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Robust evidence for mitochondrial dysfunction in neurodegenerative disorders has now been derived from cell culture and animal models, identification of human genetic defects in disease, and measurements of markers of oxidative stress and mitochondrial function in patients with neurodegenerative disease. Is this a potential target for neuroprotection? Studies of in vitro models of neuronal toxicity, and of animal models of neurodegenerative disorders, have been encouraging, demonstrating potential neuroprotective effects of compounds such as Coenzyme Q10, a potent antioxidant and component of the mitochondrial electron transport chain. Animal studies have also demonstrated how critical disturbances in cellular processes associated with mitochondrial dysfunction, such as calcium homeostasis, may be modifiable. With this data in mind, the critical question is now whether interventions predicted to enhance mitochondrial function, alleviate oxidative stress, and/or abrogate harmful downstream effects, will translate into novel therapies capable of improving patient outcomes. A number of clinical trials have now been undertaken in humans with Parkinson’s disease, Parkinson Plus disorders, Huntington’s disease, Alzheimer’s disease, Friedreich’s ataxia, and amyotrophic lateral sclerosis. Here, we examine the rationale behind these studies, describe the study outcomes, and discuss potential roadblocks in this process of bench-to-bedside translation. We also examine how recent leaps in understanding mitochondrial biology have identified novel targets for neuroprotective interventions, such as the Sirtuins, the (ARE)-Nrf2-Keap1 signaling pathway, and PGC-1α (alpha). Finally, although much farther down the road, the possibility for gene therapy is now beginning to be explored.

Parkinson's disease (PD) is a neurological disorder caused by progressive degeneration of dopaminergic neurons in the nigrostriatal area of the brain. The decrease in dopamine (DA) neurotransmitter levels in the striatum and substantia nigra pars compacta is a neurochemistry hallmark of PD. Therefore, determination of dopamine and its metabolites levels in biological samples provides an important key to understanding the neurochemistry profile of PD. This chapter describes the use of reversed-phase HPLC with electrochemical detection (ECD) for simultaneously measuring monoamine neurotransmitters, including dopamine and its metabolites, norepinephrine as well as serotonin and its metabolite. ECD provides an ultrasensitive measurement, which detects at the picogram level. One run for each sample finishes within 18 min, shows clear chromatographic peaks and a complete separation, and produces excellent precision and reproducibility. Once set up, HPLC-ECD is economic and efficient for analyzing a large number of samples. This method has been broadly used for analyzing a variety of biological samples, such as cerebrospinal fluids, plasma, microdialysis elutes, tissues, and cultured cells. In recent days, it has been reported to be able to detect the dopamine level in a single drosophila head.

Increased oxidative stress is implicated in the pathogenesis of Alzheimer's disease (AD). A large body of evidence suggests that mitochondrial dysfunction and increased reactive oxygen species occur prior to amyloid-β (Aβ) deposition. Coenzyme Q10 (CoQ10), a component of the mitochondrial electron transport chain, is well characterized as a neuroprotective antioxidant in animal models and human trials of Huntington's disease and Parkinson's disease, and reduces plaque burden in AβPP/PS1 mice. We now show that CoQ10 reduces oxidative stress and amyloid pathology and improves behavioral performance in the Tg19959 mouse model of AD. CoQ10 treatment decreased brain levels of protein carbonyls, a marker of oxidative stress. CoQ10 treatment resulted in decreased plaque area and number in hippocampus and in overlying cortex immunostained with an Aβ42-specific antibody. Brain Aβ42 levels were also decreased by CoQ10 supplementation. Levels of amyloid-β protein precursor (AβPP) β-carboxyterminal fragments were decreased. Importantly, CoQ10-treated mice showed improved cognitive performance during Morris water maze testing. Our results show decreased pathology and improved behavior in transgenic AD mice treated with the naturally occurring antioxidant compound CoQ10. CoQ10 is well tolerated in humans and may be promising for therapeutic trials in AD.

Aims: Mitochondrial damage due to Ca2+ overload-induced opening of permeability transition pores (PTP) is believed to play a role in selective degeneration of nigrostriatal dopaminergic neurons in Parkinson's disease (PD). Genetic ablation of mitochondrial matrix protein cyclophilin D (CYPD) has been shown to increase Ca2+ threshold of PTP in vitro and to prevent cell death in several in vivo disease models. We investigated the role of CYPD in a mouse model of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced PD. Results: We demonstrate that in vitro, brain mitochondria isolated from CYPD knockout mice were less sensitive to MPP+ (1-methyl-4-phenyl-pyridinium ion)-induced membrane depolarization, and free radical generation compared to wild-type mice. CYPD knockout mitochondria isolated from ventral midbrain of mice treated with MPTP in vivo exhibited less damage as judged from respiratory chain Complex I activity, State 3 respiration rate, and respiratory control index than wild-type mice, whereas assessment of apoptotic markers showed no differences between the two genotypes. However, CYPD knockout mice were significantly resistant only to an acute regimen of MPTP neurotoxicity in contrast to the subacute and chronic MPTP paradigms. Innovation: Inactivation of CYPD is beneficial in preserving mitochondrial functions only in an acute insult model of MPTP-induced dopaminergic neurotoxicity. Conclusion: Our results suggest that CYPD deficiency distinguishes the modes of dopaminergic neurodegeneration in various regimens of MPTP-neurotoxicity. Antioxid. Redox Signal. 16, 855–868.

Parkinson’s disease is a neurodegenerative movement disorder characterized by loss of midbrain dopaminergic neurons leading to motor abnormalities and autonomic dysfunctions. Despite intensive research, the etiology of Parkinson’s disease remains poorly understood leaving us with no effective therapeutic options. However, the recent identification of genes linked to heritable forms of Parkinson’s disease has revolutionized research in the field and has begun to provide new clues to disease pathogenesis. Here we discuss these recent genetic advances and highlight their significance in our quest to better understand common underlying disease mechanisms that will help us identify innovative neuroprotective therapies for Parkinson’s disease.

Oxidative damage, neuroinflammation, and mitochondrial dysfunction contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS), and these pathologic processes are tightly regulated by the Nrf2/ARE (NF-E2-related factor 2/antioxidant response element) signaling program. Therefore, modulation of the Nrf2/ARE pathway is an attractive therapeutic target for neurodegenerative diseases such as ALS. We examined two triterpenoids, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) ethylamide and CDDO trifluoroethylamide (CDDO-TFEA), that potently activate Nrf2/ARE in a cell culture model of ALS and in the G93A SOD1 mouse model of ALS. Treatment of NSC-34 cells stably expressing mutant G93A SOD1 with CDDO-TFEA upregulated Nrf2 expression and resulted in translocation of Nrf2 into the nucleus. Western blot analysis showed an increase in the expression of Nrf2/ARE-regulated proteins. When treatment started at a “presymptomatic age” of 30 days, both of these compounds significantly attenuated weight loss, enhanced motor performance, and extended the survival of G93A SOD1 mice. Treatment started at a “symptomatic age,” as assessed by impaired motor performance, was neuroprotective and slowed disease progression. These findings provide further evidence that compounds that activate the Nrf2/ARE signaling pathway may be useful in the treatment of ALS.

