The nNOS-p38MAPK pathway is mediated by NOS1AP during neuronal death.

Neuronal nitric oxide synthase (nNOS) and p38MAPK are strongly implicated in excitotoxicity, a mechanism common to many neurodegenerative conditions, but the intermediary mechanism is unclear. NOS1AP is encoded by a gene recently associated with sudden cardiac death, diabetes-associated complications, and schizophrenia (Arking et al., 2006; Becker et al., 2008; Brzustowicz, 2008; Lehtinen et al., 2008). Here we find it interacts with p38MAPK-activating kinase MKK3. Excitotoxic stimulus induces recruitment of NOS1AP to nNOS in rat cortical neuron culture. Excitotoxic activation of p38MAPK and subsequent neuronal death are reduced by competing with the nNOS:NOS1AP interaction and by knockdown with NOS1AP-targeting siRNAs. We designed a cell-permeable peptide that competes for the unique PDZ domain of nNOS that interacts with NOS1AP. This peptide inhibits NMDA-induced recruitment of NOS1AP to nNOS and in vivo in rat, doubles surviving tissue in a severe model of neonatal hypoxia-ischemia, a major cause of neonatal death and pediatric disability. The highly unusual sequence specificity of the nNOS:NOS1AP interaction and involvement in excitotoxic signaling may provide future opportunities for generation of neuroprotectants with high specificity.

A comparison of Zn2+- and Ca2+-triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition.

Intracellular Zn(2+) toxicity is associated with mitochondrial dysfunction. Zn(2+) depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn(2+)-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn(2+)-induced depolarization with the effects of Ca(2+) in single isolated rat liver mitochondria monitored with the potentiometric probe rhodamine 123. Consistent with previous work, we found that relatively low levels of Ca(2+) caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg(2+), ADP and cyclosporine A. Zn(2+) also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn(2+)-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca(2+) and Zn(2+) in a calcein-retention assay. Consistent with the well-documented ability of Ca(2+) to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn(2+)-treated mitochondria. Considered together, our results suggest that Ca(2+) and Zn(2+) depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn(2+)-induced depolarization, and that Zn(2+) is not a particularly potent mitochondrial inhibitor.

Distinct characteristics of Ca(2+)-induced depolarization of isolated brain and liver mitochondria.

Ca(2+)-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca(2+) concentrations (about 30--100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca(2+); 20 microm Ca(2+) was required to depolarize liver mitochondria. Ca(2+) did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca(2+)-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.

Direct visualization of mitochondrial zinc accumulation reveals uniporter-dependent and -independent transport mechanisms.

Current evidence suggests that zinc kills neurons by disrupting energy production, specifically by inhibiting mitochondrial function. However it is unclear if the inhibitory effect requires zinc accumulation, and if so, precisely how zinc enters mitochondria. Here, using fluorescence microscopy to visualize individual rat brain mitochondria, we detected matrix zinc uptake using the fluorophore FluoZin-3. Fluorescence increased rapidly in mitochondria treated with micromolar free zinc, and was quickly returned to baseline by membrane permeant chelation. Zinc uptake occurred through the calcium uniporter, because depolarization or uniporter blockade reduced fluorescence changes. However, increased fluorescence under these conditions suggests that zinc can enter through a uniporter-independent pathway. Fluorescence steadily declined over time and was unaffected by acidification or phosphate depletion, suggesting that zinc precipitation is not a mechanism for reducing matrix zinc. Uniporter blockade with ruthenium red also did not change the rate of zinc loss. Instead, zinc appears to exit the matrix through a novel efflux pathway not yet identified. Interestingly, dye-loaded mitochondria showed no fluorescence increase after treatment with strong oxidants, arguing against oxidant-labile intra-mitochondrial zinc pools. This study is the first to directly demonstrate zinc accumulation in individual mitochondria and provides insight about mechanisms mediating mitochondrial zinc uptake and efflux.

Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+.

