Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization.

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.

Involvement of oxidative stress in 3-nitropropionic acid neurotoxicity.

3-Nitroproprionic acid (3-NP) is a plant mycotoxin which produces selective striatal lesions in both experimental animals and in man. We previously found evidence that its neurotoxicity may be mediated by a secondary excitotoxic mechanism. In the present study we examined whether oxidative stress plays a role in the neurotoxicity of 3-NP in vivo. We examined whether the free radical spin traps alpha-phenyl-n-tert-butyl-nitrone (PBN), n-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) or 5,5-dimethyl-1-pyrroline-n-oxide (DMPO) could attenuate the neurotoxicity of 3-NP. Striatal lesions produced by systemic administration of 3-NP were protected by pretreatment with DMPO, but the toxicity of 3-NP was increased by PBN or S-PBN pretreatment. The content of 3-NP in the plasma was increased by S-PBN, but not by DMPO consistent with an effect of S-PBN on 3-NP metabolism. Lesions produced by systemic administration of 3-NP increased the production of hydroxyl free radicals (OH) in the striatum as assessed by the conversion of salicylate to 2,3 and 2,5 dihydroxybenzoic acid (DHBA). These results provide direct evidence that free radicals play a substantial role in the neurotoxicity of 3-NP induced neuronal injury.

Neuroprotective strategies for treatment of lesions produced by mitochondrial toxins: implications for neurodegenerative diseases.

Neuronal death in neurodegenerative diseases may involve energy impairment leading to secondary excitotoxicity, and free radical generation. Potential therapies for the treatment of neurodegenerative diseases therefore include glutamate release blockers, excitatory amino acid receptor antagonists, agents that improve mitochondrial function, and free radical scavengers. In the present study we examined whether these strategies either alone or in combination had neuroprotective effects against striatal lesions produced by mitochondrial toxins. The glutamate release blockers lamotrigine and BW1003C87 significantly attenuated lesions produced by intrastriatal administration of 1-methyl-4-phenylpyridinium. Lamotrigine significantly attenuated lesions produced by systemic administration of 3-nitropropionic acid. Memantine, an N-methyl-D-aspartate antagonist, protected against malonate induced striatal lesions. We previously found that coenzyme Q10 and nicotinamide, and the free radical spin trap n-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) dose-dependently protect against lesions produced by intrastriatal injection of malonate. In the present study we found that the combination of MK-801 (dizocipiline) with coenzyme Q10 exerted additive neuroprotective effects against malonate. Lamotrigine with coenzyme Q10 was more effective than coenzyme Q10 alone. The combination of nicotinamide with S-PBN was more effective than nicotinamide alone. These results provide further evidence that glutamate release inhibitors and N-acetyl-D-aspartate antagonists can protect against secondary excitotoxic lesions in vivo. Furthermore, they show that combinations of agents which act at sequential steps in the neurodegenerative process can produce additive neuroprotective effects. These findings suggest that combinations of therapies to improve mitochondrial function, to block excitotoxicity and to scavenge free radicals may be useful in treating neurodegenerative diseases.

Blockade of neuronal nitric oxide synthase protects against excitotoxicity in vivo.

Nitric oxide may be a key mediator of excitotoxic neuronal injury in the central nervous system. We examined the effects of the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) on excitotoxic striatal lesions. 7-NI significantly attenuated lesions produced by intrastriatal injections of NMDA, but not kainic acid or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) 7-NI attenuated secondary striatal excitotoxic lesions produced by the succinate dehydrogenase inhibitor malonate, and the protection was reversed by L-arginine but not by D-arginine, 7-NI produced nearly complete protection against striatal lesions produced by systemic administration of 3-nitropropionic acid (3-NP), another succinate dehydrogenase inhibitor, 7-NI protected against malonate induced decreases in ATP, and increases in lactate, as assessed by 1H magnetic resonance spectroscopy. 7-NI had no effects on spontaneous electrophysiologic activity in the striatum in vivo, suggesting that its effects were not mediated by an interaction with excitatory amino acid receptors. 7-NI attenuated increases in hydroxyl radical, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine generation in vivo, which may be a consequence of peroxynitrite formation. The present results implicate neuronal nitric oxide generation in the pathogenesis of both direct and secondary excitotoxic neuronal injury in vivo. As such they suggest that neuronal nitric oxide synthase inhibitors may be useful in the treatment of neurologic diseases in which excitotoxic mechanisms play a role.