Mitochondrial DNA (mtDNA) variants associated with Alzheimer disease (AD) and Parkinson disease (PD) were sought by restriction endonuclease analysis in a cohort of 71 late-onset Caucasian patients. A tRNAGln gene variant at nucleotide pair (np) 4336 that altered a moderately conserved nucleotide was present in 9/173 (5.2%) of the patients surveyed but in only 0.7% of the general Caucasian controls. One of these patients harbored an additional novel 12S rRNA 5-nucleotide insertion at np 956-965, while a second had a missense variant at np 3397 that converted a highly conserved methionine to a valine. This latter mutation was also found in an independent AD + PD patient, as was a heteroplasmic 16S rRNA variant at np 3196. Additional studies will be required to determine the significance, if any, of these mutations.

Basic fibroblast growth factor (bFGF) is a polypeptide that promotes neuronal survival and blocks excitatory amino acid (EAA) neurotoxicity in vitro at very low concentrations. In the present study, we examined whether systemically administered bFGF could prevent neuronal damage induced by either EAAs or hypoxia-ischemia in vivo. Neuroprotective effects were examined in a neonatal model of hypoxia-ischemia (unilateral ligation of the carotid artery followed by exposure to 8% oxygen for 1.5 h) and following intrastriatal injection of N-methyl-D-aspartate (NMDA) in 7-day-old rats. Intraperitoneal administration of a single dose of bFGF (50-300 micrograms/kg) 30 min before intrastriatal injection of NMDA showed a dose-dependent neuroprotective effect. Repeated doses of bFGF (100 micrograms/kg) both before and after intrastriatal NMDA injection produced a much greater significant protective effect than a single dose administered prior to the injection. Intraperitoneal injection of single dose of 100 micrograms/kg of bFGF 30 min before hypoxia-ischemia reduced neuronal damage by 38% (p = 0.14), while administration of bFGF at a dose of 100 micrograms/kg i.p. three times, 30 min before and 0 and 30 min after hypoxia-ischemia, significantly reduced neuronal damage by 64% (p = 0.004). Systemic administration of bFGF did not change body temperature for up to 3 h. These results show that systemic administration of bFGF can exert neuroprotective effects against both NMDA-induced excitotoxicity and hypoxia-ischemia in vivo.

Rod-like structures composed of actin and the actin-binding protein cofilin are found in Alzheimer's disease (AD) patients. However, the mechanisms underlying formation of these structures and their pathological consequences are still largely unknown. We found that microRNAs 103 and 107 repress translation of cofilin, and that reduced levels of miR-103 or miR-107 are associated with elevated cofilin protein levels and formation of rod-like structures in a transgenic mouse model of AD. These results suggest that microRNAs may play an important role in cytoskeletal pathology in AD.

In addition to mitochondria, NADPH oxidase (NOX) is a source of oxidative stress, which can induce oxidative damage in Alzheimer's disease (AD). For this reason, several groups have investigated the effect of its inhibition. In AD mice, NADPH oxidase 2 (NOX2) deficiency improved behavior and cerebrovascular function, and reduced oxidative stress. In our study, we administered the NOX inhibitor apocynin to Tg19959 mice, and found that it did not improve cognitive and synaptic deficits, and did not decrease amyloid deposition, microgliosis and hyperphosphorylated tau. However, apocynin reduced carbonyl levels in the cerebral cortex but not the hippocampus, which may have not been sufficient to ameliorate symptoms. Also, the reduction of NOX-mediated oxidative stress may not be sufficient to prevent AD, since other sources of reactive oxygen species such as mitochondria may be more important.

Caused by a polyglutamine expansion in the huntingtin protein, Huntington's disease leads to striatal degeneration via the transcriptional dysregulation of a number of genes, including those involved in mitochondrial biogenesis. Here we show that transglutaminase 2, which is upregulated in HD, exacerbates transcriptional dysregulation by acting as a selective corepressor of nuclear genes; transglutaminase 2 interacts directly with histone H3 in the nucleus. In a cellular model of HD, transglutaminase inhibition de-repressed two established regulators of mitochondrial function, PGC-1alpha and cytochrome c and reversed susceptibility of human HD cells to the mitochondrial toxin, 3-nitroproprionic acid; however, protection mediated by transglutaminase inhibition was not associated with improved mitochondrial bioenergetics. A gene microarray analysis indicated that transglutaminase inhibition normalized expression of not only mitochondrial genes but also 40% of genes that are dysregulated in HD striatal neurons, including chaperone and histone genes. Moreover, transglutaminase inhibition attenuated degeneration in a Drosophila model of HD and protected mouse HD striatal neurons from excitotoxicity. Altogether these findings demonstrate that selective TG inhibition broadly corrects transcriptional dysregulation in HD and defines a novel HDAC-independent epigenetic strategy for treating neurodegeneration.

Coenzyme Q10 (CoQ(10)), a potential neuroprotective compound, was previously investigated at a dosage of 600 mg/day in Huntington's disease (HD) patients and demonstrated a trend toward slowing disease progression. Higher CoQ(10) dosages may prove beneficial. We investigated the tolerability and blood levels associated with 1,200, 2,400, and 3,600 mg/day of CoQ(10) in HD and healthy subjects. Twenty-eight subjects (20 HD, 8 healthy) enrolled in a 20-week open-label trial. Subjects started on 1,200 mg/day of CoQ(10), increasing every 4 weeks by 1,200 mg to a maximum dosage of 3,600 mg/day. Monthly evaluations included review of adverse events and CoQ(10) blood levels. Twenty-three subjects (82%) achieved the target dosage of 3,600 mg/day. Six subjects (2 healthy, 4 HD) withdrew prematurely (gastrointestinal (GI) symptoms in 3, worsening HD in 2, and 1 because of a fall). All three serious adverse events occurred in a single subject, and were deemed unrelated to CoQ(10). The most common adverse events seen were GI symptoms. Mean (± SD) CoQ10 blood levels achieved over the course of the trial were as follows: 1.26 ± 1.27 μg/mL (baseline, n = 28), 5.59 ± 2.24 μg/mL (1,200 mg/day, week 4, n = 26), 6.38 ± 3.25 μg/mL (2,400 mg/day, week 8, n = 25), 7.49 ± 4.09 μg/mL (3,600 mg/day, week 12, n = 23), and 6.78 ± 3.36 μg/mL (3,600 mg/day, week 20, n = 20). CoQ(10) was well tolerated with over 80% of subjects achieving the target dosage. Dosages of 2,400 mg/day may provide the best balance between tolerability and blood level achieved. Further studies examining the efficacy of 2,400 mg/day are planned.

