M. Flint Beal, MD, University of Virginia 1976 - US grants

Examination of peptide neurotransmitters may lead to new insights in degenerative neurologic illnesses. The goals of this study are (1) to determine neuroanatomic pathways involving somatostatin in the basal ganglia, (2) to measure their relative contribution to the basal ganglia content of somatostatin-like immunoreactivity (SLI) as determined by radioimmunoassay (3) to study interactions between somatostatin and dopamine and its metabolites in the striatum and conversely the effects of dopamine blocking drugs on striatal SLI (4) to determine levels of somatostatin and other peptides in Parkinson's disease and to examine the normal distribution of somatostatin and other peptides within human basal ganglia (5) to measure somatostatin receptors in Huntington's disease and correlate this with the increased levels of SLI we have observed in basal ganglia in this illness. Somatostatin pathways within the basal ganglia will initially be examined by continuing our lesion studies followed by determination of striatal SLI in striatal punches by a specific radioimmunoassay. Specific pathways will be confirmed using fluorescence retrograde tracing combined with somatostatin immunocytochemistry. The interactions of dopamine and its metabolites with somatostatin will be examined using a push-pull cannula model. Dopamine, serotonin and their metabolites will be determined by high pressure liquid chromatography with electrochemical detection. The interactions of SLI with dopamine blocking drugs will be examined by administering these drugs to animals and subsequently measuring both SLI and catecholamines. Levels of SLI, substance P, neurotensin and enkephalin will be determined in both Parkinsonian brains and in detailed dissections of normal human brains. Somatostatin receptores will be analyzed in post-mortem tissue from Huntington's disease using a filtration assay with I125Tyr11 somatostatin as a tracer.

DESCRIPTION (Investigator's Abstract): Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by progressive cognitive impairment, emotional disturbances and a movement disorder. The fundamental biochemical defect underlying neuronal degeneration remains unknown, and there is no known effective therapy. We hypothesis that a defect in energy metabolism could lead to slow excitotoxic neuronal cell death. To eaamine this possibility we have utilized 1H magnetic resonance spectroscopy to determine levels of lactate as well as other neurochemicals in ND patients. In known mitochondrial disorders lactate is elevated in brain and spinal fluid. Our preliminary studies show increased levels of lactate in several brain regions in ND which correlate with the duration of disease. Our most exciting finding is the observation that treatment with coenzyme Q10, which improves mitochondrial function, significantly decreased lactate concentrations in Il of 14 treated patients. This raises the possibility that this might be a useful therapeutic strategy- in HD. The present proposal is to extend these studies. Our first specific aim is to continue to perform IN MRS to determine the regional distribution of changes in lactatc, nacetyl aspartate, creatine and choline in. HD patients as compared with normal controls. We will also carry out studies with 31P- MRS in muscle determine whether an energetic defect could be generalized in HD. Our second specific aim will be to determine if regional elevations of lactate measured by 1H-MRS or CSF lactate/pyrvvate ratios, correlate with duration of symptoms, degree of clinical neurologic impairment, ratings on standard disability scales, age of onset and bradykinetic or choreic phenotype. We will also determine if medications commonly used by HD patients have effects on lactate levels measured by 1N-MRS. Our third specific aim is to determine whether physiologic stimulation of brain regions in ND patients using photic stimulation, or sequential motor tasks result in changes in lactate or magnetic susceptibility which differ from those which occur in normal subjects.Our preliminary findings indicate that photic stimulation results in decreased lactate in occipital cortex, rather than the increases seen in normal subjects. Our last specific aim to use 1H NMS to screen for compounds which lower brain lactate in ND patients and which therefore might be attractive candidates for therapeutic trials. We will continue examining coenzyme Qio and will also examine the effects of riboflavin with nicotinamide and L-carnitine. We will also examine the effects of excitatory amino acid antagonists when these become clinically available. Our preliminary studies provide the first evidence of a possible therapy to treat the underlying disease process in ND. The present proposal has the potential of helping to elucidate the filndaniental biochemical defect underlying ND, and may lead to the development of effective the-for this disorder.