In this study we investigated fluctuations in mitochondrial membrane potential (DeltaPsim) in single isolated brain mitochondria using fluorescence imaging. Mitochondria were attached to coverslips and perfused with K+-based buffer containing 20 microM EDTA, supplemented with malate and glutamate, and rhodamine 123 for DeltaPsim determination. DeltaPsim fluctuations were triggered by mitochondrial Ca2+ uptake since they were inhibited by both ruthenium red, a Ca2+-uniporter blocker, and by high concentrations of EGTA. A very low concentration of Ca2+ (approximately 30 nM) was required to initiate the fluctuations. Both ATP and ADP reversibly inhibited DeltaPsim fluctuations, with maximal effects occurring at 100 microM. The effect of nucleotides could not be explained by the reversed mode of mitochondrial ATP-synthase, since oligomycin was not effective and nonhydrolysable analogs of ATP and ADP did not stop the fluctuations. The effects of adenine nucleotides were abolished by blockade of the adenine nucleotide translocator with carboxyatractyloside, but were insensitive to another inhibitor, bongkrekic acid. ATP-sensitive K+-channels are not involved in the mechanism of DeltaPsim fluctuations, since the inhibitor 5-hydroxydecanoate or the activator diazoxide did not affect dynamics of DeltaPsim. We suggest DeltaPsim fluctuations in brain mitochondria are not spontaneous, but are triggered by Ca2+ and are modulated by adenine nucleotides, possibly from the matrix side of the inner mitochondrial membrane.

Glucose deprivation produces a prolonged increase in sensitivity to glutamate in cultured rat cortical neurons.

In this study we investigated whether the link between mitochondrial dysfunction and deregulation of Ca(2+) homeostasis preceding excitotoxic cell death is mediated by cellular deenergization. Glycolytic and/or mitochondrial ATP synthesis was inhibited with 2-deoxy-D-glucose (2DG) and oligomycin, respectively. Changes in cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) and mitochondrial membrane potential were simultaneously measured in response to low (10 microM) glutamate concentrations, using the fluorescence dyes fura-2FF and rhodamine 123. 2DG, which blocks glycolysis and also inhibits mitochondrial respiration due to depletion of pyruvate, greatly increased and accelerated glutamate-induced elevation of [Ca(2+)](c) and mitochondrial depolarization. The 2DG-induced hypersensitivity to glutamate was observed even after 150-min washout of 2DG with glucose-containing medium, suggesting a permanent deterioration of mitochondrial function. Prior blockade of only glycolytic (2DG with pyruvate) or only mitochondrial (oligomycin) ATP synthesis did not affect neuronal sensitivity to glutamate. Collectively, these studies show that to maintain the sensitivity of neurons to glutamate at control levels at least one of the cellular sources of ATP production must be intact. Either glycolysis or oxidative phosphorylation can effectively support Ca(2+) homeostasis in cultured forebrain neurons.

Spontaneous changes in mitochondrial membrane potential in single isolated brain mitochondria.

In this study we measured DeltaPsim in single isolated brain mitochondria using rhodamine 123. Mitochondria were attached to coverslips and superfused with K(+)-based HEPES-buffer medium supplemented with malate and glutamate. In approximately 70% of energized mitochondria we observed large amplitude spontaneous fluctuations in DeltaPsim with a time course comparable to that observed previously in mitochondria of intact cells. The other 30% of mitochondria maintained a stable DeltaPsim. Some of the "stable" mitochondria began to fluctuate spontaneously during the recording period. However, none of the initially fluctuating mitochondria became stable. Upon the removal of substrates from the medium or application of small amounts of Ca(2+), rhodamine 123 fluorescence rapidly dropped to background values in fluctuating mitochondria, while nonfluctuating mitochondria depolarized with a delay and often began to fluctuate before complete depolarization. The changes in DeltaPsim were not connected to oxidant production since reducing illumination or the addition of antioxidants had no effect on DeltaPsim. Fluctuating mitochondria did not lose calcein, nor was there any effect of cyclosporin A on DeltaPsim, which ruled out a contribution of permeability transition. We conclude that the fluctuations in DeltaPsim reflect an intermediate, unstable state of mitochondria that may lead to or reflect mitochondrial dysfunction.