Iron Oxide Nanoparticles Induce Dopaminergic Damage: In vitro Pathways and In Vivo Imaging Reveals Mechanism of Neuronal Damage.

Various iron-oxide nanoparticles have been in use for a long time as therapeutic and imaging agents and for supplemental delivery in cases of iron-deficiency. While all of these products have a specified size range of ∼ 40 nm and above, efforts are underway to produce smaller particles, down to ∼ 1 nm. Here, we show that after a 24-h exposure of SHSY-5Y human neuroblastoma cells to 10 µg/ml of 10 and 30 nm ferric oxide nanoparticles (Fe-NPs), cellular dopamine content was depleted by 68 and 52 %, respectively. Increases in activated tyrosine kinase c-Abl, a molecular switch induced by oxidative stress, and neuronal α-synuclein expression, a protein marker associated with neuronal injury, were also observed (55 and 38 % percent increases, respectively). Inhibition of cell-proliferation, significant reductions in the number of active mitochondria, and a dose-dependent increase in reactive oxygen species (ROS) were observed in neuronal cells. Additionally, using a rat in vitro blood-brain barrier (BBB) model, a dose-dependent increase in ROS accompanied by increased fluorescein efflux demonstrated compromised BBB integrity. To assess translational implications, in vivo Fe-NP-induced neurotoxicity was determined using in vivo MRI and post-mortem neurochemical and neuropathological correlates in adult male rats after exposure to 50 mg/kg of 10 nm Fe-NPs. Significant decrease in T 2 values was observed. Dynamic observations suggested transfer and retention of Fe-NPs from brain vasculature into brain ventricles. A significant decrease in striatal dopamine and its metabolites was also observed, and neuropathological correlates provided additional evidence of significant nerve cell body and dopaminergic terminal damage as well as damage to neuronal vasculature after exposure to 10 nm Fe-NPs. These data demonstrate a neurotoxic potential of very small size iron nanoparticles and suggest that use of these ferric oxide nanoparticles may result in neurotoxicity, thereby limiting their clinical application.

The Carnitine Palmitoyl Transferase (CPT) System and Possible Relevance for Neuropsychiatric and Neurological Conditions.

The carnitine palmitoyl transferase (CPT) system is a multiprotein complex with catalytic activity localized within a core represented by CPT1 and CPT2 in the outer and inner membrane of the mitochondria, respectively. Two proteins, the acyl-CoA synthase and a translocase also form part of this system. This system is crucial for the mitochondrial beta-oxidation of long-chain fatty acids. CPT1 has two well-known isoforms, CPT1a and CPT1b. CPT1a is the hepatic isoform and CPT1b is typically muscular; both are normally utilized by the organism for metabolic processes throughout the body. There is a strong evidence for their involvement in various disease states, e.g., metabolic syndrome, cardiovascular diseases, and in diabetes mellitus type 2. Recently, a new, third isoform of CPT was described, CPT1c. This is a neuronal isoform and is prevalently localized in brain regions such as hypothalamus, amygdala, and hippocampus. These brain regions play an important role in control of food intake and neuropsychiatric and neurological diseases. CPT activity has been implicated in several neurological and social diseases mainly related to the alteration of insulin equilibrium in the brain. These pathologies include Parkinson's disease, Alzheimer's disease, and schizophrenia. Evolution of both Parkinson's disease and Alzheimer's disease is in some way linked to brain insulin and related metabolic dysfunctions with putative links also with the diabetes type 2. Studies show that in the CNS, CPT1c affects ceramide levels, endocannabionoids, and oxidative processes and may play an important role in various brain functions such as learning.

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.

Neuroprotective efficacy of a new brain-penetrating C-Abl inhibitor in a murine Parkinson's disease model.