oxidative stress; enzyme activity; DNA damage; Huntington's disease; neural degeneration; mitochondrial DNA; nitric oxide synthase; free radical scavengers; neurochemistry; biomarker; oxidoreductase inhibitor; ubiquinone; oxidative phosphorylation; free radical oxygen; antioxidants; nucleic acid repetitive sequence; human subject; postmortem; blood chemistry; laboratory rat; urinalysis; histopathology;

The etiology of nerve cell death in Huntington's disease (HD) and Parkinson's disease (PD) is unknown. The fundamental hypothesis in the present proposal is that a defect in energy metabolism may lead to slow excitotoxic neuronal death in these diseases. A defect in energy metabolism may lead to partial neuronal depolarization enabling voltage- dependent excitatory amino acid receptors to be activated by endogenous levels of glutamate. In the present proposal we wish to test this hypothesis and to develop toxins 3-nitropropionic acid and sodium azide procedures lesions which show specific striatal neurotoxicity. In the case of 3-nitropropionic acid the toxicity is age-dependent. We found that chronic systemic administration of low doses of 3-nitropropionic acid to middle-aged rats result in subtle lesions that share several anatomical and neurochemical features with HD. These include relative vulnerability of the striatum, increased vulnerability in older animals, sparing of aspiny neurons, and characteristic dendritic changes. The goal of this proposal is to further characterize the mechanism and specificity of striatal lesions produced by inhibitors of oxidative phosphorylation. We will study specific inhibitors of complex I (MPP+), complex II (3-nitropropionic acid and malonate) and complex IV (sodium azide), using neurochemical, neuroanatomical, and molecular biologic tools. We will examine whether the striatal degree of energy depletion using direct measurements of ATP and lactate. We will test the idea that toxicity occurs due to a secondary excitotoxic mechanism by examining the effects of excitatory amino acid antagonists, and by removing glutamatergic afferents to the striatum. We will determine whether chronic administration of 3-nitropropionic acid leads to oxidative damage to DNA and proteins, which could contribute to an ongoing degenerative process. We will carry out further studies of the age-dependence of preliminary findings indicate that intrastriatal injections of malonate, which is a competitive inhibitor of complex II, produces a different pattern of cell damage that produced by 3-nitroproprionic acid, which is an irreversible inhibitor of complex II. We will determine whether these differences are related to the degree of metabolic compromise produced by the 2 compounds. Lastly we will determine whether several agents which either increase ATP production or bypass mitochondrial defects can prevent lesions produced by these mitochondrial toxins. We will examine the effects of coenzyme Q, riboflavin with nicotinamide, 1,3, butanediol, succinate and carnitine. If these strategies are effective, they may be directly applicable to the development of new treatments for HD and PD

The etiology of neuronal degeneration in Huntington's disease and other neurodegenerative diseases may involve a complex interplay between energy impairment and oxidative damage. There is substantial evidence for metabolic dysfunction in Huntington's disease. A major advance in studying Huntington's disease was the development of transgenic models. In the present proposal we will determine whether there are defects in oxidative phosphorylation in transgenic animal models of HD. We will make direct measurements of ATP, phosphocreatine and lactate. We will measure the electron transport enzymes as well as enzymes involved in the tricarboxylic acid (TCA) cycle. We will utilize both proton and 13C glucose NMR spectroscopic studies to help to localize a defect in vivo. We will also utilize these techniques ex vivo. We will determine whether there is evidence of oxidative damage by measuring 8-hydroxy-2- deoxyguanosine, protein carbonyls, 3-nitrotyrosine, malondialdehyde and the conversion of salicylate to dihydroxybenzoic acid in tissue. We will also carry out immunocytochemical studies for oxidative markers. We will utilize microdialysis to determine whether there is evidence of increased hydroxyl radical generation in vivo. We will examine animals at 4, 8, and 12 weeks of age which are time points corresponding to a presymptomatic phase, a symptomatic phase and advanced illness. We will determine whether therapeutic interventions which may improve energy metabolism can improve both functional outcome as well as survival in transgenic HD mice. We will evaluate the effects of coenzyme Q10, nicotinamide and creatine. Lastly we will utilize mice deficient in JNK-3 kinase and mice overexpressing bcl-2 to determine whether these mice are resistant to striatal lesions produced by the mitochondrial toxins malonate or 3-nitropropionic acid (3-NP), and whether protection is associated with reductions in biochemical markers of oxidative damage. These studies will provide direct evidence whether there is a metabolic defect and oxidative damage in a transgenic animal model of HD. They also have the potential of leading to new therapeutic interventions which might be useful in the treatment of HD.

