Zbigniew Binienda

Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects of L-carnitine

Excitotoxicity and disrupted energy metabolism are major events leading to nerve cell death in neurodegenerative disorders. These cooperative pathways share one common aspect: triggering of oxidative stress by free radical formation. In this work, we evaluated the effects of the antioxidant and energy precursor, levocarnitine (l‐CAR), on the oxidative damage and the behavioral, morphological, and neurochemical alterations produced in nerve tissue by the excitotoxin and free radical precursor, quinolinic acid (2,3‐pyrindin dicarboxylic acid; QUIN), and the mitochondrial toxin, 3‐nitropropionic acid (3‐NP). Oxidative damage was assessed by the estimation of reactive oxygen species formation, lipid peroxidation, and mitochondrial dysfunction in synaptosomal fractions. Behavioral, morphological, and neurochemical alterations were evaluated as markers of neurotoxicity in animals systemically administered with l‐CAR, chronically injected with 3‐NP and/or intrastriatally infused with QUIN. At micromolar concentrations, l‐CAR reduced the three markers of oxidative stress stimulated by both toxins alone or in combination. l‐CAR also prevented the rotation behavior evoked by QUIN and the hypokinetic pattern induced by 3‐NP in rats. Morphological alterations produced by both toxins (increased striatal glial fibrillary acidic protein‐immunoreactivity for QUIN and enhanced neuronal damage in different brain regions for 3‐NP) were reduced by l‐CAR. In addition, l‐CAR prevented the synergistic action of 3‐NP and QUIN to increase motor asymmetry and depleted striatal GABA levels. Our results suggest that the protective properties of l‐CAR in the neurotoxic models tested are mostly mediated by its characteristics as an antioxidant agent.

Effect of acute exposure to 3-nitropropionic acid on activities of endogenous antioxidants in the rat brain

The response of endogenous antioxidants to acute exposure of the mitochondrial inhibitor, 3-nitropropionic acid (3-NPA), was investigated in selected rat brain regions. Rats treated with 3-NPA (30 mg/kg, s.c.) were sacrificed at 30, 60, 90 and 120 min after injection to examine the alterations in reduced glutathione levels (GSH), and activities of antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in the hippocampus (HIP), frontal cortex (FC), and caudate nucleus (CN). CAT activity increased in the HIP 90 min after 3-NPA treatment. While cytosolic copper/zinc SOD (CuZn-SOD) and mitochondrial manganese SOD (Mn-SOD) levels increased in the FC at 120 min, only the Mn-SOD increased in the CN 90 min after treatment. The activity of GPx decreased in the HIP 120 min after 3-NPA injection. When compared with the control, administration of 3-NPA led to GSH depletion in HIP within 120 min. The depletion of GSH and induction of antioxidant enzyme activities after the 3-NPA exposure suggest conditions favorable for oxidative stress.

The Protective Role of L-Carnitine against Neurotoxicity Evoked by Drug of Abuse, Methamphetamine, Could Be Related to Mitochondrial Dysfunction

Abstract: There is growing evidence that suggests that brain injury after amphetamine and methamphetamine (METH) administration is due to an increase in free radical formation and mitochondrial damage, which leads to a failure of cellular energy metabolism followed by a secondary excitotoxicity. Neuronal degeneration caused by drugs of abuse is also associated with decreased ATP synthesis. Defective mitochondrial oxidative phosphorylation and metabolic compromise also play an important role in atherogenesis, in the pathogenesis of Alzheimers disease, Parkinsons disease, diabetes, and aging. The energy deficits in the central nervous system can lead to the generation of reactive oxygen and nitrogen species as indicated by increased activity of the free radical scavenging enzymes like catalase and superoxide dismutase. The METH‐induced dopaminergic neurotoxicity may be mediated by the generation of peroxynitrite and can be protected by antioxidants selenium, melatonin, and selective nNOS inhibitor, 7‐nitroindazole. L‐Carnitine (LC) is well known to carry long‐chain fatty acyl groups into mitochondria for β‐oxidation. It also plays a protective role in 3‐nitropropioinc acid (3‐NPA)‐induced neurotoxicity as demonstrated in vitro and in vivo. LC has also been utilized in detoxification efforts in fatty acid‐related metabolic disorders.

