Mitochondrial permeability transition pore induction is linked to formation of the complex of ATPase C-subunit, polyhydroxybutyrate and inorganic polyphosphate.

Mitochondrial permeability transition pore (mPTP) opening allows free movement of ions and small molecules leading to mitochondrial membrane depolarization and ATP depletion that triggers cell death. A multi-protein complex of the mitochondrial ATP synthase has an essential role in mPTP. However, the molecular identity of the central 'pore' part of mPTP complex is not known. A highly purified fraction of mammalian mitochondria containing C-subunit of ATPase (C-subunit), calcium, inorganic polyphosphate (polyP) and polyhydroxybutyrate (PHB) forms ion channels with properties that resemble the native mPTP. We demonstrate here that amount of this channel-forming complex dramatically increases in intact mitochondria during mPTP activation. This increase is inhibited by both Cyclosporine A, an inhibitor of mPTP and Ruthenium Red, an inhibitor of the Mitochondrial Calcium Uniporter. Similar increases in the amount of complex formation occurs in areas of mouse brain damaged by ischemia-reperfusion injury. These findings suggest that calcium-induced mPTP is associated with de novo assembly of a channel comprising C-subunit, polyP and PHB.

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.