Experimental evidence suggests that oxidative and nitrative mechanisms account for much of the dopaminergic neuronal injury in Parkinson's disease (PD). The ubiquitously expressed non-receptor tyrosine kinase c-Abl is activated by oxidative stress and thus, may play a role in redox-mediated neurodegeneration. Recently, we reported that c-Abl is activated in PD and that a c-Abl inhibitor mitigated neuronal damage in a PD animal model, suggesting a novel neuroprotective therapeutic approach. In the studies presented here, we evaluated the efficacy of a potent and clinically relevant second-generation irreversible Abl kinase inhibitor, INNO-406, as a therapeutic agent for PD. Our studies reveal that INNO-406 is capable of preventing the progression of dopaminergic neuronal damage in a toxin-induced C57 mouse model of PD. Using bovine brain microvessel endothelium as an in vitro blood-brain barrier (BBB) model, we detected rapid and significant transfer of INNO-406. Additionally, pharmacokinetic analyses demonstrated significant nanomolar concentrations of INNO-406 in brain in the presence or absence of MPTP administration, however, INNO-406 did not alter the brain levels of MPP+ in MPTP-treated mice. Finally, we showed that 10 mg/kg of INNO-406 given to C57 mice for one week before MPTP treatment (4×20 mg/kg i.p., every 2 h) and then for one week after MPTP treatment decreased the loss of dopamine in the striatum by 45% and the loss of TH+ neurons in substantia nigra pars compacts by 40%. This treatment regimen also abrogated activation of c-Abl, tyrosine phosphorylation of the Abl substrate and E3-ubiquitin ligase parkin, and accumulation of the toxic parkin substrate AIMP2. We propose that compounds of the INNO-406 class of Abl inhibitors will be useful new neuroprotective drugs for the treatment of PD-like pathology in preclinical systems that should be easily translated to the clinic.

Chronic exposure to rotenone, a dopaminergic toxin, results in peripheral neuropathy associated with dopaminergic damage.

Rotenone, a widely used pesticide, causes a syndrome in rats that replicates, both pathologically and behaviorally, the symptoms of Parkinson's disease (PD). In the present study, we sought to determine if a chronic exposure to rotenone, resulting in dopaminergic loss, could also lead to peripheral neuronal damage related to motor dysfunction. Adult male Sprague-Dawley rats (n=14) were treated with rotenone (1 or 2mg/kg, s.c., once daily) on days 1, 3, 6, 8, 10, 13, 15, 17, 21, 22, and 27 to minimize mortality. Control rats received vehicle (DMSO) injections. Animals were weighed on the days of injection and monitored daily. A mortality of 21% was observed in rotenone treated rats. The motor nerve conduction velocity (MCV) was assessed using action potentials detected from the tail muscle through surface receiver electrodes installed around the distal portion of the tail. Rats exposed to rotenone often developed hind limb paresis with a significant decrease in MCV as detected in tail nerves (p<0.05). Animals were then sacrificed, either 24h after rotenone exposure on day 6 or 24h after the last dose of rotenone on day 27. The striatum and sciatic nerves were dissected on dry ice and flash-frozen and kept at -80°C until further analysis. Striatal dopamine (DA) was analyzed using HPLC-ECD and sciatic nerve pathology was analyzed for neurodegeneration. A time-dependent rotenone-induced striatal depletion of DA (60% after 7 days and 80% after 27 days) was observed. Furthermore, Neurofilament-neurofilament B, Flouro-Jade C and myelin basic protein analyses suggested a time-dependent rotenone-induced neurodegeneration in sciatic nerves. These data, for the first time, indicate an association between dopaminergic damage and peripheral motor nerve degeneration in an animal model of dopaminergic toxicity. Peripheral motor nerve dysfunction in rats following a chronic exposure to rotenone may serve not only as a relevant experimental model of motor neuropathy but also as a peripheral marker of dopaminergic neuronal damage to the central nervous system.

Molecular aspects of dopaminergic neurodegeneration: gene-environment interaction in parkin dysfunction.

Parkinson's disease (PD) is a common neurodegenerative movement disorder that is characterized pathologically by a progressive loss of midbrain dopaminergic neurons and by protein inclusions, designated Lewy bodies and Lewy neurites. PD is one of the most common neurodegenerative diseases, affecting almost 1% of the population over 60 years old. Although the symptoms and neuropathology of PD have been well characterized, the underlying mechanisms and causes of the disease are still not clear. Genetic mutations can provide important clues to disease mechanism, but most PD cases are sporadic rather than familial; environmental factors have long been suspected to contribute to the disease. Although more than 90% of PD cases occur sporadically and are thought to be due, in part, to oxidative stress and mitochondrial dysfunction, the study of genetic mutations has provided great insight into the molecular mechanisms of PD. Furthermore, rotenone, a widely used pesticide, and paraquat and maneb cause a syndrome in rats and mice that mimics, both behaviorally and neurologically, the symptoms of PD. In the current review, we will discuss various aspects of gene-environment interaction that lead to progressive dopaminergic neurodegenration, mainly focusing on our current finding based on stress-mediated parkin dysfunction.