The present proposal will investigate the role of mitochondrial dysfunction and oxidative damage in several transgenic mouse models of neurodegenerative diseases. In particular we will investigate whether mice with a deficiency in dihydrolipoamide dehydrogenase will show increased oxidative damage, increased vulnerability to mitochondrial toxins and when crossed to transgenic mouse models of Alzheimer's disease (AD), Huntington's disease (HD), spinocerebellar ataxia1 (sca1) and dentatorubropallidoluysian atrophy (DRPLA) whether this will accelerate the phenotype. The mice with a deficiency of murine dihydrolipoamide dehydrogenase show deficiencies in several mitochondrial enzymes including a 50% deficiency in both pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. They will therefore help to test the potential role of reduced levels of these enzymes in neurodegenerative diseases. We will also carry out studies of mitochondrial oxygen utilization, oxidative damage and of the mitochondrial permeability transition in transgenic mouse models of HD, SCA1 and DRPLA. Another theme of the present program project grant is that increased transglutaminase activity may contribute to neuronal dysfunction and the development of cytoplasmic and nuclear inclusions in transgenic mouse models of HD, SCA1 and DRPLA. We will therefore measure brain transglutaminase activity as well as epsilon(4-glutamyl) lysine in brains of transgenic mouse models of these illnesses in collaboration with project 4. We will also test whether tissue transglutaminase inhibitors can extend survival and block the development of the intranuclear inclusions. We will also investigate whether transgenic mice with mutations in the amyloid precursor protein exhibit metabolic abnormalities in oxidative damage. Lastly if oxidative damage plays a role in the pathogenesis of neurodegenerative diseases then crossing transgenic ADP, HD, SCA1 or DRPLA mice with transgenic mice deficient in manganese SOD or glutathione peroxidase should accelerate the phenotype. We will therefore look at defects on survival as well as on markers of oxidative damage in mice following these crosses. The proposed studies will help to elucidate the role of mitochondrial dysfunction in transgenic mouse models of both AD as well as HD, SCA1 and DRPLA. They may also lead to novel and useful treatment strategies for these disorders.

There is substantial evidence that the pathogenesis of Alzheimer's Disease (AD) may involve mitochondrial dysfunction and oxidative damage. Mitochondrial dysfunction could occur as either a consequence of primary genetic mutations or due to acquired mitochondrial DNA (mtDNA) mutations, which may be related to oxidative damage. In the present proposal, we will examine whether there is an increased incidence of mtDNA mutations in postmortem brain tissue from AD patients as compared to normal controls. We will utilize direct mtDNA sequencing as well as denaturing gelelectrophoresis to detect low frequency mutations, and we will correlate levels of 8-hydroxy-2- deoxyguanosine (OH8dG), a marker of oxidative damage to DNA. We have developed a sensitive and accurate assay for OH8dG, which is useful in examining concentrations in body fluids. We intend to utilize this assay to measure OH8dG in urine, plasma and CSF of AD patients and controls. We will make cybrids utilizing platelets obtained from well characterized AD patients. *-amyloid deposition may cause oxidative stress and/or oxidative stress may increase -amyloid production. We will examine whether transgenic mice with the APP V717F mutation have increased mtDNA mutations as assessed by direct sequencing. We will correlate this with concentrations of b-amyloid as measured by ELISA, as well as markers of oxidative damage including malondialdehyde, OH8dG and 5-nitro-gamma-tocopherol. We will examine transgenic mouse lines which are deficient in the mitochondrial free radical scavenging enzyme manganese superoxide dismutase. We will also utilize mice, which are deficient in glutathione peroxidase, which detoxifies hydrogen peroxide within mitochondria. We will determine whether these mice develop age-dependent increases in oxidative damage within mtDNA, increased numbers of mtDNA mutations and whether this correlates with increases in extractable levels of b-amyloid. These studies are designed to help to further elucidate the role of mitochondrial dysfunction and oxidative damage in normal aging and AD.