Possible Mechanism for the Neuroprotective Effects of l‐Carnitine on Methamphetamine‐Evoked Neurotoxicity

Abstract: Some of the damage to the CNS that is observed following amphetamine and methamphetamine (METH) administration is known to be linked to increased formation of free radicals. This increase could be, in part, related to mitochondrial dysfunction and/or cause damage to the mitochondria, thereby leading to a failure of cellular energy metabolism and an increase in secondary excitotoxicity. The actual neuronal damage that occurs with METH‐induced toxicity seems to affect dopaminergic cells in particular. METH‐induced toxicity is related to an increase in the generation of both reactive oxygen (hydroxyl, superoxide, peroxide) and nitrogen (nitric oxide) species. Peroxynitrite (ONOO−), which is a reaction product of either superoxide or nitric oxide, is the most damaging radical. It can be reduced by antioxidants such as selenium, melatonin, and the selective nNOS inhibitor, 7‐nitroindazole. METH‐induced toxicity has been previously shown to increase production of the peroxynitrite stress marker, 3‐nitrotyrosine (3‐NT), in vitro, in cultured PC12 cells, and also in vivo, in the striatum of adult male mice. Pre‐ and post‐treatment of mice with l‐carnitine (LC) significantly attenuated the production of 3‐NT in the striatum after METH exposure. LC is a mitochondriotropic compound in that it carries long‐chain fatty acyl groups into mitochondria for β‐oxidation. It was shown also to play a protective role against various mitochondrial toxins, such as 3‐nitropropionic acid. The protective effects of LC against METH‐induced toxicity could be related to its prevention of possible metabolic compromise produced by METH and the resulting energy deficits. In particular, LC may be maintaining the mitochondrial permeability transition (MPT) and modulating the activation of the mitochondrial permeability transition pores (mPTP), especially the cyclosporin‐dependent mPTP. The possible neuroprotective mechanism of LC against METH‐toxicity and the role of the mitochondrial respiratory chain and the generation of free radicals and their subsequent action on the MPT and mPTP are also being examined using an in vitro model of NGF‐differentiated pheochromocytoma cells (PC12). In preliminary experiments, the pretreatment of PC12 cells with LC (5 mM), added 10 min before METH (500 μM), indicated that LC enhances METH‐induced DA depletion. The role of LC in attenuating METH‐evoked toxicity is still under investigation and promises to reveal information regarding the underlying mechanisms and role of mitochondria in the triggering of cell death.

Neuroprotective role of L-carnitine in the 3-nitropropionic acid induced neurotoxicity

L-carnitine (LC) plays an important regulatory role in the mitochondrial transport of long-chain free fatty acids (FFA). 3-Nitropropionic acid (3-NPA) is known to induce cellular energy deficit and oxidative stress related neurotoxicity via an irreversible inhibition of the mitochondrial enzyme succinate dehydrogenase (SDH). Protective effects of L-carnitine on the neurotoxicity induced by 3-NPA have been shown in vitro. Here, the activities of SDH as well as the activity of the antioxidant enzymes, catalase (CAT), and superoxide dismutase (SOD) were measured in order to evaluate the protective action of LC against 3-NPA-induced neurotoxicity. Male, CD Sprague-Dawley rats, 3-month old, were injected with either 50 or 100 mg/kg of LC, i.p., 30-60 min prior to 3-NPA (30 mg/kg, s.c.) or with 3-NPA alone. Enzyme activities were assayed in caudate nucleus (CN), frontal cortex (FC), and hippocampus (HIP) post sacrifice. Increased activities of CAT and SOD were observed after treatment with 3-NPA alone. Pretreatment with low or high doses of LC was associated with attenuation of these increases equivalent to, or below, the control levels. In rats treated with 3-NPA alone, SDH activity was inhibited by 62% (CN), 50% (FC), and 65% (HIP) of controls. Pretreatment with LC prior to 3-NPA attenuated decreases of SDH activity in a dose-dependent manner. However, compared with control, the activity of SDH remained significantly lower in brain regions of treated rats despite the attenuation of inhibition by LC pretreatment (P<0.05). These data suggest protective effect of LC against 3-NPA-induced oxidative stress. It appears that the protective effect of LC against 3-NPA-induced oxidative stress is not mediated by the direct action of LC preventing the SDH inhibition but rather is achieved due to the actions of LC downstream of the SDH inhibition.

