Attenuation of acute mitochondrial dysfunction after traumatic brain injury in mice by NIM811, a non-immunosuppressive cyclosporin A analog.

Following traumatic brain injury (TBI), mitochondrial function becomes compromised. Mitochondrial dysfunction is characterized by intra-mitochondrial Ca(2+) accumulation, induction of oxidative damage, and mitochondrial permeability transition (mPT). Experimental studies show that cyclosporin A (CsA) inhibits mPT. However, CsA also inhibits calcineurin. In the present study, we conducted a dose-response analysis of NIM811, a non-calcineurin inhibitory CsA analog, on mitochondrial dysfunction following TBI in mice, and compared the effects of the optimal dose of NIM811 (10 mg/kg i.p.) against an optimized dose of CsA (20 mg/kg i.p.). Male CF-1 mice were subjected to severe TBI utilizing the controlled cortical impact model. Mitochondrial respiration was assessed from animals treated with either NIM811, CsA, or vehicle 15 min post-injury. The respiratory control ratio (RCR) of mitochondria from vehicle-treated animals was significantly (p<0.01) lower at 3 or 12 h post-TBI, relative to shams. Treatment of animals with either NIM811 or CsA significantly (p<0.03) attenuated this reduction. Consistent with this finding, both NIM811 and CsA significantly reduced lipid peroxidative and protein nitrative damage to mitochondria at 12 h post-TBI. These results showing the ability of NIM811 to fully duplicate the mitochondrial protective efficacy of CsA supports the conclusion that inhibition of the mPT may be sufficient to explain CsA's protective effects.

Differential involvement of p38 and JNK MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite degeneration in striatal neurons.

The role of p38 and c-jun-N-terminal kinases 1/2, members of the mitogen-activated protein kinase family, in mediating the toxic effects of human immunodeficiency virus-1 transactivator of transcription (Tat) and gp120 were explored in primary mouse striatal neurons in vitro. Both Tat and gp120 caused significant increases in p38 and c-jun-N-terminal kinase mitogen-activated protein kinase phosphorylation, caspase-3 activity, neurite losses and cell death in striatal neurons. Tat-induced increases in caspase-3 activity were significantly attenuated by an inhibitor of c-jun-N-terminal kinase (anthra[1,9-cd]pyrazol-6(2H)-one), but not by an inhibitor of p38 ([4-(4-fluorophenyl)-2-(4-methylsul-finylphenyl)-5-(4-pyridyl)1 H-imidazole]), mitogen-activated protein kinase. However, despite preventing increases in caspase-3 activity, c-jun-N-terminal kinase inhibition failed to avert Tat-induced neuronal losses suggesting that the reductions in caspase-3 activity were insufficient to prevent cell death caused by Tat. Alternatively, gp120-induced increases in caspase-3 activity, neurite losses and neuronal death were prevented by p38, but not c-jun-N-terminal kinase, mitogen-activated protein kinase inhibition. Our findings suggest that gp120 induces neuronal dysfunction and death through actions at p38 mitogen-activated protein kinase, while Tat kills neurons through actions that are independent of p38 or c-jun-N-terminal kinase mitogen-activated protein kinase, or through the concurrent activation of multiple proapoptotic pathways.

Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation.

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

Ceramide inhibition of mammalian phospholipase D1 and D2 activities is antagonized by phosphatidylinositol 4,5-bisphosphate.