Involvement of free radicals in excitotoxicity in vivo.

Recent evidence has linked excitotoxicity with the generation of free radicals. We examined whether free radical spin traps can attenuate excitotoxic lesions in vivo. Pretreatment with N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) significantly attenuated striatal excitotoxic lesions in rats produced by N-methyl-D-aspartate (NMDA), kainic acid, and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). In a similar manner, striatal lesions produced by 1-methyl-4-phenylpyridinium (MPP+), malonate, and 3-acetylpyridine were significantly attenuated by either S-PBN or alpha-phenyl-N-tert-butylnitrone (PBN) treatment. Administration of S-PBN in combination with the NMDA antagonist MK-801 produced additive effects against malonate and 3-acetylpyridine toxicity. Malonate injections resulted in increased production of hydroxyl free radicals (.OH) as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (DHBA). This increase was significantly attenuated by S-PBN, consistent with a free radical scavenging effect. S-PBN had no effects on malonate-induced ATP depletions and had no significant effect on spontaneous striatal electrophysiologic activity. These results provide the first direct in vivo evidence for the involvement of free radicals in excitotoxicity and suggest that antioxidants may be useful in treating neurologic illnesses in which excitotoxic mechanisms have been implicated.

Coenzyme Q10 and nicotinamide and a free radical spin trap protect against MPTP neurotoxicity.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) produces Parkinsonism in both experimental animals and in man. MPTP is metabolized to 1-methyl-4-phenylpridinium, an inhibitor of mitochondrial complex I. MPTP administration produces ATP depletions in vivo, which may lead to secondary excitotoxicity and free radical generation. If this is the case then agents which improve mitochondrial function or free radical scavengers should attenuate MPTP neurotoxicity. In the present experiments three regimens of MPTP administration produced varying degrees of striatal dopamine depletion. A combination of coenzyme Q10 and nicotinamide protected against both mild and moderate depletion of dopamine. In the MPTP regimen which produced mild dopamine depletion nicotinamide or the free radical spin trap N-tert-butyl-alpha-(2-sulfophenyl)-nitrone were also effective. There was no protection with a MPTP regimen which produced severe dopamine depletion. These results show that agents which improve mitochondrial energy production (coenzyme Q10 and nicotinamide) and free radical scavengers can attenuate mild to moderate MPTP neurotoxicity.

Coenzyme Q10 and nicotinamide block striatal lesions produced by the mitochondrial toxin malonate.

A potential mechanism of neuronal injury in neurodegenerative diseases is a defect in energy metabolism that may lead to slow excitotoxic neuronal death. Consistent with this possibility, we showed that specific inhibitors of the electron transport chain produce excitotoxic lesions in vivo. In the present study we examined whether agents that improve energy metabolism can block lesions produced by the mitochondrial toxin malonate. Striatal lesions produced by the complex II inhibitor malonate were blocked in a dose-dependent manner by oral pretreatment with coenzyme Q10. Administration of nicotinamide by Alzet pump for 1 week attenuated malonate-induced lesions, but riboflavin had no effect. Administration of nicotinamide intraperitoneally just prior to and following induction of the lesions produced dose-dependent neuroprotection. A combination of coenzyme Q10 with nicotinamide was more effective than either compound alone, as shown by both lesion size and magnetic resonance imaging in vivo. Both coenzyme Q10 and nicotinamide blocked adenosine triphosphate depletions and lactate increases. These results confirm that mitochondrial toxins produce striatal excitotoxic lesions by a mechanism involving energy depletion in vivo. Furthermore, they suggest novel neuroprotective strategies that may be useful in the treatment of both mitochondrial encephalopathies and neurodegenerative diseases.

