA on primary spinal neuronal death in vitro. Cultured spinal cord neurons were transfected with CLS1 siRNA (r) for 24 h. I CL content was assayed using acridine orange 10-nonyl bromide (NAO, Invitrogen). Knocking down CL synthase, the key enzyme of de novo CL biosynthesis, with CLS1 siRNA significantly decreased CL. J–O Activated caspase-3/7 was examined using CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen). Apoptotic cells with activated caspase-3/7 showed bright green nuclei (arrows). Bar = 100 μm. P Bar graph showed that CLS1 siRNA significantly induced neuronal apoptosis, evidenced by increased number of activated caspase-3/7 cells. **P < 0.01 (Student t-test, n = 6/group). Data represent the mean ± s.e.m. from 3 independent experiments. Q–S Effects of exogenous CL on mitochondrial dysfunction and spinal neuronal death in the scratch injury model in vitro. Cultured spinal cord neurons were pre-incubated with CL liposomes for 30 min before scratch injury. Q Scratch injury-induced neuronal death was significantly reversed by exogenous CL in a dose-dependent manner. R, S Exogenous CL (100 µM) also significantly reversed scratch injury-induced CL loss (R) and decreased mitochondrial membrane potential (MMP) (S). Veh, vehicle; *P < 0.05, **P < 0.01 (One-way ANOVA, Tukey’s multiple comparisons test, n = 6/group). Data represent the mean ± s.e.m. from 3 independent experiments.