Ceramides inhibit phospholipase D (PLD) activity in several mammalian cell types. These effects have been related to preventing activation by ARF1, RhoA, and protein kinase C-alpha and -beta and therefore indicate that PLD1 is inhibited. In the present work, we investigated the effects of ceramides in inhibiting both PLD1 and PLD2 and the interaction with another activator, phosphatidylinositol 4,5-bisphosphate (PIP2). PLD1 and PLD2 were overexpressed separately in Sf9 insect cells using baculovirus vectors. In our cell-free system, PLD1 activity was inhibited completely by C2-ceramide at sub-optimum concentrations of PIP2 (3 and 6 microM), whereas at supra-optimum PIP2 concentrations (18 and 24 microM) C2-ceramide did not inhibit PLD1 activity. Partially purified PLD2 exhibited an absolute requirement for PIP2 when the activity was measured using Triton X-100 micelles. Ceramides inhibited PLD2 activity, and this inhibition was decreased as PIP2 concentrations increased. However, C2-ceramide also reversibly inhibited the activity of PLD1 and PLD2 mutants in which binding of PIP2 was decreased, indicating that ceramides are interacting with the catalytic core of the mammalian PLDs. By contrast, C2-ceramide failed to produce a significant inhibition of PLDs from bacteria and plants. Our results provide a novel demonstration that ceramides reversibly inhibit mammalian PLD2 as well as PLD1 activities and that both of these actions are more pronounced when PIP2 concentrations are rate-limiting.

Activation of LA-N-2 cell phospholipase D by amyloid beta protein (25-35).

Amyloid beta protein is the major protein component of neuritic plaques found in the brain of Alzheimer's disease. The activation of phospholipase D by amyloid beta protein (25-35), quisqualate and phorbol 12, 13-dibutyrate was investigated in LA-N-2 cells by measuring phosphatidylethanol formation. The activation of phospholipase D by quisqualate and APP (25-35) was calcium-independent. The AbetaP (25-35) and quisqualate activation of phospholipase D appeared to be mediated through a pertussis toxin-sensitive GTP-binding protein. Phospholipase D activation by AbetaP (25-35), quisqualate and phorbol dibutyrate was not blunted by the protein kinase C inhibitors, staurosporine, H-7 and RO-31-8220. However, it was abolished by overnight exposure to phorbol dibutyrate. This activation of phospholipase D was prevented by the tyrosine kinase inhibitor, genistein but not by tyrophostin A. Several excitatory amino acid antagonists were tested for their ability to prevent the phospholipase D activation by quisqualate and AbetaP (25-35). Only NBQX was effective with an IC50 of 75 microM for AbetaP (25-35) and quisqualate. Activation of phospholipase D by AbetaP or quisqualate was absent in LA-N-2 cells previously desensitized by quisqualate or AbetaP (25-35), but the activation by phorbol dibutyrate was unaltered. The responsiveness to AbetaP and quisqualate in previously desensitized cells reappeared subsequent to a period of resensitization. The observations with the antagonist NBQX, and the desensitization and resensitization experiments, are consistent with a receptor occupancy mediated activation of phospholipase D by quisqualate and by AbetaP (25-35).

(-)Nicotine inhibits the activations of phospholipases A2 and D by amyloid beta peptide.

It has been established that amyloid beta peptide (AbetaP) activates phospholipase A2, phospholipase C and phospholipase D of LA-N-2 cells and other cell types. Nicotine in addition to being a cholinergic agonist, may be neuroprotective. We have investigated the ability of (-)nicotine to blunt the phospholipase activations by AbetaP in LA-N-2 cells. (-)Nicotine inhibits the AbetaP activation of phospholipase A2, with an IC50 of 76 microM and of phospholipase D with an IC50 of 252 microM. (-)Nicotine did not blunt the AbetaP activation of phospholipase C. These inhibitions of AbetaP activations were not observed with (+)nicotine or cotinine. The (-)nicotine inhibition of AbetaP activation of these two phospholipases was unaffected by hexamethonium and D-tubocurarine. There was no inhibition of the phospholipase A2 activity present in homogenates of LA-N-2 cells. Exposure of LA-N-2 cells to (-)nicotine for 2 h resulted in the blockade of phospholipase A2 activation by kainate and AbetaP but did not affect the ability of quisqualate and AbetaP to activate phospholipase D. These data suggest that if the nicotine inhibition of AbetaP activations is receptor occupancy mediated then it is by an atypical receptor type.

Amyloid beta protein (25-35) stimulation of phospholipase C in LA-N-2 cells.