[unreadable] DESCRIPTION (provided by applicant): The pathogenesis of Huntington's Disease (HD) is as yet unknown but there is substantial evidence that both altered gene transcription as well as mitochondrial dysfunction play an important role. There is evidence that huntingtin binds to transcription factors which results in decreased expression of genes which may play a critical role in neuronal survival. A secondary consequence of this appears to be impaired oxidative phosphorylation and increased generation of reactive oxygen species. In our prior grant, we showed that there was impaired oxidative phosphorylation in transgenic mouse models of Huntington's disease, and that this was associated with increased oxidative damage. We also showed that agents such as creatine and coenzyme Q, which improve cellular bioenergetics, exert neuroprotective effects in transgenic mouse models of Huntington's disease. In the present proposal, we intend to extend these studies to two further unique transgenic mouse models of Huntington's disease. We will determine whether there is mitochondrial dysfunction and oxidative damage in a knock-in mouse model developed by MacDonald and colleagues. These mice are a very accurate genetic model of Huntington's disease. We will also examine the tetracycline-off model developed by Yamamoto and colleagues to determine whether there is mitochondrial dysfunction and oxidative damage with the gene turned on, which then resolves once the gone is turned off. We will carry out similar studies with an inducible cell culture model. We will investigate whether histone deacetylase (HDAC) inhibitors exert neuroprotective effects by altering gene transcription in transgenic mouse models of Huntington's disease. We will examine whether a phosphodiesterase IV inhibitor can exert neuroprotective effects in transgenic mouse models of HD by increasing cyclic AMP levels, leading to increased CREB transcriptional activity, and whether this improves mitochondrial function. Our prior studies showed that combinations of agents, which target different disease mechanisms in Huntington's disease, may exert additive neuroprotective effects. We will, therefore, examine whether a combination of creatine or coenzyme Q with either a HDAC inhibitor or a phosphodiesterase IV inhibitor can exert additive neuroprotective effects. [unreadable] [unreadable]

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, which affects motor neurons of the spinal cord and cerebral cortex, leading to progressive paralysis and premature death. While the majority of the cases are sporadic and due to unknown causes, about 5-10% are familial (FALS), and of those approximately 25% are associated with mutations in the gene for Cu/Zn superoxide dismutase (SOD1). These mutations are the first identified cause of ALS, and their finding has allowed the development of a transgenic mouse model of the disease. Abnormal mitochondria and impaired mitochondrial respiratory chain function have been reported in motor neurons of patients with sporadic and ALS, as well as in the mouse models. A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical formation in the mitochondria and damage to the mitochondrial DNA (mtDNA) could be causally involved in motor neuron death in ALS. Indeed, several studies have postulated the involvement of mtDNA abnormalities in motor neuron degeneration. To define the role of mitochondrial alterations in the pathogenesis of ALS, we propose the following studies. 1) Analyze mitochondrial functions in brain and spinal cord in ALS transgenic mice at various stages of disease and in cultured cells expressing mutant human SOD1. Analyze the mtDNA from ALS transgenic mice in cybrids constructed with synaptosomes from brain and spinal cords. 2) Study the structural properties and the functional effects of SOD1 localized to mitochondria in transgenic animals and in cultured neuroblastoma cells expressing wild type and mutated h SOD1. 3) Analyze respiratory chain functions and mtDNA in individual motor neurons from ALS patients. We think that a better comprehension of the molecular basis of mitochondrial dysfunction in ALS will help us to understand the mechanisms responsible for the motor neuron loss and, in some cases may even identify the primary cause of the disease, with a potential impact on therapeutic strategies.

