Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways.

In view of evidence that increased consumption of epicatechin (E) and quercetin (Q) may reduce the risk of stroke, we have measured the effects of combining E and Q on mitochondrial function and neuronal survival following oxygen-glucose deprivation (OGD). Relative to mouse cortical neuron cultures pretreated (24h) with either E or Q (0.1-10µM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3µM) increased spare respiratory capacity but only E+Q (0.3µM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in cyclic AMP response element binding protein (CREB) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1α). Consistent with these findings, E+Q (0.1 and 1.0µM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0µM), but not E (3.0µM), elevated cytosolic calcium (Ca(2+)) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3µM), but not Q (0.1 and 0.3µM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-N(G)-nitroarginine methyl ester (1.0µM) blocked neuroprotection by E (0.3µM) or Q (1.0µM). Oral administration of E+Q (75mg/kg; once daily for 5days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca(2+)-mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance which confer profound protection against ischemic injury.

Drp1 is dispensable for apoptotic cytochrome c release in primed MCF10A and fibroblast cells but affects Bcl-2 antagonist-induced respiratory changes.

BACKGROUND AND PURPOSE: Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and is thought to promote Bax/Bak-induced cytochrome c release during apoptosis. Conformationally active Bax, Bak and Bax/Bak-activating BH3-only proteins, such as Bim, are restrained by anti-apoptotic Bcl-2 proteins in cells that are 'primed for death'. Inhibition of Bcl-2/Bcl-xL/Bcl-w by the antagonist ABT-737 causes rapid apoptosis of primed cells. Hence, we determined whether Drp1 is required for cytochrome c release, respiratory alterations and apoptosis of cells that are already primed for death. EXPERIMENTAL APPROACH: We tested the Drp1 inhibitor mdivi-1 for inhibition of cytochrome c release in MCF10A cells primed by Bcl-2 overexpression. We measured ATP synthesis-dependent, -independent and cytochrome c-limited maximal oxygen consumption rates (OCRs) and cell death of immortalized wild-type (WT) and Drp1 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with ABT-737. KEY RESULTS: Mdivi-1 failed to attenuate ABT-737-induced cytochrome c release. ABT-737 decreased maximal OCR measured in the presence of uncoupler in both WT and Drp1 KO MEF, consistent with respiratory impairment due to release of cytochrome c. However, Drp1 KO MEF were slightly less sensitive to this ABT-737-induced respiratory inhibition compared with WT, and were resistant to an initial ABT-737-induced increase in ATP synthesis-independent O2 consumption. Nevertheless, caspase-dependent cell death was not reduced. Pro-apoptotic Bax was unaltered, whereas Bak was up-regulated in Drp1 KO MEF. CONCLUSIONS AND IMPLICATIONS: The findings indicate that once fibroblast cells are primed for death, Drp1 is not required for apoptosis. However, Drp1 may contribute to ABT-737-induced respiratory changes and the kinetics of cytochrome c release.

'Mild Uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress.

Cultured rat cerebellar granule neurons were incubated with low nanomolar concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) to test the hypothesis that 'mild uncoupling' could be neuroprotective by decreasing oxidative stress. To quantify the uncoupling, respiration and mitochondrial membrane potential (Deltapsi(m)) were determined in parallel as a function of FCCP concentration. Deltapsi(m) dropped by less than 10 mV before respiratory control was lost. Conditions for the valid estimation of matrix superoxide levels were determined from the rate of oxidation of the matrix-targeted fluorescent probe MitoSOX. No significant change in the level of matrix superoxide could be detected on addition of FCCP while respiratory control was retained, although cytoplasmic superoxide levels measured by dihydroethidium oxidation increased. 'Mild uncoupling' by 30 nmol/L FCCP did not alleviate neuronal dysregulation induced by glutathione depletion and significantly enhanced that due to menadione-induced oxidative stress. Low protonophore concentrations enhanced N-methyl-d-aspartate receptor-induced delayed calcium deregulation consistent with a decrease in the spare respiratory capacity available to match the bioenergetic demand of chronic receptor activation. It is concluded that the 'mild uncoupling' hypothesis is not supported by this model.

Postnatal brain development and neural cell differentiation modulate mitochondrial Bax and BH3 peptide-induced cytochrome c release.

Bax mediates cytochrome c release and apoptosis during neurodevelopment. Brain mitochondria that were isolated from 8-day, 17-day, and adult rats displayed decreasing levels of mitochondrial Bax. The amount of cytochrome c released from brain mitochondria by a peptide containing the BH3 cell death domain decreased with increasing age. However, approximately 60% of cytochrome c in adult brain mitochondria could be released by the BH3 peptide in the presence of exogenous human recombinant Bax. Mitochondrial Bax was downregulated in PC12S neural cells differentiated with nerve growth factor, and mitochondria isolated from these cells demonstrated decreased sensitivity to BH3-peptide-induced cytochrome c release. These results demonstrate that immature brain mitochondria and mitochondria from undifferentiated neural cells are particularly sensitive to cytochrome c release mediated by endogenous Bax and a BH3 death domain peptide. Postnatal developmental changes in mitochondrial Bax levels may contribute to the increased susceptibility of neurons to pathological apoptosis in immature animals.

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability.

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.

Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria.

This study tested the hypothesis that mitochondrial precursor targeting peptides can elicit the release of cytochrome c from both liver and brain mitochondria by a mechanism distinct from that mediated by the classical, Ca2+-activated permeability transition pore. Human cytochrome oxidase subunit IV signal peptide (hCOXIV1-22) at concentrations from 15 to 100 microM induced swelling, a decrease in membrane potential, and cytochrome c release in both types of mitochondria. Although cyclosporin A and bongkrekic acid were without effect, dibucaine, propanolol, dextran, and the uncoupler FCCP were each able to inhibit signal peptide-induced swelling and cytochrome c release. Adenylate kinase was coreleased with cytochrome c, arguing against a signal peptide-induced cytochrome c-specific pathway of efflux across the outer membrane. Taken together, the data indicate that a human mitochondrial signal peptide can evoke the release of cytochrome c from both liver and brain mitochondria by a unique permeability transition that differs in several characteristics from the classical mitochondrial permeability transition.