Effects of Metabolic Modifiers Such as Carnitines, Coenzyme Q10, and PUFAs against Different Forms of Neurotoxic Insults: Metabolic Inhibitors, MPTP, and Methamphetamine

Abstract: A number of strategies using the nutritional approach are emerging for the protection of the brain from damage caused by metabolic toxins, age, or disease. Neural dysfunction and metabolic imbalances underlie many diseases, and the inclusion of metabolic modifiers may provide an alternative and early intervention approach that may prevent further damage. Various models have been developed to study the impact of metabolism on brain function. These have also proven useful in expanding our understanding of neurodegeneration processes. For example, the metabolic compromise induced by inhibitors such as 3‐nitropropionic acid (3‐NPA), rotenone, and 1‐methyl‐4‐phenylpyridinium (MPP+) can cause neurodegeneration in animal models and these models are thought to simulate the processes that may lead to diseases such as Huntingtons and Parkinsons diseases. These inhibitors of metabolism are thought to selectively kill neurons by inhibiting various mitochondrial enzymes. However, the eventual cell death is attributed to oxidative stress damage of selectively vulnerable cells, especially highly differentiated neurons. Various studies indicate that the neurotoxicity resulting from these types of metabolic compromise is related to mitochondrial dysfunction and may be ameliorated by metabolic modifiers such as l‐carnitine (L‐C), creatine, and coenzyme Q10, as well as by antioxidants such as lipoic acid, vitamin E, and resveratrol. Mitochondrial function and cellular metabolism are also affected by the dietary intake of essential polyunsaturated fatty acids (PUFAs), which may regulate membrane composition and influence cellular processes, especially the inflammatory pathways. Cellular metabolic function may also be ameliorated by caloric restriction diets. L‐C is a naturally occurring quaternary ammonium compound that is a vital cofactor for the mitochondrial entry and oxidation of fatty acids. Any factors affecting L‐C levels may also affect ATP levels. This endogenous compound, L‐C, together with its acetyl ester, acetyl‐l‐carnitine (ALC), also participates in the control of the mitochondrial acyl‐CoA/CoA ratio, peroxisomal oxidation of fatty acids, and production of ketone bodies. A deficiency of carnitine is known to have major deleterious effects on the CNS. We have examined L‐C and its acetylated derivative, ALC, as potential neuroprotective compounds using various known metabolic inhibitors, as well as against drugs of abuse such as methamphetamine.

Oral administration of 3,4-methylenedioxymethamphetamine (MDMA) produces selective serotonergic depletion in the nonhuman primate.

MDMA (3,4-methylenedioxymethamphetamine) has been reported to produce serotonergic depletion in nonhuman primates at doses as low as 2.5 mg/kg (1-2 times the typical human dose). The current study evaluated the dose-response relationships of MDMA (1.25-20.0 mg/kg) using regional concentrations of serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), and home cage behavior as endpoints. Adult female rhesus monkeys (n = 16) were treated orally with 0, 1.25, 2.5, or 20.0 mg/kg MDMA twice daily for 4 consecutive days. Eighteen behaviors were measured in the home cage prior to, during, and after MDMA treatment. One month after the last dose, the animals were sacrificed and brains dissected into several regions for neurochemical analyses. 5-HT and 5-HIAA were analyzed via HPLC/EC. The lower doses of MDMA (1.25 and 2.5 mg/kg) did not significantly alter 5-HT or 5-HIAA concentrations in any brain region except hippocampus in which 5-HT concentrations were decreased after 2.5 mg/kg. MDMA at 20.0 mg/kg significantly decreased 5-HT and 5-HIAA concentrations in several cortical and midbrain structures. However, 5-HT and 5-HIAA concentrations in brain stem and hypothalamus were not significantly altered after any dose of MDMA. Combined with previous data from this laboratory, these results indicate that the decreased concentrations of 5-HT and 5-HIAA in selected brain regions show a selective dose-response relationship for MDMA-induced neurotoxicity as measured by serotonergic depletion in the nonhuman primate.