Buprenorphine modulates methamphetamine-induced dopamine dynamics in the rat caudate nucleus.

Methamphetamine (METH) abuse and addiction present a major problem in the United States and globally. Oxidative stress associated with exposure to METH mediates to the large extent METH-evoked neurotoxicity. While there are currently no medications approved for treating METH addiction, its pharmacology provides opportunities for potential pharmacotherapeutic adjuncts to behavioral therapy in the treatment of METH addiction. Opioid receptor agonists can modulate the activity of dopamine neurons and could, therefore, modify the pharmacodynamic effects of METH in the dopaminergic system. Efficacy of the adjunctive medication with buprenorphine has been demonstrated in the treatment of cocaine addiction extending beyond opiate addiction. We investigated the interactions of morphine (10 mg/kg) and buprenorphine (0.01 and 10 mg/kg) with METH (2 mg/kg) affecting striatal dopaminergic transmission. The extracellular concentration of dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were determined using brain microdialysis coupled with high performance liquid chromatography with electrochemical detection (HPLC-ED) in the caudate nucleus of adult, awake, male Sprague-Dawley rats. Compared to METH alone, extracellular DA release was prolonged for 140 min without changes in DA peak-effect by combined treatment with morphine/METH. Morphine did not change DOPAC efflux evoked by METH. On the other hand, both buprenorphine doses attenuated the METH-induced DA peak-effect. However, whereas high buprenorphine dose extended DA outflow for 190 min, the low-dose abbreviated DA release. High buprenorphine dose also shortened METH-induced decrease in DOPAC efflux. Data confirm that opiates modulate dopaminergic neurotransmission evoked by METH. Alteration of dopaminergic response to METH challenge under buprenorphine may suggest effectiveness of buprenorphine treatment in METH addiction.

Neuroprotective strategies in drug abuse-evoked encephalopathy.

Encephalopathy is evidenced as an altered mental state with various neurological symptoms, such as memory and cognitive problems. The type of a substance-evoked encephalopathy will depend on the drug, substance, or combination being abused. The categories into which we could place the various abused substances could be tentatively divided into stimulants, amphetamines, hallucinogens, narcotics, inhalants, anesthetics, anabolic steroids, and antipsychotics/antidepressants. Other factors that may underlie encephalopathy, such as infectious agents, environmental, and other factors have also to be taken into account. Drugs of abuse can be highly toxic to the CNS following acute, but more so in chronic exposure, and can produce significant damage to other organs, such as the heart, lungs, liver, and kidneys. The damage to these organs may be at least partially reversible when drug abuse is stopped but CNS damage from repeated or prolonged abuse is often irreversible. The major pathways for the organ and CNS toxicity could be related to ischemic events as well as increased cell damage due to metabolic or mitochondrial dysfunction resulting in increased excitotoxicity, reduced energy production, and lowered antioxidant potential. These susceptibilities could be strengthened by the use of antioxidants to combat free radicals (e.g., vitamin E, lipoic acid); trying to improve energy generation by using mitochondriotropic/metabolic compounds (e.g., thiamine, coenzyme Q10, carnitine, riboflavin); by reducing excitotoxicity (e.g., glutamate antagonists) and other possible strategies, such as robust gene response, need to be investigated further. The drug-abuse-evoked encephalopathy still needs to be studied further to enable better preventative and protective strategies.

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.

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.

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.

Metabolic syndrome in drug abuse.

