Faculty Bibliography

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.

BACKGROUND: Calcium signaling plays a key role in the regulation of multiple processes in mammalian mitochondria, from cellular bioenergetics to the induction of stress-induced cell death. While the total concentration of calcium inside the mitochondria can increase by several orders of magnitude, the concentration of bioavailable free calcium in mitochondria is maintained within the micromolar range by the mitochondrial calcium buffering system. This calcium buffering system involves the participation of inorganic phosphate. However, the mechanisms of its function are not yet understood. Specifically, it is not clear how calcium-orthophosphate interactions, which normally lead to formation of insoluble precipitates, are capable to dynamically regulate free calcium concentration. Here we test the hypothesis that inorganic polyphosphate, which is a polymerized form of orthophosphate, is capable to from soluble complexes with calcium, playing a significant role in the regulation of the mitochondrial free calcium concentration. METHODS: We used confocal fluorescence microscopy to measure the relative levels of mitochondrial free calcium in cultured hepatoma cells (HepG2) with variable levels of inorganic polyphosphate (polyP). RESULTS: The depletion of polyP leads to the significantly lower levels of mitochondrial free calcium concentration under conditions of pathological calcium overload. These results are coherent with previous observations showing that inorganic polyphosphate (polyP) can inhibit calcium-phosphate precipitation and, thus, increase the amount of free calcium. CONCLUSIONS: Inorganic polyphosphate plays an important role in the regulation of mitochondrial free calcium, leading to its significant increase. GENERAL SIGNIFICANCE: Inorganic polyphosphate is a previously unrecognized integral component of the mitochondrial calcium buffering system.

Aggregation of alpha-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of alpha-synuclein on calcium signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that alpha-synuclein induces an increase in basal intracellular calcium in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that alpha-synuclein monomers induce irregular ionic currents, while alpha-synuclein oligomers induce rare discrete channel formation. Despite the ability for monomeric alpha-synuclein to affect calcium signalling, it is only the oligomeric form of alpha-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular calcium, which prevented the alpha-synuclein induced calcium dysregulation. The findings of this study confirm that alpha-synuclein interacts with membranes to affect calcium signalling in a structure-specific manner and the oligomeric beta sheet rich alpha-synuclein species ultimately leads to calcium dysregulation and calcium dependent cell death.

Mitochondrial permeability transition pore (mPTP) is a large channel located in the mitochondrial inner membrane. The opening of mPTP during pathological calcium overload leads to the membrane depolarization and disruption of ATP production. mPTP activation has been implicated as a central event during the process of stress-induced cell death. mPTP is a supramolecular complex composed of many proteins. Recent studies suggest that mitochondrial ATPase plays the central role in the formation of mPTP. However, the structure of the central conducting pore part of mPTP (mPTPore) remains elusive. Here we review current models proposed for the mPTPore and involvement of polyP in its formation and regulation. We discuss the underestimated role of polyP as an effector and a putative structural component of the mPTPore. We propose the hypothesis that inclusion of polyP can explain such properties of mPTP activity as calcium activation, selectivity and voltage-dependence.

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 cortical neuron cultures pretreated (24 h) with either E or Q (0.1-10 muM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3 muM) increased spare respiratory capacity but only E+Q (0.3 muM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in CREB (cAMP response element-binding protein) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1alpha). Consistent with these findings, E+Q (0.1 and 1.0 muM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0 muM), but not E (3.0 muM), elevated cytosolic calcium (Ca2+) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3 muM), but not Q (0.1 and 0.3 muM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-NG-Nitroarginine methyl ester (1.0 muM) blocked neuroprotection by E (0.3 muM) or Q (1.0 muM). Oral administration of E+Q (75 mg/kg; once daily for 5 days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca2+- mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance that confer profound protection against ischemic injury.

AIMS: The mitochondrial permeability transition pore (mPTP) plays a central role for tissue damage and cell death during ischemia-reperfusion. We investigated the contribution of mitochondrial inorganic polyphosphate (polyP), a potent activator of Ca2+-induced mPTP opening, towards mPTP activation and cardiac cell death in ischemia-reperfusion. METHODS AND RESULTS: A significant increase in mitochondrial Ca2+ ([Ca2+]m), ROS generation, mitochondrial membrane potential depolarization (DeltaPsim) and mPTP activity, but no cell death was observed after 20 min of ischemia. The [Ca2+]m increase during ischemia was partially prevented by the mitochondrial Ca2+ uniporter inhibitor Ru360 and completely abolished by the combination of Ru360 and the ryanodine receptor type 1 blocker dantrolene suggesting two complimentary Ca2+ uptake mechanisms. In the absence of Ru360 and dantrolene mPTP closing by polyP depletion or cyclosporine A decreased mitochondrial Ca2+ uptake suggesting that during ischemia Ca2+ can enter mitochondria through mPTP. During reperfusion a burst of endogenous polyP production coincided with a decrease in [Ca2+]m, a decline in superoxide generation and an acceleration of hydrogen peroxide (H2O2) production. H2O2 increase correlated with restoration of mitochondrial pHm and increase in cell death. mPTP opening and cell death on reperfusion were prevented by antioxidants Trolox and MnTBAP. Enzymatic polyP depletion did not affect mPTP opening during reperfusion, but increased ROS generation and cell death, suggesting that polyP plays a protective role in cellular stress response. Conclusions: Transient Ca2+/polyP-mediated mPTP opening during ischemia may serve to protect cells against cytosolic Ca2+ overload, while ROS/pH-mediated sustained mPTP opening on reperfusion induces cell death.

The transient receptor potential ion channel of the melastatin subfamily, TRPM8, is a major cold receptor in the peripheral nervous system. Along with the sensory neurons, the TRPM8 protein is highly expressed in the prostate epithelial cells, and this expression is regulated by androgens. Here we investigated the expression and intracellular localization of the TRPM8 channel in relationship to androgens. We performed experiments using human prostate tissues obtained from healthy individuals and patients with prostate cancer at various stages of the disease as well as in cultured cells. Using an immunohistochemistry approach, we detected an intensive colocalization pattern of the TRPM8 protein with endogenous androgens in all tissues tested, suggesting possible interactions. Co-immunoprecipitation experiments performed using cultured prostate epithelial cells, prostate cancer cells, and HEK-293 cells stably expressing TRPM8 further confirmed direct binding of the steroid hormone, testosterone, to the TRPM8 protein. Applications of picomolar concentrations of testosterone to the primary human prostate cells, endogenously expressing TRPM8, elicited Ca(2+) responses and channel currents, and those were inhibited in the presence of TRPM8 antagonist, N-(2-aminoethyl)-N-(4-(benzyloxy)-3-methoxybenzyl)thiophene-2-carboxamide hydrochloride. These results indicate that the TRPM8 channel is physically associated with testosterone and suggest that, in addition to a genomic role, testosterone plays a role in direct regulation of the TRPM8 channel function.

Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits.

The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) - sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix.