Ashraf Virmani

Food, Nutrigenomics, and Neurodegeneration—Neuroprotection by What You Eat!

Diet in human health is no longer simple nutrition, but in light of recent research, especially nutrigenomics, it is linked via evolution and genetics to cell health status capable of modulating apoptosis, detoxification, and appropriate gene response. Nutritional deficiency and disease especially lack of vitamins and minerals is well known, but more recently, epidemiological studies suggest a role of fruits and vegetables, as well as essential fatty acids and even red wine (French paradox), in protection against disease. In the early 1990s, various research groups started considering the use of antioxidants (e.g., melatonin, resveratrol, green tea, lipoic acid) and metabolic compounds (e.g., nicotinamide, acetyl-l-carnitine, creatine, coenzyme Q10) as possible candidates in neuroprotection. They were of course considered on par with snake oil salesman (women) at the time. The positive actions of nutritional supplements, minerals, and plant extracts in disease prevention are now mainstream and commercial health claims being made are subject to regulation in most countries. Apart from efficacy and finding, the right dosages, the safety, and especially the level of purification and lack of contamination are all issues that are important as their use becomes widespread. From the mechanistic point of view, most of the time these substances replenish the body’s deficiency and restore normal function. However, they also exert actions that are not sensu stricto nutritive and could be considered pharmacological especially that, at times, higher intake than recommended (RDA) is needed to see these effects. Free radicals and neuroinflammation processes underlie many neurodegenerative conditions, even Parkinson’s disease and Alzheimer’s disease. Curcumin, carotenoids, acetyl-l-carnitine, coenzyme Q10, vitamin D, and polyphenols and other nutraceuticals have the potential to target multiple pathways in these conditions. In summary, augmenting neuroprotective pathways using diet and finding new natural substances that can be more efficacious, i.e., induction of health-promoting genes and reduction of the expression of disease-promoting genes, could be incorporated into neuroprotective strategies of the future.

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.

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.

Evidence for the Involvement of Carnitine-Dependent Long-Chain Acyltransferases in Neuronal Triglyceride and Phospholipid Fatty Acid Turnover

Abstract: This study focuses on the potential involvement of carnitine palmitoyltransferase (CRT) on the phospholipid and triglyceride fatty acid turnover in neurons. This category of enzymes, which has been identified in several rat brain tissues, is well known for its role in modulating cellular fatty acid oxidation. Neuronal cell cultures from rat brain cortex incorporated radioactive palmitate or oleate into phospholipids and triglycerides. The largest fraction of radioactive fatty acids was recovered in phosphatidyl‐ choline followed by triglycerides and, to a lesser extent, phosphatidylethanolamine. CPT activity measured in neuronal lysates obtained from neurons treated with 40 μM 2‐tetradecylglycidic acid (TDGA) was almost completely abolished. Furthermore, between 2 and 10 μM TDGA CPT activity dropped more rapidly than between 10 and 40 μM. When the cells were pretreated with TDGA, the incorporation process of either radioactive fatty acid into triglycerides was dose‐dependently suppressed. Radioactive fatty acid incorporation into phosphatidylcholine was significantly decreased in cells treated with TDGA. In contrast, phosphatidylethanolamine reacylation was essentially not affected by the CpT inhibitor. Similar results on the fatty acid incorporation into triglycerides and phospholipids were observed with neurons treated with palmitoyl‐dl‐ aminocarnitine (PAC), a reversible CPT inhibitor, which does not consume free CoA. These effects do not seem to be the result of an inhibitory activity toward one of the steps involved in the acylation‐deacylation process of triglycerides or phospholipids, as cellular lysates from TDGA‐treated cells or lysates containing PAC incorporated radioactive fatty acids at rates comparable to controls. Our results suggest that CRT may be an important partner in the pathway of phospholipid and triglyceride fatty acid turnover in neurons.

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.

Links between nutrition, drug abuse, and the metabolic syndrome.