Drug abuse is associated with significant health risk. Whether drug abusers are at a higher risk of suffering the metabolic syndrome is not widely known. The metabolic syndrome is a cluster of metabolic abnormalities, including hyperinsulinemia, hypertension, dyslipidemia, and abdominal obesity, and is probably triggered by initial imbalances at the cellular level in various critical metabolic pathways. These initially small metabolic imbalances are believed to cascade with time and lead to larger problems. Some indications that drug abuse may increase the risk of the metabolic syndrome include the following: Drug-abusing patients have higher rates of diabetes complications. Substance abuse is a significant contributing factor for treatment noncompliance in diabetes. Nutrition education can enhance substance abuse treatment outcomes. Each type of drug/substance abuse has a unique profile of toxicity. For example, the amphetamines generally affect the cardiovascular and neurological systems, worsening the risk factors for the metabolic syndrome. Methamphetamine (meth) abusers suffer cognitive deficits and abnormal metabolic activity, which affect nutritional status. This condition is further worsened by a drastic reduction in oral health in meth abusers, resulting in improper chewing and, therefore, digestion. Nutritional deficiency in combination with drug abuse would increase the risk of developing the metabolic syndrome by increasing cell damage, augmenting excitotoxicity, reducing energy production, and lowering the antioxidant potential of the cells. Another potential risk factor in the development of the metabolic syndrome is genetic vulnerability, especially in combination with drug abuse and nutritional deficiencies. The strategies available to treat this problem include pharmacological agents as well as dietary antioxidants. Such measures may be useful in reducing drug abuse-related toxicity that may lead to the metabolic syndrome.

Effects of L-carnitine pretreatment in methamphetamine and 3-nitropropionic acid-induced neurotoxicity.

Adult, male Sprague-Dawley rats were injected with 3-ni-tropropionic acid (3-NPA) at 30 mg/kg or methamphetamine (METH) at 20 mg/kg alone or following pretreatment with L-cartnitine (LC) at 100 mg/kg. Rectal temperature was measured before and 4 h following treatment. Animals were sacrificed at 4 h posttreatment. Monoamine neurotransmitters, dopamine (DA) and serotonin (5-HT), and their metabolites were analyzed in the striatum using high-performance liquid chromatography method coupled with electrochemical detection (HPLC/ED). Transcripts of several genes related to DA metabolism were quantified using real time reverse transciption polymerase chain reaction (RT-PCR). Core temperature decreased significantly after 3-NPA acid and increased in METH-treated rats (P < 0.05). Temperature change at 4 h exhibited a significant LC effect for 3-NPA, preventing hypothermia (P < 0.05) and no effect for METH. Concentration of DA and 5-HT, and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA), increased significantly in 3-NPA and decreased in METH-treated rats. An increase in DOPAC/DA turnover and serotonin observed after 3-NPA was abolished in LC-/3-NPA-treated rats. In both 3-NPA- and METH-treated rats, LC prevented an increase in DA receptor D(1) gene expression. It appears that carnitine effect preventing hypothermia after 3-NPA treatments may be related not only to its mitochondriotropic actions but also to inhibitory effect on the DA and 5-HT systems activated after the exposure to 3-NPA. The same effect observed at the transcriptional level, at least for the DA receptor D(1), may account for protection against METH toxicity.

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

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.

Co-regulation of dopamine D1 receptor and uncoupling protein-2 expression in 3-nitropropionic acid-induced neurotoxicity: neuroprotective role of L-carnitine.

This study tested the hypothesis that the expression of uncoupling proteins (UCPs) and dopamine (DA) system genes is responsive to 3-nitropropionic acid (3-NPA) neurotoxic effects and to the neuroprotective effects of the mitochondrial enhancer, L-carnitine (LC), in the rat striatum. Inactivation of mitochondrial succinate dehydrogenase (SDH) by 3-NPA results in hypoxic brain damage. Hypoxic conditions induce uncoupling protein-2 (UCP-2). An increase in UCP-2 expression may lead to a decrease in production of reactive oxygen species (ROS) associated with energy depletion. However, this adaptive response can also lead to a reduction of ATP that may further contribute to energy deficit and mitochondrial dysfunction. Here, male adult Sprague-Dawley rats (n=5/group) were injected either with saline or 3-NPA at 30 mg/kg, s.c. alone or 30 min after pre-treatment with LC (100mg/kg, i.p.). Rectal temperature was monitored before treatment and 4h following 3-NPA administration. Animals were sacrificed 4h post-treatment. Total RNA was isolated from the striatum and transcripts of UCP-2, UCP-4 and UCP-5 genes, as well as genes related to dopamine metabolism, such as DA D(1) and D(2) receptors, tyrosine hydroxylase (TH), monoamine oxidase-B (MAO-B), and vesicular monoamine transporter-2 (VMAT-2), were measured using real-time reverse transcription polymerase chain reaction (RT-PCR). While core temperature decreased significantly in 3-NPA-treated rats, LC significantly inhibited the hypothermic effect of 3-NPA (p<0.05). 3-NPA caused a significant increase in UCP-2 and DA D(1) receptor gene expression in the striatum and both effects were attenuated by pre-treatment with LC. Since LC maintains the ATP/ADP ratio and was previously shown to be neuroprotective against 3-NPA toxicity, the modulation of UCP-2 expression by LC suggests that LC counteracts energy dissipation and thus prevents the negative effects of ATP decline on DA neurotransmission.

