Possible Involvement of 2',3'-Cyclic Nucleotide-3'-Phosphodiesterase in the Protein Phosphorylation-Mediated Regulation of the Permeability Transition Pore.

Calcium as a secondary messenger regulates the phosphorylation of several membrane-bound proteins in brain and liver mitochondria. Regulation of the activity of different protein kinases and phosphatases by Ca2+ occurs through its binding with calmodulin. The protein phosphorylation is strongly dependent on the Ca2+-induced mitochondrial permeability transition pore (mPTP) opening. 2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNPase) was phosphorylated by protein kinases A and C. CNPase and melatonin (MEL) might interact with calmodulin. The effects of the calmodulin antagonist calmidazolium and the inhibitor of protein kinase A H89 on mPTP opening in rat brain mitochondria of male Wistar rats were investigated. In addition, the role of CNPase, serine/threonine kinases, and MEL in the mPTP opening was examined. The anti-CNPase antibody added to rat brain mitochondria (RBM) reduced the content of CNPase in mitochondria. The threshold [Ca2+] decreased, and mitochondrial swelling was accelerated in the presence of the anti-CNPase antibody. H89 enhanced the effect of anti-CNPase antibody and accelerated the swelling of mitochondria, while CmZ abolished the effect of anti-CNPase antibody under mPTP opening. The levels of phospho-Akt and phospho-GSK3ß increased, while the MEL content did not change. It can be assumed that CNPase may be involved in the regulation of these kinases, which in turn plays an important role in mPTP functioning.

2',3'-Cyclic nucleotide 3'-phosphodiesterase as a messenger of protection of the mitochondrial function during melatonin treatment in aging.

The process of aging is considered to be tightly related to mitochondrial dysfunction. One of the causes of aging is an increased sensitivity to the induction of mitochondrial permeability transition pore (mPTP) opening in the inner membrane of mitochondria. Melatonin, a natural antioxidant, is a hormone produced by the pineal gland. The role of melatonin whose level decreases with aging is well understood. In the present study, we demonstrated that long-term treatment of aged rats with melatonin improved the functional state of mitochondria; thus, the Ca2+ capacity was enhanced and mitochondrial swelling was deaccelerated in mitochondria. Melatonin prevented mPTP and impaired the release of cytochrome c and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) from mitochondria of both young and aged rats. Our data suggest that melatonin retains СNPase inside mitochondria, thereby providing the protection of the protein against deleterious effects of 2',3'-cAMP in aging.

Effect of the CRAC Peptide, VLNYYVW, on mPTP Opening in Rat Brain and Liver Mitochondria.

The translocator protein (TSPO; 18 kDa) is a high-affinity cholesterol-binding protein located in the outer membrane of mitochondria. A domain in the C-terminus of TSPO was characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide may participate in the regulation of mitochondrial membrane permeability. Herein, we report the effect of the synthetic CRAC peptide, VLNYYVW, on mitochondrial permeability transition pore (mPTP) opening. It was found that the CRAC peptide alone prevents the mPTP from opening, as well as the release of apoptotic factors (cytochrome c, AIF, and EndoG) in rat brain mitochondria (RBM). Co-incubation of CRAC, together with the TSPO drug ligand, PK 11195, resulted in the acceleration of mPTP opening and in the increase of apoptotic factor release. VLNYYVW did not induce swelling in rat liver mitochondria (RLM). 3,17,19-androsten-5-triol (19-Atriol; an inhibitor of the cholesterol-binding activity of the CRAC peptide) alone and in combination with the peptide was able to stimulate RLM swelling, which was Ca2+- and CsA-sensitive. Additionally, a combination of 19-Atriol with 100 nM PK 11195 or with 100 µM PK 11195 displayed the opposite effect: namely, the addition of 19-Atriol with 100 µM PK 11195 in a suspension of RLM suppressed the Ca2+-induced swelling of RLM by 40%, while the presence of 100 nM PK 11195 with 19-Atriol enhanced the swelling of RLM by 60%. Taken together, these data suggest the participation of the TSPO's CRAC domain in the regulation of permeability transition.

Mitochondrial 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) interacts with mPTP modulators and functional complexes (I-V) coupled with release of apoptotic factors.

We previously reported that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) is present in rat brain and liver mitochondria, in the outer membrane and mitoplasts. Substrates of CNP, 2',3'-cAMP and 2',3'-cNADP, were found to accelerate opening of mitochondrial permeability transition pore (mPTP). In purified non-synaptic mitochondria, CNP was observed to co-immunoprecipitate with main modulators of mPTP, i.e. VDAC, ANT, and cyclophilin D, as well as with tubulin and COX IV. Using Blue Native Electrophoresis, with following Western blot, CNP was revealed to associate with functional inner membrane mitochondrial complexes I-V. In Ca(2+) -overloaded mitochondria, association of CNP with complexes I-V was decreased. Cyclosporine A increased the association of CNP with complexes I and III, supporting the idea of the involvement of these complexes in mPTP function. 2',3'-cAMP enhanced CNP dissociation from complexes I, III, IV and V in Ca(2+)-overloaded mitochondria (i.e. when pore is opened). Association of CNP with complexes I, III, IV, and V was shown in mitochondria isolated from brain, liver and heart. Stimulation of the opening of the non-selective pore in mitochondria correlated with CNP release from mitochondria in parallel with release of cytochrome c, AIF and Endo G. In Ca(2+)-overloaded mitochondria, 2',3'-cAMP further accelerated the release of AIF, Endo G and CNP, but did not alter cytochrome c release. These results provide strong evidence that CNP, one of the possible regulators of mPTP complex, might be involved in the control of respiration and energy production in mitochondria. This reveals a new function of CNP outside the myelin structure.