The amyloid beta protein (25-35) stimulated appearance of 3H-inositol phosphates from [3H]inositol-prelabeled LA-N-2 cells was investigated. This stimulation was unaltered by extra- and intracellular calcium chelators in a calcium-free medium or by several protein kinase inhibitors. This phospholipase C stimulation by amyloid beta protein appeared to be pertussis toxin sensitive. It is possible that this phospholipase C stimulation by amyloid beta protein is a receptor-mediated process. This possibility is based on two related observations. The stimulation is ablated by the presence of conventional antagonists for metabotropic, adrenergic, and bombesin agonists. The IC50 values were 12 microM for propranolol, 15 microM for AP-3, and 25 nM for [Tyr4,D-Phe12]bombesin. Additional support comes from results of desensitization and resensitization experiments. Amyloid beta protein stimulation of phospholipase C was absent from LA-N-2 cells previously treated with norepinephrine, trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), bombesin, or amyloid beta peptide. In a similar manner, LA-N-2 cells previously treated with amyloid beta protein were no longer responsive to norepinephrine, t-ACPD, or bombesin. The responsiveness to amyloid beta protein returned, subsequent to a period of resensitization for the individual agonists. It is suggested that this observed amyloid beta protein stimulation of phospholipase C may be responsible for the elevated quantity of inositol seen in the brains of Alzheimer's disease patients.

Membrane depolarization in LA-N-1 cells. The effect of maitotoxin is Ca(2+)- and Na(+)-dependent.

We investigated the influence of ion compositions on the membrane potential in LA-N-1 human neuroblastoma cells using bisoxonol as a potential-sensitive fluorescent dye. The ability of K+, ouabain, veratridine, and maitotoxin to induce membrane depolarization was evaluated. Increasing concentrations of K+ ions from 10 to 50 mM caused a dose-dependent increase of bisoxonol fluorescence, which was completely independent on Na+ and Ca2+. Ouabain (5 mM), an inhibitor of the Na+, K(+)-ATPase, failed to induce membrane depolarization. Veratridine (40 and 100 microM), a Na+ channel activator, only in the presence of 10 micrograms of Leiurus scorpion venom reduced the membrane potential. Maitotoxin (MTX) from 3 to 10 ng/mL depolarized LA-N-1 cells in a dose-dependent manner, and produced a rapid and sustained increase of intracellular free calcium monitored by means of fluorescent probe fura-2. The MTX-induced depolarization and the increase in cytosolic free calcium concentration were dependent on extracellular Ca2+ ions. On the other hand, Na+ ions also seem to be, although only partially, implicated in the MTX effects, since both the blockade of tetrodotoxin (TTX)-sensitive voltage-operated Na+ channels and the removal of Na+ ions were able to reduce the depolarization. In conclusion, our data indicate that the depolarizing action of MTX on LA-N-1 cells is Ca(2+)- and Na(+)-dependent, although the latter only partially, and that this effect is dependent on Ca2+ influx into the cells likely through a voltage-insensitive calcium-entry system.

Activation of LA-N-2 cell phospholipases by single alanine substitution analogs of amyloid beta peptide (25-35).

A series of single alanine substituted analogs of amyloid beta peptide (25-35) were tested for their ability to activate the phospholipases of cultured LA-N-2 cells. Substitution of alanine for the amino acids 29-34 prevented the activation of phospholipases A2 and D. In addition substitution of alanine at 28 prevented phospholipase D but not phospholipase A2 activation. All the alanine substitutions, except for positions 33 and 35, blunted phospholipase C activations. There were no activations by scrambled amyloid beta peptide.

Indomethacin and nordihydroguaiaretic acid inhibition of amyloid beta protein (25-35) activation of phospholipases A2 and D of LA-N-2 cells.

Amyloid beta protein (25-35) stimulates the phospholipase A2, C and D activation of LA-N-2 cells. Nordihydroguaiaretic acid reduced the phospholipase D activation by 30% (P < 0.008) and indomethacin reduced the phospholipase A2 activation by 58% (P < .001). There were no reductions of the amyloid beta protein activations by acetylsalicylic acid (ASA), gentisic acid, sulindac sulfone and acetaminophen. The activation of phospholipase C by amyloid beta protein was unaffected by these compounds.