There is a substantial body of literature, which has demonstrated that creatine has neuroprotective effects both in vitro and in vivo. Creatine can protect against excitotoxicity as well as against β-amyloid toxicity in vitro. We carried out studies examining the efficacy of creatine as a neuroprotective agent in vivo. We demonstrated that creatine can protect against excitotoxic lesions produced by N-methyl-D: -aspartate. We also showed that creatine is neuroprotective against lesions produced by the toxins malonate and 3-nitropropionic acid (3-NP) which are reversible and irreversible inhibitors of succinate dehydrogenase, respectively. Creatine produced dose-dependent neuroprotective effects against MPTP toxicity reducing the loss of dopamine within the striatum and the loss of dopaminergic neurons in the substantia nigra. We carried out a number of studies of the neuroprotective effects of creatine in transgenic mouse models of neurodegenerative diseases. We demonstrated that creatine produced an extension of survival, improved motor performance, and a reduction in loss of motor neurons in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). Creatine produced an extension of survival, as well as improved motor function, and a reduction in striatal atrophy in the R6/2 and the N-171-82Q transgenic mouse models of Huntington's disease (HD), even when its administration was delayed until the onset of disease symptoms. We recently examined the neuroprotective effects of a combination of coenzyme Q10 (CoQ10) with creatine against both MPTP and 3-NP toxicity. We found that the combination of CoQ and creatine together produced additive neuroprotective effects in a chronic MPTP model, and it blocked the development of alpha-synuclein aggregates. In the 3-NP model of HD, CoQ and creatine produced additive neuroprotective effects against the size of the striatal lesions. In the R6/2 transgenic mouse model of HD, the combination of CoQ and creatine produced additive effects on improving survival. Creatine may stabilize mitochondrial creatine kinase, and prevent activation of the mitochondrial permeability transition. Creatine, however, was still neuroprotective in mice, which were deficient in mitochondrial creatine kinase. Administration of creatine increases the brain levels of creatine and phosphocreatine. Due to its neuroprotective effects, creatine is now in clinical trials for the treatment of Parkinson's disease (PD) and HD. A phase 2 futility trial in PD showed approximately a 50% improvement in Unified Parkinson's Disease Rating Scale at one year, and the compound was judged to be non futile. Creatine is now in a phase III clinical trial being carried out by the NET PD consortium. Creatine reduced plasma levels of 8-hydroxy-2-deoxyguanosine in HD patients phase II trial and was well-tolerated. Creatine is now being studied in a phase III clinical trial in HD, the CREST trial. Creatine, therefore, shows great promise in the treatment of a variety of neurodegenerative diseases.

We investigated the ability of AMP-activated protein kinase (AMPK) to activate PPARgamma coactivator-1alpha (PGC-1alpha) in the brain, liver and brown adipose tissue (BAT) of the NLS-N171-82Q transgenic mouse model of Huntington's disease (HD). In the striatum of the HD mice, the baseline levels of PGC-1alpha, NRF1, NRF2, Tfam, COX-II, PPARdelta, CREB and ERRalpha mRNA and mitochondrial DNA (mtDNA), were significantly reduced. Administration of the creatine analog beta guanidinopropionic acid (GPA) reduced ATP and PCr levels and increased AMPK mRNA in both the cerebral cortex and striatum. Treatment with GPA significantly increased expression of PGC-1alpha, NRF1, Tfam and downstream genes in the striatum and cerebral cortex of wild-type (WT) mice, but there was no effect on these genes in the HD mice. The striatum of the untreated HD mice showed microvacuolation in the neuropil, as well as gliosis and huntingtin aggregates, which were exacerbated by treatment with GPA. GPA treatment produced a significant increase in mtDNA in the cerebral cortex and striatum of WT mice, but not in HD mice. The HD mice treated with GPA had impaired activation of liver PGC-1alpha and developed hepatic steatosis with accumulation of lipids, degeneration of hepatocytes and impaired activation of gluconeogenesis. The BAT in the HD mice showed vacuolation due to accumulation of neutral lipids, and age-dependent impairment of UCP-1 activation and temperature regulation. Impaired activation of PGC-1alpha, therefore, plays an important role in the behavioral phenotype, metabolic disturbances and pathology of HD, which suggests the possibility that agents that enhance PGC-1alpha function will exert therapeutic benefits in HD patients.

A large body of evidence from postmortem brain tissue and genetic analysis in humans, as well as biochemical and pathological studies in animal models of neurodegeneration suggest that mitochondrial dysfunction is a key pathological mechanism in Parkinson's Disease (PD). Mitochondrial dysfunction leads to oxidative stress, damage to mitochondrial DNA, mitochondrial DNA deletions, altered mitochondrial morphology, alterations in mitochondrial fission and fusion and ultimately neuronal demise. Therapeutic approaches targeting mitochondrial dysfunction and oxidative damage, therefore, hold great promise in PD. A number of agents, which target energy metabolism, are presently in therapeutic trials in PD. Both creatine and Coenzyme Q10 (CoQ10) are being tested in phase III clinical trials. In addition, preclinical studies in animal models have shown efficacy of mitochondrial-targeted antioxidants and the SS peptides. A promising approach for increasing antioxidant defenses is to transcriptionally increase the activity of the Nrf2/ARE pathway, which activates transcription of anti-inflammatory and antioxidant genes. A number of agents including sulforaphane, curcumin and triterpenoids have been shown to activate this pathway and to produce neuroprotective effects. Lastly, newly identified therapeutic targets include peroxisomal proliferator activated receptor gamma-coactivator (PGC-1alpha) and sirtuins. These pathways provide promise for future therapeutic developments in the treatment of PD.

An increasing body of evidence has implicated excitoxicity as a mechanism of neuronal death in both acute and chronic neurological diseases. A major recent advance has been the successful cloning and expression of the non-NMDA, NMDA, and metabotropic glutamate receptors. The cellular mechanisms responsible for cell death following activation of these receptors are still being clarified. A recent advance in conceptualizing excitotoxicity is the notion that a slow excitotoxic process may occur as a consequence of either a receptor abnormality or an impairment of energy metabolism. It is possible that such a mechanism may occur in neurodegenerative illnesses. Recent therapeutic studies have focused on glycine site antagonists and on the efficacy of non-NMDA antagonists in ischemia.