DESCRIPTION (provided by applicant):Huntington's disease (HD) is progressive and fatal neurological disorder which is caused by a CAG repeat expansion in the gene coding for protein of unknown function, huntington. Although major advances have been made in understanding the biology of the illness, as yet there is no effective treatment. A number of different mechanisms are thought to contribute to disease pathogenesis. Several new therapeutic agents have been identified which target several of these disease mechanisms. These include agents, which modulate gene transcription, ameliorate mitochondrial dysfunction, which have anti oxidative and anti inflammatory effects and which modulate apoptic cell death. In the present proposal we plan to continue studies of novel therapeutic agents targeting different disease mechanism which may lead to new insights into classes of therapeutic compounds which may prove to effective in treating HD patients. In some cases we have previously identified therapeutic efficacy with these targets. In the latter stages of this project we will also examine the effects of combinations of therapies, which may result increased levels of neuroprotection. We will study therapies in both transgenic mice with an N-terminal fragment of huntington (R6/2), as well as a full-length transgenic mouse model in which the human full-length huntington gene has been incorporated into a bacterial artificial chromosome with an expanded 226 CAG repeat region (BAC 226Q). These mice show robust phenotype, as well as cell loss, and therefore appear to be a very useful model for screening therapeutic agents. We are therefore proposing a series of experiments that will examine a number of therapeutic targets for HD. We believe that these studies will provide critical pre-clinical data to determine the agents, which have the most promise for use as neuroprotective agents in clinical drug trials in HD patients.

laboratory mouse; mitochondrial disease /disorder; pathologic process

Parkinson's Disease (PD) is a progressive, neurodegenerative disease, which affects over 1,000,000 Americans. In an NIH-supported phase II trial, our group demonstrated that treatment of patients with early, untreated PD with high dosages of coenzyme Q10 (300, 600 and 1200 mg/d) was safe and well tolerated and that there was a positive trend for treatment to slow the progressive impairment as measured by the Unified Parkinson Disease Rating Scale (UPDRS). In pre-specified secondary analyses, we noted that the highest dosage of coenzyme Q10 was the most effective dosage and that treatment with coenzyme Q10 helped the PD patients maintain independence, as measured by Schwab and England Scale. In the proposed study, we will conduct a prospective, randomized, placebo-controlled, double-blind phase clinical trial of coenzyme Q10 to attempt to confirm and extend the results of our phase II study. We propose to enroll 600 subjects who have early PD and do not yet require treatment with dopaminergic agents. Subjects will be randomly assigned to receive placebo, coenzyme Q10 1200 mg/d or 2400 mg/d, evaluated at screening, baseline, months 1, 4, 8, 12, and 16 visits and assessed with the UPDRS. The investigator will determine whether the subject has reached disability requiring treatment with a dopaminergic agent. Our primary analysis will undertake to confirm the benefit found in our phase II study by analyzing the change in total UPDRS score to the point that the subjects reach disability requiring treatment with a dopaminergic agent or to the 16 month visit. We will also analyze the Schwab and England Scale and the PD Quality of Life Scale data collected to the point that the subjects reach disability requiring treatment with a dopaminergic agent or to the 16 month visit. We will determine the mean plasma coenzyme Q10 levels at baseline and at visits 1, 8 and 16 months, and determine whether the mean correlates with reduction in worsening of the total UPDRS score.

