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.

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.

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.

An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies.

An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC50 ranging from 1.14 to 2.13 µg ml⁻¹. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC50 values of 22, 15 and 34 ng ml⁻¹, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC50 of 0.38 ng ml⁻¹. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.

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.