In aging, the vulnerability of rat brain mitochondria is enhanced due to reduced level of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP) and subsequently increased permeability transition in brain mitochondria in old animals.

Aging is accompanied by progressive dysfunction of mitochondria associated with a continuous decrease of their capacity to produce ATP. Mitochondria isolated from brain of aged animals show an increased mitochondrial permeability transition pore (mPTP) opening. We recently detected new regulators of mPTP function in brain mitochondria, the enzyme 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and its substrates 2', 3'-cAMP and 2', 3'-cNADP, and the neuronal protein p42(IP4). Here, we compared parameters of mPTP opening in non-synaptic brain mitochondria isolated from young and old rats. In mitochondria from old rats (>18 months), mPTP opening occurred at a lower threshold of Ca(2+) concentration than in mitochondria from young rats (<3 months). mPTP opening in mitochondria from old rats was accelerated by 2', 3'-cAMP, which further lowered the threshold Ca(2+) concentration. In non-synaptic mitochondria from old rats, the CNP level was decreased by 34%. Lowering of the CNP level in non-synaptic mitochondria with aging was accompanied by decreased levels of voltage-dependent anion channel (VDAC; by 69%) and of p42(IP4) (by 59%). Thus, reduced levels of CNP in mitochondria could lead to a rise in the concentration of the mPTP promoter 2', 3'-cAMP. The level of CNP and p42(IP4) and, probably VDAC, might be essential for myelination and electrical activity of axons. We propose that in aging the reduction in the level of these proteins leads to mitochondrial dysfunction, in particular, to a decreased threshold Ca(2+) concentration to induce mPTP opening. This might represent initial steps of age-related mitochondrial dysfunction, resulting in myelin and axonal pathology.

Carbenoxolone induces permeability transition pore opening in rat mitochondria via the translocator protein TSPO and connexin43.

Ca(2+)-induced permeability transition pore (mPTP) opening in isolated rat brain mitochondria is promoted through targeting of connexin43. After a threshold Ca(2+) load, mitochondrial membrane potential drops and efflux of accumulated Ca(2+) from the mitochondrial matrix occurs, indicating the mPTP opening. Specific antibodies were used to assess the role of the translocator protein (18kDa; TSPO) and connexin43 in swelling of isolated rat liver and brain mitochondria induced by carbenoxolone and the endogenous TSPO ligand protoporphyrin IX. Mitochondrial membrane potential, Ca(2+) transport and oxygen consumption were determined using selective electrodes. All the parameters were detected simultaneously in a chamber with the selective electrodes. The phosphorylation state of mitochondrial protein targets was assessed. We report that Ca(2+)-induced mitochondrial swelling was strengthened in the presence of both carbenoxolone and protoporphyrin IX. The carbenoxolone- and protoporphyrin IX-accelerated mPTP induction in brain mitochondria was completely prevented by antibodies specific for the mitochondrial translocator protein (TSPO). The anti-TSPO antibodies were more effective than anti-сonnexin43 antibodies. Moreover, carbenoxolone-stimulated phosphorylation of mitochondrial proteins was inhibited by anti-TSPO antibodies. Taken together, the data suggests that, in addition to acting via connexion43, carbenoxolone may exert its effect on mPTP via mitochondrial outer membrane TSPO.

Identification of phosphorylated form of 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium.

In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca(2+) as a second messenger. One of the proteins, the 46 kDa phosphoprotein was found to be highly phosphorylated when Ca(2+)-induced permeability transition pore (mPTP) was opened in rat brain mitochondria (RBM). In the present study the 46 kDa phosphoprotein was identified as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) after purification by 2D diagonal electrophoresis following mass spectrometric analysis and Western blot probed with anti-CNP antibody. CNPase was discovered in immunoprecipitates of mitochondria, phosphorylated under both conditions (control and with opened mPTP). Status phosphorylation of CNPase was found to be higher in the inmmunoprecipiates of calcium-overloaded RBM. The phospohoserine and phosphotyrosine residues were detected in phosphorylated 46 kDa band (CNPase) as well as in CNPase immunoprecipitates indicating possible participation of tyrosine and serine protein kinases in phosphorylation of CNPase in mitochondria. The levels of phospo-Ser and phospho-Tyr were increased in RBM with mPTP opened. It was found that CNPase substrate, 2',3'-cAMP (5 µM) and, a non-competitive CNPase inhibitor, atractyloside (5 µM), were able to increase the level of CNPase phosphorylation in calcium-overloaded mitochondria, while CsA (mPTP blocker) was able to strong suppress the phosphorylation of the enzyme. Collectively, our results provide evidence that Ca(2+)-stimulated and mPTP-associated CNPase phosphorylation might be an important stage of mPTP regulation in mitochondria, revealing a new function of CNPase outside of myelin structure.