Huntington's disease is a progressive neurodegenerative disease in which the basal ganglia are preferentially affected. Recent evidence, however, suggests involvement of the cerebral cortex as well, with sparing of neurochemically defined subsets of gamma-aminobutyric acid (GABA)-ergic interneurons. In the present study, we examined changes in concentrations of the amino acid neurotransmitters GABA, glutamate, and aspartate in nine cortical regions from 23 patients with advanced Huntington's disease and 12 control brains. GABA concentrations were significantly increased in eight of the nine regions, consistent with a sparing of GABAergic local circuit neurons in the context of progressive cortical atrophy. Small but significant increases in glutamate were found in six of the nine regions, while aspartate levels were generally unaffected. Striate cortex (Brodmann's area 17) showed the most profound increases in GABA and glutamate. We also investigated the effects of powdering the excitotoxins N-methyl-D-aspartate (NMDA) or kainic acid onto the dura of rats. The resulting lesions were examined at 1 week and 6 months. The NMDA-induced lesions showed striking sparing of parvalbumin-positive neurons (a subset of GABAergic interneurons), and this sparing was reflected in neurochemical measurements of GABA; kainic acid lesions failed to display this selectivity. Somatostatin, cholecystokinin, and vasoactive intestinal polypeptide concentrations were spared by the NMDA-induced lesions, and substance P levels were significantly increased. These results provide evidence that NMDA excitotoxic lesions of cerebral cortex can produce a selective pattern of neuronal damage similar to that which occurs in Huntington's disease.

In the present experiment unilateral intrastriatal injections of aminooxyacetic acid (1, 2.5, 5 μmol) to freely moving animals and pentobarbital-anesthetized rats produced contralateral jerks and dose-dependent mortality, but no barrel rotation. At 10–12 days there were no significant differences in exploratory activity, passive avoidance behavior, and elevated plus-maze test in aminooxyacetic acid-treated animals as compared with controls. However freely moving animals microinjcctcd with aminooxyacetic acid (but not the pentobarbital-pretreated group) had impaired learning activity in an active avoidance conditioning test, and showed reduced striatal concentrations of substance P and GABA. Intrastriatal injections of aminooxyacetic acid therefore result in both acute and chronic behavioral changes which are attenuated by pentobarbital anesthesia.

The cerebral distribution and regulation of excitatory amino acid levels may play a crucial role in neuronal development. In the present study we examined concentrations of the endogenous excitatory amino acid antagonist kynurenic acid and related substances during development in fetal and neonatal rat brain and fetal non-human primate cerebral cortex. Kynurenic acid concentrations in rat fetal whole brain were significantly increased 4-5 fold prenatally, then declined rapidly at 1 day after birth, and reached adult concentrations at 7 days after birth. L-Kynurenine concentrations were also markedly increased prior to birth and then declined to adult concentrations at 1 day after birth. L-Tryptophan was increased 3 fold before birth, and decreased to adult concentrations 1 day after birth. In contrast concentrations of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid and homovanillic acid increased 1 day prior to birth and continued to increase following birth. Fetal baboon cerebral cortex showed significant increases in kynurenic acid concentrations both pre-term and near-term as compared with adult concentrations. These results show that marked changes in kynurenic acid concentrations occur prior to and following birth. It is possible that high levels of kynurenic acid prior to birth inhibit neurite branching and development of excitatory synapses, which then develop rapidly in parallel with the decrease in kynurenic acid levels.

The effects of 1-methyl-4-phenylpyridinium (MPP+) were studied in rat striatum. Using freeze-clamp, microwave, and water-suppressed proton chemical shift magnetic resonance imaging techniques, MPP+ resulted in marked increases in lactate and a depletion of ATP for up to 48 h after the injections. MPP+ produced dose-dependent depletions of dopamine, serotonin, gamma-aminobutyric acid, and substance P that were partially blocked at 1 week by prior decortication or completely blocked by MK-801 at 24 h. The lesions showed relative sparing of somatostatin-neuropeptide Y neurons, consistent with N-methyl-D-aspartate (NMDA) excitotoxicity. MPP+ produces impairment of oxidative phosphorylation in vivo, which may result in membrane depolarization with persistent activation of NMDA receptors and excitotoxic neuronal degeneration. An impairment of energy metabolism may therefore underlie slow excitotoxic neuronal death in neurodegenerative diseases.

β-Amyloid (1–40) has recently been shown to exhibit both neurotoxic and neurotrophic properties. The putatively active moiety is β-(25–35), which has a structural homology to the human tachykinin substance P. Substance P, which preferentially binds to neurokinin 1 (NK1) receptors in the central nervous system (CNS), has been shown to block the neurotoxic effects of β-amyloid (1–40). These data suggest that effects of β-amyloid may be mediated by an NK1 receptor-mediated process. However, in the present study, we demonstrate that β-(25–35) is unable to competitively inhibit the binding of 0.15 nM 125I-substance P from rat CNS NK1 receptors. Therefore, the mechanisms of action of β-amyloid neurotoxic effects are probably not to be mediated through a NK1 receptor-mediated process. © 1992 Wiley-Liss, Inc.

Background The risk of Parkinson disease (PD) and its rate of progression may decline with increasing concentration of blood urate, a major antioxidant. Objective To determine whether serum and cerebrospinal fluid concentrations of urate predict clinical progression in patients with PD. Design, Setting, and Participants Eight hundred subjects with early PD enrolled in the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) trial. The pretreatment urate concentration was measured in serum for 774 subjects and in cerebrospinal fluid for 713 subjects. Main Outcome Measures Treatment-, age-, and sex-adjusted hazard ratios (HRs) for clinical disability requiring levodopa therapy, the prespecified primary end point of the original DATATOP trial. Results The HR of progressing to the primary end point decreased with increasing serum urate concentrations (HR for highest vs lowest quintile = 0.64; 95% confidence interval [CI], 0.44-0.94; HR for a 1-SD increase = 0.82; 95% CI, 0.73-0.93). In analyses stratified by α-tocopherol treatment (2000 IU/d), a decrease in the HR for the primary end point was seen only among subjects not treated with α-tocopherol (HR for a 1-SD increase = 0.75; 95% CI, 0.62-0.89; vs HR for those treated = 0.90; 95% CI, 0.75-1.08). Results were similar for the rate of change in the Unified Parkinson's Disease Rating Scale score. Cerebrospinal fluid urate concentration was also inversely related to both the primary end point (HR for highest vs lowest quintile = 0.65; 95% CI, 0.44-0.96; HR for a 1-SD increase = 0.89; 95% CI, 0.79-1.02) and the rate of change in the Unified Parkinson's Disease Rating Scale score. As with serum urate concentration, these associations were present only among subjects not treated with α-tocopherol. Conclusions Higher serum and cerebrospinal fluid urate concentrations at baseline were associated with slower rates of clinical decline. The findings strengthen the link between urate concentration and PD and the rationale for considering central nervous system urate concentration elevation as a potential strategy to slow PD progression. Published online October 12, 2009 (doi:10.1001/archneurol.2009.247).

Meredith Spindler1, M Flint Beal1,2, Claire Henchcliffe1,21Department of Neurology, 2Department of Neuroscience, Weill Medical College of Cornell University, New York, NY, USAAbstract: Coenzyme Q10 (CoQ10) is an essential cofactor in the mitochondrial respiratory chain, and as a dietary supplement it has recently gained attention for its potential role in the treatment of neurodegenerative disease. Evidence for mitochondrial dysfunction in neurodegenerative disorders derives from animal models, studies of mitochondria from patients, identification of genetic defects in patients with neurodegenerative disease, and measurements of markers of oxidative stress. Studies of in vitro models of neuronal toxicity and animal models of neurodegenerative disorders have demonstrated potential neuroprotective effects of CoQ10. With this data in mind, several clinical trials of CoQ10 have been performed in Parkinson’s disease and atypical Parkinson’s syndromes, Huntington’s disease, Alzheimer disease, Friedreich’s ataxia, and amyotrophic lateral sclerosis, with equivocal findings. CoQ10 is widely available in multiple formulations and is very well tolerated with minimal adverse effects, making it an attractive potential therapy. Phase III trials of high-dose CoQ10 in large sample sizes are needed to further ascertain the effects of CoQ10 in neurodegenerative diseases.Keywords: coenzyme Q10, neurodegenerative disease, Parkinson’s disease, Huntington’s disease, mitochondrial dysfunction

Background: Mutations in LRRK2 gene represent the most common known genetic cause of Parkinson's disease (PD). Methodology/Principal Findings: We used metabolomic profiling to identify biomarkers that are associated with idiopathic and LRRK2 PD. We compared plasma metabolomic profiles of patients with PD due to the G2019S LRRK2 mutation, to asymptomatic family members of these patients either with or without G2019S LRRK2 mutations, and to patients with idiopathic PD, as well as non-related control subjects. We found that metabolomic profiles of both idiopathic PD and LRRK2 PD subjects were clearly separated from controls. LRRK2 PD patients had metabolomic profiles distinguishable from those with idiopathic PD, and the profiles could predict whether the PD was secondary to LRRK2 mutations or idiopathic. Metabolomic profiles of LRRK2 PD patients were well separated from their family members, but there was a slight overlap between family members with and without LRRK2 mutations. Both LRRK2 and idiopathic PD patients showed significantly reduced uric acid levels. We also found a significant decrease in levels of hypoxanthine and in the ratios of major metabolites of the purine pathway in plasma of PD patients. Conclusions/Significance: These findings show that LRRK2 patients with the G2019S mutation have unique metabolomic profiles that distinguish them from patients with idiopathic PD. Furthermore, asymptomatic LRRK2 carriers can be separated from gene negative family members, which raises the possibility that metabolomic profiles could be useful in predicting which LRRK2 carriers will eventually develop PD. The results also suggest that there are aberrations in the purine pathway in PD which may occur upstream from uric acid. © 2009 Johansen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

A large body of evidence suggests the importance of inflammation and oxidative or nitrosative stress in Alzheimer's disease (AD) pathogenesis. Inflammatory stimuli upregulate transcription of inducible nitric oxide synthase (iNOS), which can lead to the production of nitric oxide and other reactive nitrogen species. We previously found that genetic deletion of iNOS in mice overexpressing the amyloid precursor protein (APP) and presenilin-1 (PS1) reduced mortality, nitrosative stress, amyloid plaque burden, microgliosis, astrocytosis, and peri-plaque tau phosphorylation. We therefore examined the effects of N6-(1-iminoethyl)-L-lysine (L-NIL), a pharmacological iNOS inhibitor, or d-NIL, its enantiomeric control, in a transgenic mouse model of amyloid deposition. Tg19959 mice carry human APP with two mutations and develop amyloid plaques and memory impairment starting at 3-4 months of age. Mice were given L-NIL or D-NIL in the drinking water from 1 month of age and assessed behaviorally and histopathologically at 8 months of age. We found that L-NIL administration reduced disinhibition in the elevated plus maze, improved spatial memory performance in the Morris water maze, and decreased cortical amyloid deposition as well as microglial activation in 8-month-old Tg19959 mice. These findings are consistent with previous reports demonstrating that iNOS inhibition ameliorates AD pathogenesis.

Background Parkinson's disease (PD) is the most common movement disorder. Extrapyramidal motor symptoms stem from the degeneration of the dopaminergic pathways in patient brain. Current treatments for PD are symptomatic, alleviating disease symptoms without reversing or retarding disease progression. Although the cause of PD remains unknown, several pathogenic factors have been identified, which cause dopaminergic neuron (DN) death in the substantia nigra (SN). These include oxidative stress, mitochondrial dysfunction, inflammation and excitotoxicity. Manipulation of these factors may allow the development of disease-modifying treatment strategies to slow neuronal death. Inhibition of DJ-1A, the Drosophila homologue of the familial PD gene DJ-1, leads to oxidative stress, mitochondrial dysfunction, and DN loss, making fly DJ-1A model an excellent in vivo system to test for compounds with therapeutic potential. Results In the present study, a Drosophila DJ-1A model of PD was used to test potential neuroprotective drugs. The drugs applied are the Chinese herb celastrol, the antibiotic minocycline, the bioenergetic amine coenzyme Q10 (coQ10), and the glutamate antagonist 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo[f]-quinoxaline (NBQX). All of these drugs target pathogenic processes implicated in PD, thus constitute mechanism-based treatment strategies. We show that celastrol and minocycline, both having antioxidant and anti-inflammatory properties, confer potent dopaminergic neuroprotection in Drosophila DJ-1A model, while coQ10 shows no protective effect. NBQX exerts differential effects on cell survival and brain dopamine content: it protects against DN loss but fails to restore brain dopamine level. Conclusion The present study further validates Drosophila as a valuable model for preclinical testing of drugs with therapeutic potential for neurodegenerative diseases. The lower cost and amenability to high throughput testing make Drosophila PD models effective in vivo tools for screening novel therapeutic compounds. If our findings can be further validated in mammalian PD models, they would implicate drugs combining antioxidant and anti-inflammatory properties as strong therapeutic candidates for mechanism-based PD treatment.