Identification of rat hippocampal mRNAs altered by the mitochondrial toxicant, 3-NPA.

3-Nitropropionic acid (3-NPA) is a model mitochondrial inhibitor that causes selective neurodegeneration in brain. 3-NPA-induced neurodegeneration occurs via a secondary neurotoxicity, caused initially by ATP depletion and redox changes in the cell. It is known that the hippocampal degeneration caused by mitochondrial dysfunction affects learning and memory, cognitive functions commonly disturbed in neurodegenerative diseases. The 3-NPA- treated animal model can be used to study molecular mechanisms underlying selective degeneration in the brain. In this study, a microarray approach was utilized to define changes in the expression of 530 genes in the rat hippocampus after acute exposure to 3-NPA at 30 mg/kg, sc. The microarray data were collected at 30 min, 2 h, and 4 h post-3-NPA. Statistical modeling using an ANOVA mixed model applied to Van der Waerden scores of rank-transformed intensity data was used to assign statistical significance to 44 transcripts. These transcripts represent genes associated with energy metabolism, calcium homeostasis, the cytoskeleton, neurotransmitter metabolism, and other cellular functions. Changes in the transcripts of genes encoding 2 transporters [blood-brain specific anion transporter (Slco1c1) and sodium-dependent inorganic phosphate cotransporter (Slc17a7)] were confirmed by real-time RT-PCR. In conclusion, this study identified 2 new potential targets for enhancement of neuroprotection or inhibition of neurodegeneration associated with ATP depletion in the hippocampus.

L-carnitine and neuroprotection in the animal model of mitochondrial dysfunction.

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.

Effects of metabolic modifiers such as carnitines, coenzyme Q10, and PUFAs against different forms of neurotoxic insults: metabolic inhibitors, MPTP, and methamphetamine.

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 Huntington's and Parkinson's 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.

The differential JunB responses to inhibition of succinate dehydrogenase in rat hippocampus and liver.

The inhibitor of mitochondrial enzyme succinate dehydrogenase, 3-nitropropionic acid (3-NPA), induces cellular energy deficit followed by oxidative stress, secondary excitotoxicity and neuronal degeneration. The fast activation of Jun and Fos proteins and other proteins encoding inducible transcription factors (ITFs) occurs in most tissues upon exposure to a variety of stressors including exposure to mitochondrial inhibitors. However, the consequences of this activation can differ dramatically in different organs. For example, while activation of the same ITFs may lead to apoptosis and necrosis in neurons it may stimulate liver regeneration. Here, we report the alterations in mRNAs levels of c-Fos, JunB, and Krox20 proteins induced in the rat brain and liver by the acute exposure to 3-NPA at 30 mg/kg, s.c. While the increase of c-fos transcripts was observed in both the hippocampus and liver, the junb transcript increased in the hippocampus but decreased in the liver. No changes were observed in krox-20 mRNA in the hippocampus. Interestingly, there was a large variation in krox-20 mRNA levels in the liver among animals within the same experimental group. In conclusion, out of the three ITFs transcripts examined here junb may activate different pathways depending on the tissue as indicated by differential responses to mitochondrial inhibition in the hippocampus and liver.

Role of mitochondrial dysfunction in neurotoxicity of MPP+: partial protection of PC12 cells by acetyl-L-carnitine.

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.