Potential role of subunit c of F0F1-ATPase and subunit c of storage body in the mitochondrial permeability transition. Effect of the phosphorylation status of subunit c on pore opening.

Phosphorylated and non-phosphorylated forms of the F0F1-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca2+ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca2+. Collectively, the data allowed us to suppose that subunit c F0F1-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status.

Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43.

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 µM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.

The mitochondria permeability transition pore complex in the brain with interacting proteins - promising targets for protection in neurodegenerative diseases.

Mitochondria increasingly attract attention as control points within the mechanisms of neuronal death. Mitochondria play a central role in swinging the balance in favor of either survival or death of brain tissue. Cell death in vertebrates proceeds mostly via the mitochondrial pathway of apoptosis. Permeability transition pore (PTP) development in mitochondria is a decisive stage of apoptosis. Therefore, regulation of the permeability of both outer and inner mitochondrial membranes helps to induce neuroprotection. Through PTP control, mitochondria can to a large degree manage the intracellular calcium homeostasis, and thus control the potent death cascade initiated by excess calcium. Here we summarize the evidence for the role of mitochondria in brain cell death. We describe the involvement of the 18-kDa translocator protein (TSPO; previously called peripheral benzodiazepine receptor), and of two new mitochondrial proteins, that is, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and p42(IP4) (also designated centaurin alpha1; ADAP 1), in the control of the PTP. Furthermore, ligands of TSPO, as well as substrates of CNP, are possible modulators of PTP function. This scenario of control and regulation of PTP function might provide multiple important targets, which are suitable for developing protective strategies for neurons and non-neuronal brain cells in therapies of neurodegenerative diseases.

Ca2+-dependent permeability transition regulation in rat brain mitochondria by 2',3'-cyclic nucleotides and 2',3'-cyclic nucleotide 3'-phosphodiesterase.

Recent evidence indicates that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a marker enzyme of myelin and oligodendrocytes, is also present in neural and nonneural mitochondria. However, its role in mitochondria is still completely unclear. We found CNP in rat brain mitochondria and studied the effects of CNP substrates, 2',3'-cyclic nucleotides, on functional parameters of rat brain mitochondria. 2',3'-cAMP and 2',3'-cNADP stimulated Ca(2+) overload-induced Ca(2+) release from mitochondrial matrix. This Ca(2+) release under threshold Ca(2+) load correlated with membrane potential dissipation and mitochondrial swelling. The effects of 2',3'-cyclic nucleotides were suppressed by cyclosporin A, a potent inhibitor of permeability transition (PT). PT development is a key stage in initiation of apoptotic mitochondria-induced cell death. 2',3'-cAMP effects were observed on the functions of rat brain mitochondria only when PT was developed. This demonstrates involvement of 2',3'-cAMP in PT regulation in rat brain mitochondria. We also discovered that, under PT development, the specific enzymatic activity of CNP was reduced. Thus we hypothesize that suppression of CNP activity under threshold Ca(2+) load leads to elevation of 2',3'-cAMP levels that, in turn, promote PT development in rat brain mitochondria. Similar effects of 2',3'-cyclic nucleotides were observed in rat liver mitochondria. Involvement of CNP in PT regulation was confirmed in experiments using mitochondria from CNP-knockdown oligodendrocytes (OLN93 cells). CNP reduction in these mitochondria correlated with lowering the threshold for Ca(2+) overload-induced Ca(2+) release. Thus our results reveal a new function for CNP and 2',3'-cAMP in mitochondria, being a regulator/promotor of mitochondrial PT.

The brain-specific protein, p42(IP4) (ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+.

In brain, p42(IP4) (centaurin-alpha1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf-GTPase activating proteins) is mainly expressed in neurons. p42(IP4) operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42(IP4) is localized in mitochondria, isolated from rat brain and from cells transfected with p42(IP4). In rat brain mitochondria we additionally found interaction of p42(IP4) with 2', 3'-cyclic nucleotide 3'-phosphodiesterase and alpha-tubulin by pull-down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42(IP4) is predominantly associated with the intermembrane space and the inner membrane. This localization of p42(IP4) indicates that p42(IP4) might have a still unknown mitochondrial function. We studied whether p42(IP4) is involved in Ca(2+)-induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42(IP4) in mitochondria. In mitochondria isolated from p42(IP4)-transfected mouse neuroblastoma cells, over-expression of p42(IP4) significantly decreased Ca(2+) capacity and lag time for Ca(2+) retention. Thus, we suggest that p42(IP4) is involved in the regulation of Ca(2+) transport in mitochondria. We propose that p42(IP4) promotes Ca(2+)-induced permeability transition pore opening and thus destabilizes mitochondria.

The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria.

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria.

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F0F1-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741-744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca2+-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (delta psiM) and the Ca2+-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca2+ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.