Background Missense mutations and multiplications of the α-synuclein gene cause autosomal dominant familial Parkinson's disease (PD). α-Synuclein protein is also a major component of Lewy bodies, the hallmark pathological inclusions of PD. Therefore, α-synuclein plays an important role in the pathogenesis of familial and sporadic PD. To model α-synuclein-linked disease in vivo, transgenic mouse models have been developed that express wild-type or mutant human α-synuclein from a variety of neuronal-selective heterologous promoter elements. These models exhibit a variety of behavioral and neuropathological features resembling some aspects of PD. However, an important deficiency of these models is the observed lack of robust or progressive nigrostriatal dopaminergic neuronal degeneration that is characteristic of PD. Results We have developed conditional α-synuclein transgenic mice that can express A53T, E46K or C-terminally truncated (1–119) human α-synuclein pathological variants from the endogenous murine ROSA26 promoter in a Cre recombinase-dependent manner. Using these mice, we have evaluated the expression of these α-synuclein variants on the integrity and viability of nigral dopaminergic neurons with age. Expression of A53T α-synuclein or truncated αSyn119 selectively in nigrostriatal pathway dopaminergic neurons for up to 12 months fails to precipitate dopaminergic neuronal loss in these mice. However, αSyn119 expression in nigral dopaminergic neurons for up to 12 months causes a marked reduction in the levels of striatal dopamine and its metabolites together with other subtle neurochemical alterations. Conclusion We have developed and evaluated novel conditional α-synuclein transgenic mice with transgene expression directed selectively to nigrostriatal dopaminergic neurons as a potential new mouse model of PD. Our data support the pathophysiological relevance of C-terminally truncated α-synuclein species in vivo. The expression of αSyn119 in the mouse nigrostriatal dopaminergic pathway may provide a useful model of striatal dopamine depletion and could potentially provide a presymptomatic model of PD perhaps representative of the earliest derangements in dopaminergic neuronal function observed prior to neuronal loss. These conditional α-synuclein transgenic mice provide novel tools for evaluating and dissecting the age-related effects of α-synuclein pathological variants on the function of the nigrostriatal dopaminergic pathway or other specific neuronal populations.

In Alzheimer’s disease (AD), oxidative stress is present early and contributes to disease pathogenesis. We previously reported that in Tg19959 transgenic AD mice, partial deficiency of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) exacerbated amyloid pathology. We therefore asked whether MnSOD overexpression would prove beneficial against AD pathogenesis, by studying the offspring of Tg19959 mice crossed with MnSOD-overexpressing mice. At 4 mo of age, there was a 2- to 3-fold increase in MnSOD protein levels in Tg19959-MnSOD mice compared to Tg19959 littermates. Tg19959-MnSOD mice also had a 50% increase in catalase protein levels, a 50% decrease in levels of oxidized protein, and a 33% reduction in cortical plaque burden compared to Tg19959 littermates. Spatial memory was impaired and synaptophysin levels were decreased in Tg19959 mice compared to wild-type littermates, but memory and synaptophysin levels were restored to wild-type levels in Tg19959-MnSOD littermates. These benefits occurred without changes in sodium dodecyl sulfate-soluble or formic acid-soluble Aβ pools or Aβ oligomers in Tg19959-MnSOD mice compared to Tg19959 littermates. These data demonstrate that facilitation of the mitochondrial antioxidant response improves resistance to Aβ, slows plaque formation or increases plaque degradation, and markedly attenuates the phenotype in a transgenic AD mouse model.—Dumont, M., Wille, E., Stack, C., Calingasan, N. Y., Beal, M. F., Lin, M. T. Reduction of oxidative stress, amyloid deposition, and memory deficit by manganese superoxide dismutase overexpression in a transgenic mouse model of Alzheimer’s disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. We created a LRRK2 transgenic mouse model that recapitulates cardinal features of the disease: an age-dependent and levodopa-responsive slowness of movement associated with diminished dopamine release and axonal pathology of nigrostriatal dopaminergic projection. These mice provide a valid model of Parkinson's disease and are a resource for the investigation of pathogenesis and therapeutics.

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased alpha-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and alpha-synclein aggregation.

The effects of aging on levels of neurotransmitters were determined in two regions of the cerebral cortex in rhesus monkeys (Macaca mulatta). Choline acetyltransferase (ChAT) activity as well as somatostatin, neuropeptide Y, and substance P immunoreactivities were analyzed in the right caudal cingulate gyrus and in the left and right inferior occipital poles in five age groups: 4-6 years; 8-11 years; 20-25 years; 26-29 years; and 31-34 years. Neuroactive amino acids and markers for monoamine transmitters were analyzed only in the youngest (4-6 years) and oldest (31-34 years) animals. Across the five age groups studied. ChAT activity as well as somatostatin and neuropeptide Y immunoreactivities were significantly decreased bilaterally in occipital poles of the 31- to 34-year-old group. There were no significant age-related differences in substance P immunoreactivity. In 4-6-year-old vs. 31-34-year-old monkeys, levels of amino acid neurotransmitters were unchanged. However, there were significant reductions in norepinephrine, serotonin and its metabolites, kynurenine, and 4-hydroxyphenyllactic acid in occipital poles of the 31- to 34-year-old monkeys. No significant neurochemical changes were detected in the cingulate cortex. These findings demonstrate that aged nonhuman primates show reductions in cortical markers for a variety of neurotransmitters, including acetylcholine, somatostatin, neuropeptide Y, norepinephrine, and serotonin but that these changes do not occur uniformly in the neocortex.

Many of the characteristic neuroanatomical and neurochemical features of Huntington's disease (HD) are produced in experimental animals by an intrastriatal injection of the endogenous N- methyl- d -aspartate receptor agonist quinolinic acid (QUIN). Conceivably, a chronic over-production of QUIN in brain could be involved in the pathogenesis of HD. To investigate this hypothesis, concentrations of QUIN were measured both in cerebrospinal fluid (CSF) and postmortem tissue from patients with HD and neurologically normal age-matched controls. CSF QUIN concentrations were slightly lower in patients with HD, however the changes were not significant. Mean concentrations of QUIN tended to be lower in HD putamen, dentate nucleus and several cortical regions, although significant reductions were found only in Brodmann areas 17, 20 and 28. The mechanisms responsible for these small reductions in brain QUIN concentrations remain to be determined. These results do not support the hypothesis that a chronic increase of QUIN production is responsible for neurodegeneration in HD.

Parkinson's disease (PD) is a common and often devastating neurodegenerative disease affecting up to one million individuals in the United States alone. Multiple lines of evidence support mitochondrial dysfunction as a primary or secondary event in PD pathogenesis; a better understanding, therefore, of how mitochondrial function is altered in vivo in brain tissue in PD is a critical step toward developing potential PD biomarkers. In vivo study of mitochondrial metabolism in human subjects has previously been technically challenging. However, proton and phosphorus magnetic resonance spectroscopy ((1)H and (31)P MRS) are powerful noninvasive techniques that allow evaluation in vivo of lactate, a marker of anaerobic glycolysis, and high energy phosphates, such as adenosine triphosphate and phosphocreatine, directly reflecting mitochondrial function. This article reviews previous (1)H and (31)P MRS studies in PD, which demonstrate metabolic abnormalities consistent with mitochondrial dysfunction, and then presents recent (1)H MRS data revealing abnormally elevated lactate levels in PD subjects.