[unreadable] DESCRIPTION (provided by applicant): There is increasing evidence that oxidative damage may contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer's Disease (AD). A number of recent studies have suggested that oxidative damage may precede and be causally linked to the deposition of beta-amyloid. Conversely, beta-amyloid may induce oxidative damage. There are two major classes of oxidants and biological systems; reactive oxygen intermediates and reactive nitrogen intermediates. A number of studies have shown that there are increased reactive oxygen intermediates in AD postmortem brain tissue as assessed by oxidative damage markers. There also appears to be increased reactive nitrogen intermediates as assessed by biochemical and immunocytochemical measurements of 3-nitrotyrosine. Our data, which has been confirmed by others, shows that there is an increase in inducible nitric oxide synthase (NOS2) immunoreactivity within neurons in both AD postmortem brain tissue, as well as in transgenic mouse models. Genetic reduction of NOS2 activity markedly reduced beta-amyloid deposition in a transgenic mouse model of AD. The goals of the present application are to utilize two compounds, which can block reactive oxygen species and reactive nitrogen intermediates. We will utilize coenzyme Q10 and L-iminoethyl-L-lysine (L-NIL) in transgenic mouse models of AD. CoQ10 is a cofactor of the electron transport gene, which has strong antioxidant properties. It is extremely well tolerated in human subjects and is under clinical development for treatment of Parkinson's Disease, Huntington's Disease and amyotrophic lateral sclerosis. We will also look at the effects of L-iminoethyl-L-lysine, which is a relatively specific inhibitor of NOS2. We will determine whether treatment with either CoQ10 or L-NIL can exert neuroprotective effects against beta-amyloid deposition and oxidative damage, and improve memory in transgenic mouse models of AD. If we can demonstrate significant effects of CoQ10 and L-NIL, this could lead to rapid development of new therapies for slowing the progression of AD. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The New York Academy of Sciences (NYAS) is planning a major 3 1/2-day conference entitled "Mitochondria and Oxidative Stress in Neurodegenerative Disorders" to be held September 26-29, 2007 at the New York Academy of Sciences, New York, NY. The conference is being organized by Gary E. Gibson, PhD, Professor of Neuroscience, Weill Medical College of Cornell University, and Burke-Cornell Medical Research Institute, Rajiv R. Ratan, MD, PhD, Burke Professor of Neurology and Neuroscience, Weill Medical College of Cornell University, Executive Director, Burke-Cornell Medical Research Institute, and M. Flint Beal, MD, Professor and Chairman, Department of Neurology and Neuroscience, Weill Medical College of Cornell University. Our understanding of the interaction of mitochondria with other cellular organelles, with transcription and in the ability to detect oxidative modification of macromolecules has improved significantly in past decade. Moreover the roles of mitochondria and oxidative stress are better defined in the pathophysiology of neurodegenerative disorders. The goal of the meeting is to determine what we need to know to move towards better therapies. This conference will combine basic, clinical and translational research in a forum designed to provide the most current information on aspects of mitochondrial function and its relationship to age-related neurodegenerative diseases and their treatment. Specific objectives of this conference are to: 1) develop a better understanding of the role of mitochondrial in the nervous system; 2) discuss the involvement of mitochondria in specific neurodegenerative disorders; 3) address therapeutic approaches to treating these diseases; 4) allow students, fellows and junior investigators to be involved in the program and to foster their ability to form networks amongst themselves and with more senior investigators and 5) disseminate the proceedings by print and electronic means so that a wider audience can benefit from the insights shared. The age-related onset and progression of neurodegenerative disorders such as Alzheimer's disease, Huntington's disease and Parkinson's disease underscores a relationship between aging, mitochondrial impairment and oxidative stress. The development of effective therapies requires that we understand mitochondrial function, structure, intracellular and intercellular oxidant signaling, oxidative stress and the control of genes that code mitochondrial components. This conference will provide a forum for discussion of the state-of-the-art in oxidative stress and its relationship to neurodegenerative diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a devastating neurodegenerative disorder characterized by a loss of substantia nigra pars compacta neurons, which currently affects 1.5 million people in the United States. Although rare mutations associated with familial forms of PD have been identified most cases occur sporadically. While the causes of PD are unknown, a critical role of environmental factors either alone, or in combination with genetic susceptibilities is implicated in disease pathogenesis. Major pathologic mechanisms that lead to neurodegenerative phenotype of nigrostriatal dopaminergic neurons include mitochondrial dysfunction, oxidative damage, impairments of key cell survival signaling, and activation of inherent cell death pathways. Epidemiological studies have identified pesticides as potential environmental exposures that influence the risk of PD. Our preliminary data suggest that oral administration of pesticides in mice cause degeneration of nigrostriatal dopaminergic neurons, striatal loss of dopamine and its metabolites, pathologic accumulation of 1-synuclein and oxidative damage. We hypothesize that administration of these pesticides cause neurodegeneration due to mitochondrial dysfunction, and by activation or impairment of signal transduction pathways that are detrimental for normal functioning of dopaminergic neurons. Additionally, these pathologic mechanisms may lead to exacerbation of neurodegeneration in transgenic mouse models of familial PD. Three specific aims are proposed to test the hypothesis. Aim 1 will study the identification and validation of key cell signaling pathways that lead to nigrostriatal dopaminergic neurodegeneration due to pesticide exposure in mice. Aim 2 will study the role of transcriptional regulation of key cell signaling pathways such as Nrf2/ARE, PGC-1 alpha and SIRT1 in addition to specific signal transduction pathways identified and validated in Aim 1 on mitochondrial function and neuropathological features involved in disease development due to pesticide exposure in mice. Aim 3 will examine the role of pesticide exposures in transgenic mouse models of familial PD to assess mitochondrial function, cell signaling pathways leading to exacerbation of nigrostriatal dopaminergic neurons and behavioral abnormalities. These studies will provide us with novel mechanistic insights to events leading to disease development in both sporadic and familial forms of PD due to pesticide exposure and will enable us identify potential links between gene environment interactions in PD. PUBLIC HEALTH RELEVANCE: This study proposes to identify novel signal transduction pathways in wild type and transgenic mice harboring familial PD mutations for onset and development of Parkinson's disease (PD) due to exposure of mitochondria targeted pesticide. The study will enrich and refine our understanding of pesticide-induced cell damage pathways observed in sporadic and familial PD and identify new target(s) for intervention in PD pathogenesis.

