Yu. L. Baburina

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca2+ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.

Age-related effect of melatonin on permeability transition pore opening in rat brain mitochondria

Aging is accompanied by mitochondrial dysfunction related with lowering of the respiratory complex activity and decrease of ATP synthesis, as well as by an enhancement of oxidative stress and increased sensitivity to mitochondrial permeability transition pore (mPTP) opening in mitochondral triggering the programmed cell death. In the present work we studied the effect of natural antioxidant (melatonin) on parameters of mPTP detected in non-synaptic mitochondria isolated from the brain of young and old rats (3 and 18 months, resp.) with different melatonin treatments; namely, melatonin was either directly applied to the mitochondrial suspension or chronically administered to rats with drinking water. The data obtained have shown that mitochondria isolated from brain of old rats were more susceptive to induction of mPTP. Melatonin added directly to suspension of brain mitochondria isolated from young rats demonstrated a proapoptotic effect. A prolonged chronical treatment with melatonin of old rats produced an anti-apoptotic protective effect. Non-synaptic mitochondria isolated from the brain of old rats treated with melatonin were more resistant to the mPTP opening and demonstrated the activation of respiration of mitochondria as compared to the untreated rats.

Effect of melatonin on stress-induced opening of non-selective pore in mitochondria from brain of young and old rats

Oxidative stress is the major factor affecting different organs during aging. Mitochondria are considered to be a source of endogenous oxidants, concentrations of which can be maintained at a low level by antioxidant enzymes. At present, the aging process is considered to be tightly related with mitochondrial dysfunction, one of the reasons of which might be an increased sensitivity to induction of permeability transition pore in the inner membrane of mitochondria. Currently, the role of melatonin, concentration of which is lowered with aging, is widely examined. In the present study, the effect of melatonin on characteristics of stress-induced mPTP opening in mitochondria isolated from young and old rats, treated or not treated with melatonin, has been examined. Oxidative stress was induced by 1 or 100 μM cumene hydroperoxide. It was found that chronic treatment of old rats with melatonin caused suppression of mPTP opening in mitochondria. Melatonin was found to be able to prevent the cumene hydroperoxide-induced swelling of mitochondria isolated from melatonin-treated rats.

2′,3′-cyclic nucleotide phosphodiesterase (CNPase) as a target in neurodegenerative diseases

We review the structure, characteristics, post-translational modifications, and functioning of 2′,3′-cyclic nucleotide phosphodiesterase (CNPase) localized in the CNS myelin and Schwann cells, as well as previously unknown properties of this protein, which was identified in brain mitochondria. We summarize the data on the localization of CNPase in the cell, the specificity of its substrates, the characteristics of the N- and C-terminals of the molecule, and its interactions with proteins-partners and nucleic acids. We describe the possible role of CNPase in ageing and neurodegenerative diseases, such as Alzheimer’s disease and multiple sclerosis, with the main emphasis on the role of mitochondria in these processes.

The identification of phosphorylated forms of myelin basic protein associated with mitochondria

Current data demonstrate that neurodegenerative processes are associated with mitochondrial dysfunction. One of the causes of mitochondrial dysfunction is increased permeability of the inner membrane and the formation of the so-called non-selective mitochondrial permeability transition pore (mPTP), which is recognized as an early stage of programmed cell death. One of the factors of neurodegeneration is disruption of the interaction between glial cells and axons. We recently demonstrated that the myelin protein 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase), which is localized in mitochondria, is involved in the regulation of the mPTP. Here, we show that two phosphoproteins with molecular weights of 21.5 and 17 kDa increase their phosphorylation state after pore opening. Using two-dimensional electrophoresis followed by mass spectrometry, we identified these proteins as phosphorylated isoforms of the myelin basic protein (MBP). We found that association of the 21.5 and 17 kDa isoforms of MBP increased under conditions of an opened pore. Thus, one more myelin protein is probably involved in the regulation of the mPTP.