Resveratrol, a polyphenol found in red wine, peanuts, soy beans, and pomegranates, possesses a wide range of biological effects. Since resveratrol's properties seem ideal for treating neurodegenerative diseases, its ability to diminish amyloid plaques was tested. Mice were fed clinically feasible dosages of resveratrol for forty-five days. Neither resveratrol nor its conjugated metabolites were detectable in brain. Nevertheless, resveratrol diminished plaque formation in a region specific manner. The largest reductions in the percent area occupied by plaques were observed in medial cortex (-48%), striatum (-89%) and hypothalamus (-90%). The changes occurred without detectable activation of SIRT-1 or alterations in APP processing. However, brain glutathione declined 21% and brain cysteine increased 54%. The increased cysteine and decreased glutathione may be linked to the diminished plaque formation. This study supports the concept that onset of neurodegenerative disease may be delayed or mitigated with use of dietary chemo-preventive agents that protect against beta-amyloid plaque formation and oxidative stress.

We aimed to investigate oxidative stress biomarkers in a cross-sectional pilot study of 50 participants with sporadic ALS (SALS) compared to 46 control subjects. We measured urinary 8-oxodeoxyguanosine (8-oxodG), urinary 15-F(2t)-isoprostane (IsoP), and plasma protein carbonyl by ELISA methods. We also determined if ELISA measurement of 8-oxodG could be validated against measures from high-pressure liquid chromatography coupled with electrochemical detection, the current standard method. We found that 8-oxodG and IsoP levels adjusted for creatinine were significantly elevated in SALS participants. These differences persisted after age and gender were controlled in regression analyses. These markers are highly and positively correlated with each other. 8-oxodG measured by the two techniques from the same urine sample were positively correlated (p

There is increasing evidence linking mitochondrial dysfunction to neurodegenerative diseases. Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Mutations in mitochondrial DNA and oxidative stress both contribute to ageing, which is the greatest risk factor for neurodegenerative diseases. This is the case in Alzheimer's disease, in which there is evidence that both beta-amyloid and the amyloid precursor protein may directly interact with mitochondria, leading to increased free radical production. In the case of Huntington's disease (HD), recent evidence suggests that the coactivator PGC1alpha, a key regulator of mitochondrial biogenesis in respiration, is down-regulated in patients with HD and in several animal models of this neurodegenerative disorder. In Parkinson's disease, the autosomal recessive genes parkin, DJ1 and PINK1 are all linked to either oxidative stress or mitochondrial dysfunction. In amyotrophic lateral sclerosis, there is strong evidence that mutant superoxide dismutase directly interacts with the outer mitochondrial membrane as well as the intermembrane space and matrix. Therefore, an impressive number of disease specific proteins interact with mitochondria. Therapies that target basic mitochondrial processes such as energy metabolism in free radical generation, or specific interactions of disease-related protein with mitochondria, hold great promise.

We have previously demonstrated that the active form of matrix metalloproteinase-3 (actMMP-3) is released from dopamine(DA)rgic neurons undergoing apoptosis. Herein, whether actMMP-3 might be generated intracellularly, and if so, whether it is involved in apoptosis of DArgic neurons itself was investigated in primary cultured DArgic neurons of wild-type, MMP-3 knockout animals, and CATH.a cells. During apoptosis, gene expression of MMP-3 is induced, specifically among the various classes of MMPs, generating the proform (55 kDa) which is subsequently cleaved to the catalytically active actMMP-3 (48 kDa) involving a serine protease. Intracellular actMMP-3 activity is directly linked to apoptotic signaling in DArgic cells: (i) Pharmacologic inhibition of enzymatic activity, repression of gene expression by siRNA, and gene deficiency all lead to protection; (ii) pharmacologic inhibition causes attenuation of DNA fragmentation and caspase 3 activation, the indices of apoptosis; and (iii) inhibition of the pro-apoptotic enzyme c-Jun N-terminal protein kinase leads to repression of MMP-3 induction. Under the cell stress condition, MMP-3 is released as actMMP-3 rather than the proform (proMMP-3), and catalytically active MMP-3 added to the medium does not cause cell death. Thus, actMMP-3 seems to have a novel intracellular role in apoptotic DArgic cells and this finding provides an insight into the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is a progressive and chronic neurodegenerative disorder, characterized by progressive loss of dopaminergic neurons in substantia nigra. The etiology and pathogenesis of PD is still elusive, however, a large body of evidence suggests a prominent role of oxidative stress, inflammation, apoptosis, mitochondrial dysfunction and proteosomal dysfunction in the pathogenesis of PD. Due to multifactorial nature of the disease, currently available drug therapy cannot halt / slow down the disease progression, and only provides symptomatic relief. Peroxisome proliferator-activated receptor (PPAR), a member of nuclear receptor superfamily, regulates development, tissue differentiation, inflammation, mitochondrial function, wound healing, lipid metabolism and glucose metabolism. Recently, several PPAR agonists were shown to exert neuroprotective activity against oxidative damage, inflammation and apoptosis in several neurodegenerative disorders including Alzheimer's, Parkinson's, Huntington's, amyotrophic lateral sclerosis and multiple sclerosis. Similarly, regular intake of PPAR activating non-steroidal anti-inflammatory drugs such as indomethacin and ibuprofen was associated with reduced incidence and progression of neurodegenerative disorders in several epidemiological studies. In this article, we review studies relating to the neuroprotective effect of PPAR agonists in in vitro and in vivo models of PD. Similarly, the pharmacological mechanism in neuroprotective actions of PPAR agonists is also reviewed. In conclusion, PPAR agonists exert neuroprotective actions by regulating the expression of a set of genes involved in cell survival processes, and could be a therapeutic target in debilitating neurodegenerative illnesses such as PD.

The development of biomarkers for the diagnosis and monitoring disease progression in Parkinson's disease (PD) is of great importance since diagnosis based on clinical parameters has a considerable error rate. In this study, we utilized metabolomic profiling using high performance liquid chromatography coupled with electrochemical coulometric array detection (LCECA) to look for biomarkers in plasma useful for the diagnosis of PD. We examined 25 controls and 66 PD patients. We also measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels as a marker of oxidative damage to DNA. We initially examined the profiles of unmedicated PD subjects compared to controls to rule out confounding effects of symptomatic medications. We found a complete separation of the two groups. We then determined the variables, which played the greatest role in separating the two groups and applied them to PD subjects taking dopaminergic medications. Using these parameters, we achieved a complete separation of the PD patients from controls. 8-OHdG levels were significantly increased in PD patients, but overlapped controls. Two other markers of oxidative damage were measured in our LCECA profiles. Uric acid was significantly reduced while glutathione was significantly increased in PD patients. These findings show that metabolomic profiling with LCECA coulometric array has great promise for developing biomarkers for both the diagnosis, as well as monitoring disease progression in PD.