DESCRIPTION (provided by applicant): This application addresses broad challenge area (03): Validation and Specific Challenge Topic 03-AT-102: Antioxidant biomarkers. Development and validation of biomarkers of oxidative stress that could be used to assess the antioxidant effects of dietary supplements in vivo. Parkinson's Disease (PD) as well as other neurodegenerative diseases are associated with oxidative damage. In PD, there is an early reduction in reduced glutathione in the substantia nigra, as well as increases in markers of lipid, protein and DNA oxidation. A number of laboratories have reported increases in plasma and cerebrospinal fluid biomarkers of oxidative stress in PD. We recently found reduced uric acid and increased 8-hydroxy-2-deoxyguanosine (8-OHdG) in plasma of PD patients. We are presently carrying out a phase III clinical trial of the antioxidant nutritional supplement coenzyme Q10 (CoQ10) in PD. Six hundred newly diagnosed unmedicated PD subjects are being randomized to placebo, 1200mg of CoQ10 or 2400 mg of CoQ10 daily. Patients are assessed using the Unified Parkinson's Disease Rating Scale (UPDRS) every 3 months until they require dopaminergic therapy or they complete 16 months. We propose to measure several markers of oxidative damage at baseline, 1, 8 and 16 months of therapy. We will measure plasma levels of 8-OHdG, malondialdehyde, ascorbic acid, uric acid, oxidized and reduced CoQ10, and oxidized and reduced glutathione. All samples will be examined using metabolomic profiling using HPLC with coulometric array detection. This results in detection of up to 2000 small molecules, which are electrochemically active. The measurements of individual peaks will be correlated with those of established markers of oxidative damage such as 8-OHdG, malondialdehyde and the ratio of oxidized/reduced glutathione, to see if more sensitive and specific novel biomarkers of oxidative stress can be identified. The biomarkers will be correlated with clinical improvements in PD patients as assessed using UPDRS scores. These studies will establish the relative utility of existing biomarkers of oxidative damage, and may lead to the development of novel biomarkers, which will be useful in assessing the efficacy of the antioxidant effects of dietary supplements in vivo, and will help in assessing their effectiveness with respect to human health. As such, these studies may be useful in establishing the effectiveness of a large number of dietary interventions, which may impact human health. Public Health Relevance: This study proposes to identify biomarkers of oxidative stress to facilitate in vivo study of the antioxidant effects of the dietary supplement coenzyme Q10 (CoQ10). We intend to comprehensively evaluate a panel of established markers of oxidative damage and to utilize metabolic profiling to identify novel markers of oxidative stress. We will examine the relative sensitivity of these biomarkers in relationship to clinical efficacy of CoQ10 in a phase III clinical trial in Parkinson's Disease. The development of useful biomarkers to assess oxidative stress in relationship to human health, has the potential of having a major impact in the development of therapies to treat and prevent neurodegenerative diseases. These biomarkers may be useful in assessing therapeutic interventions in numerous other human illnesses in which oxidative stress plays an important role.

? DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disease leading to both cognitive as well as motor deficits, and severe disability. The disease is caused by an unstable CAG repeat expansion which leads to a polyglutamine stretch within the protein huntingtin. The toxic effects of mutant huntingtin result in transcriptional dysregulation as well as mitochondrial dysfunction and oxidative damage. There is a deficiency of PGC-1alpha, a transcriptional coactivator which controls mitochondrial biogenesis and expression of antioxidant enzymes. There is also a deficiency of SIRT3, which is a protein deacetylase whose expression within mitochondria is dependent on PGC-1alpha. We propose validating both PGC-1alpha and SIRT3 as therapeutic targets for the treatment of HD. We will determine whether crossing both R6/2 and KI-zQ175 mice with SIRT3 overexpressing mice will produce neuroprotective effects. We will administer nicotinamide riboside (NR) in the diet as a means of activating SIRT1 and SIRT3, and determine whether this produces neuroprotective effects in both the R6/2 and the BACHD transgenic mouse models of HD. Lastly we will determine whether administration of the panPPAR agonist fenofibrate, or the RXR agonist bexarotene either alone or in combination will increase PGC-1alpha expression and exert neuroprotective effects in R6/2 and BACHD transgenic mice. These studies will validate PGC-1alpha and SIRT3 as therapeutic targets, and will determine whether NR, fenofibrate and bexarotene are suitable for further preclinical development, and ultimately for clinical trials to establish efficacy as neuroprotective therapies for HD.

Mitochondrial dysfunction and oxidative damage play an important role in the pathogenesis of Alzheimer's disease (AD), and metabolic disturbances, such as reduced glucose utilization precede the ?-amyloid and tau pathology. We hypothesize that therapeutics targeting and correcting metabolic dysfunction will be efficacious in treating AD. PGC-1? enhances both mitochondrial biogenesis and expression of antioxidant enzymes, which produces neuroprotective effects both in vitro and in vivo. Sirtuins are NAD+ dependent enzymes which play important roles in regulating metabolism. SIRT3 is of particular interest since it is localized to mitochondria where it activates enzymes of energy metabolism, and protects against reactive oxygen species (ROS) by increasing MnSOD and mitochondrial glutathione concentrations, and it inhibits activation of the mitochondrial permeability transition by deacetylating cyclophilin D. We will examine whether crossing transgenic mice with increased SIRT3 expression will exert neuroprotective effects in transgenic mouse models with increased amyloid deposition (Tg19959 and APP-NL/G/F), or with increased tau phosphorylation and NFTs (P301S). We will also determine whether treatment with nicotinamide riboside (NR), which increases brain and mitochondrial NAD+ and activates SIRT1 and SIRT3, will produce neuroprotective effects in Tg19959, APP-NL/G/F or P301S transgenic mice. The peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors is a group of ligand modulated transcription factors that regulate gene expression of metabolic pathways, including PGC-1?. We will determine whether the PPAR? agonist palmitoylethanolamide (PEA), the PPAR? agonist pioglitazone or the panPPAR agonist fenofibrate which modulate energy metabolism and inflammation, are neuroprotective in transgenic mouse models of AD with either APP or tau mutations. Levels of thiamine and the thiamine dependent enzymes ?-ketoglutarate dehydrogenase and transketolase are reduced in AD. Lastly, we will determine whether the lipid soluble thiamine analogue benfotiamine can reduce oxidative stress and inflammation, and is neuroprotective in transgenic mouse models of AD with either APP or tau mutations. We will utilize microPET and MRI to determine the time course of the development of impaired glucose metabolism amyloid plaques (PIB) and tau (T807), which areas show the most susceptibility, and whether these can be altered by therapeutic interventions including NR, PPAR agonists and benfotiamine. These experiments are highly significant since they may lead to novel treatments to slow or halt the cognitive impairment and neurodegenerative processes which occur in AD and related dementias.