Neuroprotective Effects of l‐Carnitine in Induced Mitochondrial Dysfunction

Abstract: The neuroprotective action of l‐carnitine (LC) in the rat model of 3‐nitropropionic acid (3‐NPA)‐induced mitochondrial dysfunction was examined. 3‐NPA is known to produce decreases in neuronal ATP levels via inhibition of the succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain. SDH is involved in reactions of the Krebs cycle and oxidative phosphorylation, and its inhibition leads to both necrosis and apoptosis. LC enhances mitochondrial metabolism and, together with its acetylated form, acetyl‐l‐carnitine (ALC), via the LC‐ALC‐mediated transfer of acetyl groups, plays an important modulatory role in neurotransmitter signal transduction pathways and gene expression in neuronal cells. In the study described here, adult male Sprague‐Dawley rats were injected with 3‐NPA alone or treated with LC prior to 3‐NPA administration. Pretreatment with LC totally prevented the 3‐NPA‐induced decrease in brain temperature measured using temperature probes implanted intracranially. It appears that the protective effects of LC against 3‐NPA‐induced neurotoxicity are achieved via compensatory enhancement of several pathways of mitochondrial energy metabolism. The results of this and previous studies conducted by our division in the 3‐NPA model of mitochondrial dysfunction demonstrate that 3‐NPA may be employed in vivo to evaluate enhancers of mitochondrial function that might exert neuroprotective effects.

Introducing Black-Gold II, a highly soluble gold phosphate complex with several unique advantages for the histochemical localization of myelin

A novel gold phosphate complex called Black-Gold II with improved myelin staining properties has been developed. It differs from its predecessor, Black-Gold, in that it is highly water soluble at room temperature. This unique physical property confers a number of advantages for the high resolution staining of myelinated fibers. Specifically, it 1) allows for easier solution preparation, eliminating the need for extended heating or sonicating; 2) produces a more uniform and consistent tracer concentration, resulting in more consistent staining and 3) can be used at a 50% higher concentration, resulting in faster and more intense staining without the need for subsequent treatment with gold chloride intensifiers. To characterize the stain, both normal rat brains as well as those exposed to the neurotoxins kainic acid or methamphetamine were examined. The study also incorporates the first application of such stains to examine peripheral nerves of control and acrylamide-exposed rats.

Role of Mitochondrial Dysfunction in Neurotoxicity of MPP:+: Partial Protection of PC12 Cells by Acetyl‐l‐Carnitine

Abstract: The damage to the central nervous system that is observed after administration of either methamphetamine (METH) or 1‐methyl‐4‐phenylpyridinium (MPP+), the neurotoxic metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), is known to be linked to dopamine (DA). The underlying neurotoxicity mechanism for both METH and MPP+ seem to involve free radical formation and impaired mitochondrial function. The MPP+ is thought to selectively kill nigrostriatal dopaminergic neurons by inhibiting mitochondrial complex I, with cell death being attributed to oxidative stress damage to these vulnerable DA neurons. In the present study, MPP+ was shown to significantly inhibit the response to MTT by cultured PC12 cells. This inhibitory action of MPP+ could be partially reversed by the co‐incubation of the cells with the acetylated form of carnitine, acetyl‐l‐carnitine (ALC). Since at least part of the toxic action of MPP+ is related to mitochondrial inhibition, the partial reversal of the inhibition of MTT response by ALC could involve a partial restoration of mitochondrial function. The role carnitine derivatives, such as ALC, play in attenuating MPP+ and METH‐evoked toxicity is still under investigation to elucidate the contribution of mitochondrial dysfunction in mechanisms of neurotoxicity.