Somatostatin may play a role in several neurodegenerative diseases. Somatostatin concentrations are depleted in cerebral cortex in both Alzheimer's disease and in the dementia that accompanies Parkinson's disease. Somatostatin neurons in both illnesses are markedly dystrophic and may be reduced in number. In Huntington's disease, somatostatin concentrations are increased in the basal ganglia, as is the density of somatostatin neurons. The precise role of somatostatin changes in the pathophysiology of these illnesses requires further study.

A depletion of large cholinergic neurons in the nucleus basalis of Meynert is a consistent finding in Alzheimer's disease (AD). The nucleus basalis of Meynert also contains interneurons and afferents that may modulate its functioning. In the present study we examined neurochemical markers for neuropeptides, amino acid neurotransmitters, and monoaminergic neurotransmitters in postmortem samples of the nucleus basalis in 16 control subjects and 30 patients with AD. There were no significant changes in glutamate, aspartate, taurine, gamma-aminobutyric acid (GABA), and catecholamines; however, concentrations of serotonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophol were significantly reduced. Choline acetyltransferase activity was significantly reduced, consistent with previous reports. Galanin immunoreactivity was significantly increased twofold in the patients with AD, but there were no significant changes in substance P, somatostatin, or neuropeptide Y immunoreactivity. Since galanin inhibits acetylcholine release, and produces cognitive deficits in animals, increased galanin immunoreactivity in the nucleus basalis of Meynert in AD may contribute to the cognitive deficits that characterize the illness.

In the present study we investigated the relative vulnerability of neuronal subsets in the striatum to ischemia that was induced by bilateral transient ligation of the common carotid arteries in gerbils. After 4 days of survival, brains were evaluated using histochemical methods (NADPH-diaphorase and silver degeneration procedures) and neurochemical methods with radioimmunoassays for somatostatin-, neuropeptide Y-, and substance P-like immunoreactivity and measurements of amino acids using high-pressure liquid chromatography with electrochemical detection. NADPH-diaphorase-positive neurons were strikingly preserved in the ischemic dorsolateral portion of the striatum, in which there was severe neuronal loss. There was no significant depletion of NADPH-diaphorase-positive neurons in the striatum or cerebral cortex. Concentrations of neuropeptide Y-like and somatostatin-like immunoreactivity were unchanged despite a significant 25% depletion of substance P-like immunoreactivity and gamma-aminobutyric acid. Ischemic brain damage may be mediated by a neurotoxic effect of glutamate acting at the N-methyl-D-aspartate (NMDA) receptor. Previous studies of NMDA excitotoxin lesions in rat striatum have shown a sparing of neurons containing NADPH-diaphorase, somatostatin, and neuropeptide Y. The similar sparing of these neurons following ischemic lesions in gerbil striatum provides further evidence that NMDA receptor activation may play a role in ischemic injury.

Excitatory amino acids may play a role in the pathogenesis of cell death in neurodegenerative diseases, including Huntington's disease (HD). In an attempt to develop a tissue culture model for HD, the toxicity of glutamate was examined in primary striatal cultures derived from newborn rats. Morphological criteria were used to determine the toxic effects of glutamate in 6-, 12-, and 18-day-old cultures which were examined before and after 1-3 h of exposure to glutamate. Although younger cultures demonstrated little susceptibility to glutamate relative to controls, the number of neurons in older cultures was significantly depleted in the presence of glutamate. Glutamate toxicity was dose-dependent, with an ED50 of approximately 300 microns glutamate, and a maximal effect was observed within 3 h of initial exposure. Affected neurons demonstrated somal swelling within 1 h of glutamate exposure and disruption of neuritic processes and somal integrity within 3 h. Cell death was significantly increased by raising the extracellular calcium concentration and could be decreased by the addition of magnesium to the incubation medium. Moreover, the N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid, showed a toxicity profile similar to that of glutamate. The NMDA receptor competitive antagonist, 2-amino-5-phosphonovalerate (APV) significantly reduced toxicity, albeit incompletely. An additional component of glutamate mediated toxicity in striatal cultures could be explained by activation of non-NMDA receptor subtypes. These in vitro studies indicate that glutamate is toxic to a subset of mature striatal neurons in the absence of a glutamatergic afferent input, and that this toxicity is mediated partially by the NMDA receptor, with an additional component due to non-NMDA receptors.

There is increasing evidence linking mitochondrial dysfunction to neurodegenerative diseases. Mitochondria are critical regulators of cell death, a key feature of neurodegeneration. Mutations in mitochondrial DNA and oxidative stress both contribute to ageing, which is the greatest risk factor for neurodegenerative diseases. This is the case in Alzheimer's disease, in which there is evidence that both beta-amyloid and the amyloid precursor protein may directly interact with mitochondria, leading to increased free radical production. In the case of Huntington's disease (HD), recent evidence suggests that the coactivator PGC1alpha, a key regulator of mitochondrial biogenesis in respiration, is down-regulated in patients with HD and in several animal models of this neurodegenerative disorder. In Parkinson's disease, the autosomal recessive genes parkin, DJ1 and PINK1 are all linked to either oxidative stress or mitochondrial dysfunction. In amyotrophic lateral sclerosis, there is strong evidence that mutant superoxide dismutase directly interacts with the outer mitochondrial membrane as well as the intermembrane space and matrix. Therefore, an impressive number of disease specific proteins interact with mitochondria. Therapies that target basic mitochondrial processes such as energy metabolism in free radical generation, or specific interactions of disease-related protein with mitochondria, hold great promise.

Coenzyme Q10 (CoQ10) is a promising agent for neuroprotection in neurodegenerative diseases. We tested the effects of various doses of two formulations of CoQ10 in food and found that administration in the diet resulted in significant protection against loss of dopamine (DA), which was accompanied by a marked increase in plasma concentrations of CoQ10. We further investigated the neuroprotective effects of CoQ10, reduced CoQ10 (ubiquinol), and CoQ10 emulsions in the (MPTP) model of Parkinson's disease (PD). We found neuroprotection against MPTP induced loss of DA using both CoQ10, and reduced CoQ10, which produced the largest increases in plasma concentrations. Lastly, we administered CoQ10 in the diet to test its effects in a chronic MPTP model induced by administration of MPTP by Alzet pump for 1 month. We found neuroprotective effects against DA depletion, loss of tyrosine hydroxylase neurons and induction of alpha-synuclein inclusions in the substantia nigra pars compacta. The finding that CoQ10 is effective in a chronic dosing model of MPTP toxicity, is of particular interest, as this may be more relevant to PD. These results provide further evidence that administration of CoQ10 is a promising therapeutic strategy for the treatment of PD.