3-Acetylpyridine produces age-dependent excitotoxic lesions in rat striatum.

The effects of 3-acetylpyridine (3-AP) were studied in rat striatum. Striatal injections of 3-AP produced dose-dependent lesions. The lesion size was significantly increased in 4- and 12-month-old rats compared to 1-month-old rats. Coinjection of the competitive N-methyl-D-aspartate (NMDA) antagonist 2-amino-5-phosphonovaleric acid (APV) or systemic administration of the noncompetitive NMDA antagonist MK-801, the competitive NMDA antagonist LY274614, or the glutamate release inhibitor lamotrigine partially but significantly attenuated striatal lesion volume. Consistent with an NMDA receptor-mediated excitotoxic effect, histologic studies showed that 3-AP lesions result in relative sparing of NADPH-diaphorase neurons. Using freeze clamp, 3-AP resulted in a marked depletion of ATP. Two-dimensional water-suppressed proton chemical shift magnetic resonance imaging showed a striatal depletion of the neuronal marker N-acetylaspartate but no focal increase in lactate during the first 3 h after intrastriatal 3-AP injections. Pretreatment with fructose-1,6-biphosphate attenuated the lesion volume significantly, which may be due to its ability to serve as a substrate for glycolytic metabolism, with resulting ATP production. The results of the present studies support the hypothesis that 3-AP produces an impairment of energy metabolism due to its substitution for niacinamide in the formation of NAD(P). Furthermore, 3-AP toxicity may involve a secondary excitotoxic mechanism mediated by NMDA receptors.

Systemic or local administration of azide produces striatal lesions by an energy impairment-induced excitotoxic mechanism.

Sodium azide is an inhibitor of cytochrome oxidase which produces selective striatal lesions in both rodents and primates. In the present study we investigated the neurochemical and histologic effects of both intrastriatal and systemic administration of sodium azide, as well as the age dependence and mechanism of the lesions. Intrastriatal administration of sodium azide produced dose-dependent lesions. Neurochemical and histologic evaluation showed that markers of both spiny projection neurons (GABA, substance P) and aspiny interneurons (somatostatin, neuropeptide Y, NADPH-diaphorase) were equally affected. Subacute systemic administration of sodium azide resulted in lesions with a similar neurochemical profile; however, in contrast to intrastriatal injections there was sparing of dopaminergic striatal afferents. Prior decortication significantly attenuated lesions produced by intrastriatal administration of sodium azide, consistent with an excitotoxic process. Chronic administration of sodium azide for 1 month lead to striatal neuropathological changes. Lesions produced by intrastriatal administration of sodium azide in 1-, 4-, and 12-month-old animals showed age dependence. Both freeze-clamp measurements and chemical-shift magnetic resonance spectroscopy confirmed that sodium azide impairs oxidative phosphorylation in the striatum following either intrastriatal or systemic administration. These results show that the striatum is particularly vulnerable to oxidative stress produced by sodium azide, and that it produces striatal lesions by a secondary excitotoxic mechanism.

Aminooxyacetic acid striatal lesions attenuated by 1,3-butanediol and coenzyme Q10.

We previously showed that intrastriatal administration of aminooxyacetic acid (AOAA) produces striatal lesions by a secondary excitotoxic mechanism associated with impairment of oxidative phosphorylation. In the present study, we show that and the specific complex I inhibitor rotenone produces a similar neurochemical profile in the striatum, consistent with an effect of AOAA on energy metabolism. Lesions produced by AOAA were dose-dependently blocked by MK-801, with complete protection against GABA and substance P depletions at a dose of 3 mg/kg. AOAA lesions were significantly attenuated by pretreatment with either 1,3-butanediol or coenzyme Q10, two compounds which are thought to improve energy metabolism. These results provide further evidence that AOAA produces striatal excitotoxic lesions as a consequence of energy depletion and they suggest therapeutic strategies which may be useful in neurodegenerative diseases.