Age-dependent changes of mitochondrial functions in Ca2+-induced opening of permeability transition pore

Mitochondria are intracellular organelles, which provide cells with energy and participate in multiple processes of cell vital functions. Within one of the numerous theories of aging, dysfunction of mitochondria is considered to lead to tissue degeneration and induce the initial stage in developing of degenerative diseases. Since mitochondria play a clue role in apoptosis/necrosis processes, it was suggested that dysfunction of mitochondria observed under aging is related with disturbance of programmed cell death regulation. In the present study, a comparative examination of parameters of the functional states of mitochondria isolated from young (2–3-months old) and old (20–22-months old) rats under conditions of opening of unselective pore (PTP, permeability transition pore) has been performed. Ca2+ accumulation rate in mitochondria isolated from old rats was found to be decreased by 25–30%, threshold calcium concentration was lowered to 50%, and the swelling of mitochondria loaded by calcium was stimulated 3–4-fold. Production of reactive oxygen species (ROS) has been also determined in these mitochondria. In old mitochondria superoxide anion level was increased. In addition, H2O2 content was found to be 2 times higher in mitochondria with PTP opened. Using electron microscopy method, a decreased amount of cristae in mitochondria was revealed under aging.

Melatonin Modulates Phosphorylation of 2′,3′-Cyclic Nucleotide-3′-Phosphodiesterase in the Presence of Protoporphyrin IX in the Brain Mitochondria of Rats during the Functioning of the Non-Specific Mitochondrial Pore

Melatonin (N-acetyl-5-methoxytryptamine), a neuroendocrine hormone of the pineal gland, participates in the modulation of the mitochondrial nonspecific pore (mitochondrial permeability transition pore, mPTP). According to the results we obtained, protoporphyrin IX (PPIX, a ligand of the TSPO translocator protein) induces the opening of the mPTP in brain mitochondria; melatonin slows the induction of the mPTP in rat brain mitochondria incubated with PPIX. Induction of the mPTP activates protein kinases/protein phosphatases that are involved in the regulation of protein phosphorylation. The TSPO modulates protein phosphorylation; in the presence of PPIX, the phosphorylation of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) increases. Here, we showed an increase in the degree of phosphorylation of CNPase in PPIX-treated rat brain mitochondria, a melatonin-induced decrease in level of CNPase phosphorylation in mitochondria incubated with PPIX; hence, we propose that melatonin participates in TSPOmodulated protein phosphorylation.

Detection of Protein Kinase A and C Target Proteins in Rat Brain Mitochondria

Phosphorylation of some membrane-bound proteins in the mitochondria of rat liver and brain is regulated by Ca2+ and cAMP acting as secondary messengers. These proteins are the main myelin components: 46 kDa 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP) and two isoforms of the myelin basic protein (MBP) with molecular weights of 17 and 21.5 kDa, which we have identified previously and found outside myelin in rat brain mitochondria. The phosphorylation level of CNP and both MBP isoforms increases when the mitochondrial permeability transition pore (mPTP) is opened. It is known that protein kinases A and C in heart mitochondria are directly bound to mPTP regulator proteins and are able to modulate the pore function. It is shown in this study that the inhibitors of protein kinases A (H-89) and C (staurosporin, Go 6976, and GF 109203 X) decrease the phosphorylation level of CNP and two MBP isoforms allowing us to assume that they are the targets of the signaling protein kinases A and C.

The effects of isoquinoline carboxamide and melatonin on the differentiation of N1Е-115 mouse neuroblastoma cells (clone C-1300) and on the expression of the TSPO translocation protein and 2’,3’-cyclonucleotide-3’-phosphodiesterase in these cells

We studied the effect of isoquinoline carboxamide (PK11195) applied alone or in combination with melatonin on the differentiation of N1E-115 mouse neuroblastoma cells (clone C-1300). PK11195 is a synthetic ligand of the mitochondrial translocator protein (TSPO), which is one of the mitochondrial proteins that are responsible for the opening of the mitochondrial pore (mPTP); expression of this protein is enhanced in different types of cancer cells. PK11195 is considered as a potential anticancer drug. It has been shown that PK11195 at a nontoxic/subtoxic concentration induces the differentiation of the N1Е-115 mouse neuroblastoma cells and suppresses cell proliferation; the magnitude of this effect coincides with the effect induced by melatonin at a nontoxic concentration. The combination of PK11195 with melatonin did not intensify its effect as a differentiation inducer. Based on the results of Western blot analysis, it has been hypothesized that the differentiation of N1Е-115 neuroblastoma cells is associated with the expression of mitochondrial 2’,3’-cyclonucleotide-3’-phosphodiesterase but not with the expression of the mitochondrial translocator protein TSPO.