Abstract:  Nutritional deficiency in combination with drug abuse may increase risk of developing the metabolic syndrome by augmenting cell damage, excitotoxicity, reducing energy production, and lowering the antioxidant potential of the cells. We have reviewed here the following points: effects of drugs of abuse on nutrition and brain metabolism; effects of nutrition on actions of the drugs of abuse; drug abuse and probability of developing metabolic syndrome; role of genetic vulnerability in nutrition/drug abuse and brain damage; and the role of neuroprotective supplements in drug abuse. Nutrition education is an essential component of substance abuse treatment programs and can enhance substance abuse treatment outcomes. The strategies available, in particular the nutritional approach to protect the drug abusers from the metabolic syndrome and other diseases are discussed.

l‐Carnitine and Neuroprotection in the Animal Model of Mitochondrial Dysfunction

Abstract: We have shown previously that pretreatment with l‐carnitine (LC) prior to 3‐nitropropionic acid (3‐NPA) exposure, while not significantly attenuating succinate dehydrogenase (SDH) inhibition, prevented hypothermia and oxidative stress. The plant and fungal toxin, 3‐NPA, acts as an inhibitor of mitochondrial function via irreversible inactivation of the mitochondrial inner membrane enzyme, SDH. Inhibition of SDH disturbs electron transport, leading to cellular energy deficits and oxidative stress‐related neuronal injury. In the study presented here, a neurohistological method was applied to examine the mitochondriotropic effect of LC pretreatment against 3‐NPA‐induced neurotoxicity. Twenty adult male Sprague‐Dawley rats randomly divided into two groups (n= 10/group) were injected twice with 3‐NPA at 30 mg/kg sc, at 2 days apart, or received LC pretreatment at 100 mg/kg, at 30‐40 min before 3‐NPA administration. Rats in both groups were perfused 7 days later and their brains harvested. Degenerating neurons were identified and localized via the fluorescent marker Fluoro‐Jade B. Data analysis showed that LC was protective against 3‐NPA‐induced toxicity, as reflected by both reduced mortality and significantly reduced neuronal degeneration.

Neuroprotective effect of L-carnitine in the 3-nitropropionic acid (3-NPA)-evoked neurotoxicity in rats.

A plant and fungal toxin, 3-NPA, acts as an inhibitor of mitochondrial function via irreversible inactivation of the mitochondrial inner membrane enzyme, succinate dehydrogenase (SDH). Inhibition of SDH disturbs electron transport and leads to cellular energy deficits and neuronal injury. We have shown that pretreatment with l-carnitine, while not significantly attenuating SDH inhibition, prevented hypothermia and oxidative stress-associated increased activity of free radical-scavenging enzymes. Here, a neurohistological method was applied to examine the effect of carnitine pretreatment against 3-NPA-induced neurotoxicity. Twenty adult male Sprague-Dawley rats were randomly divided into two groups (n = 10/group). Rats in the first group were injected twice with 3-NPA at 30 mg/kg s.c., 2 days apart, and the second group of animals received l-carnitine pretreatment at 100 mg/kg 30-40 min before 3-NPA administration. Rats in both groups were perfused 7 days later and their brains harvested. Degenerating neurons were identified and localized via the fluorescent marker Fluoro-Jade B. In the three animals that survived 3-NPA dosing, one exhibited no pathology, one exhibited moderate unilateral damage to the striatum, and the third exhibited extensive bilateral neuronal degeneration in multiple forebrain regions. In the seven surviving animals that received l-carnitine prior to 3-NPA insult, six exhibited no lesions, while one exhibited a modest unilateral lesion in the striatum. It appears that l-carnitine is protective against 3-NPA-induced toxicity, as reflected by both reduced mortality and significantly reduced neuronal degeneration.

Assessment of 3-nitropropionic acid-evoked peripheral neuropathy in rats: Neuroprotective effects of acetyl-l-carnitine and resveratrol

Oxidative stress and secondary excitotoxicity, due to cellular energy deficit, are major factors playing roles in 3-nitropropionic acid (3-NPA) induced mitochondrial dysfunction. Acute or chronic exposure to 3-NPA also leads to neuronal degeneration in different brain regions. The present study quantitatively assessed peripheral neuropathy induced by chronic exposure to 3-NPA in rats. The neuroprotective abilities of two antioxidants, acetyl-l-carnitine and resveratrol, were investigated as well. Rats were exposed for up to four weeks to 3-NPA alone or 3-NPA combined with acetyl-l-carnitine or resveratrol, administered peripherally. The experimental outcome was evaluated by neurophysiological, histological, and morphometric analyses. Rats exposed to 3-NPA developed hind limb paresis. Furthermore, a significant decrease in motor nerve conduction velocity (MCV) was detected in tail nerves and axonal degeneration in sciatic nerves (p<0.05). Treatment with resveratrol prevented the functional effects of 3-NPA exposure, whereas treatment with acetyl-l-carnitine, preventing paresis, was not effective to MCV and morphological changes. These data suggest that resveratrol is a good candidate for treatment of metabolic neuropathy. The experimental outcome of this study shows that chronic treatment with 3-NPA in rats is relevant in development of an experimental model of toxic neuropathy.