David Keith Simon - US grants

Parkinson's disease (PD) is a common neurodegenerative disease of unknown cause. As many as 1.4% of persons over 55 years old and 4.3% of persons over 85 years old are affected. Evidence is accumulating that defective energy metabolism plays a major role in PD. A great deal of interest has recently focused on the mechanisms by which impaired energy metabolism might lead to cell death. Both oxidative stress and excitotoxicity have been implicated. Though there is evidence to support these hypothesis, it has yet to be firmly established that either oxidative stress or excitotoxicity due to impaired energy metabolism plays a causative role in idiopathic PD. These issues will be addressed in this proposal by studying markers of oxidative stress and analyzing neuroprotective measures in vitro in cytoplasmic hybrids ("cybrids") of patients with PD. Cybrids are formed using platelet mitochondria DNA to repopulate a human neuroblastoma cell line containing no mitochondrial DNA. This allows the study of the functional significance of defects in mitochondrial DNA while controlling for potential nuclear DNA factors that might influence mitochondrial function. The specific aims of his proposal are as follows: 1)to analyze the PD cybrids and aged-matched controls for evidence of oxidative damage to DNA, proteins, and lipids by measuring 8-hydroxy-2-deoxyguanosine, protein carbonyls and 3-nitrotyrosine, and malondialdehyde, respectively, and to correlate these findings with clinical features of disease. 2) to determine whether exposure of the PD affected cybrid cells to the mitochondrial toxin MPP+ is associated with increased oxidative damage. MPP is the active metabolite of MPTP, a neurotoxic compound commonly used in animal models of PD. 3) to analyze enhancers of mitochondrial function, antioxidants, inhibitors of nitric oxide synthase, and inhibitors of poly- ADP-ribosylation for their ability to protect against intrinsic oxidative damage and cell death as well as that induced by MPP+ in these PD cybrids. Though several symptomatic therapies exist, no current treatments have been shown to be effective in slowing the progression of PD. The proposed studies will assess the role of oxidative stress due to impaired mitochondrial function, and provide an in vitro assay for testing novel therapeutic strategies that may ultimately slow the process off neuronal degeneration in PD.

DESCRIPTION (provided by applicant): Mitochondrial complex I (CI) activity appears to play a key role in the pathogenesis of Parkinson?s disease (PD). Cl activity is impaired in the substantia nigra (SN) in PD, and Cl inhibitors induce parkinsonism when systemically administered in animals. Indirect evidence suggests a role for mitochondrial DNA (mtDNA) mutations. However, detection of these mutations may not be possible by standard screening techniques, particularly if they are present at low mutational burdens. We hypothesize that numerous acquired mutations, each individually present at a low mutational burden, could reach a sufficient aggregate burden to cause mitochondrial dysfunction. Such mutations are hypothesized to result from oxidative damage to mtDNA. Brain levels of 8-hydroxy-2?deoxyguanosine (OH8dG), a marker of oxidative DNA damage associated with point mutations, are 16-fold higher in mtDNA than in nuclear DNA, increase with aging, and increase further in PD. We have developed a protocol for detecting mtDNA mutations present at extremely low mutational burdens. We propose to determine the frequency of oxidative stress-induced mtDNA mutations in frontal cortex and substantia nigra in controls and in PD. Using Laser Capture Microdissection (Arcturus), we will examine the specific subpopulation of neurons susceptible in PD. Single-cell PCR will allow us to address the question of accumulation of individual acquired mutations within single neurons. We also propose to establish an in vitro system for analyzing the induction of oxidative stress-induced mutations in dividing and in post-mitotic cells by exposure to hydrogen peroxide or to agents that inhibit mtDNA repair. Collaborations have been established which will make the proposed studies possible. These studies will lay the foundation for future studies addressing the role of acquired mtDNA mutations in aging and in neurodegenerative diseases.

DESCRIPTION (provided by applicant): No agent has been demonstrated unequivocally to have clinically significant neuroprotective efficacy in Parkinson's disease (PD) patients;that is to slow or stop the ongoing loss of dopaminergic neurons and synapses. On the other hand, numerous agents have shown clear neuroprotective efficacy in vitro and in various in vivo models of PD, providing hope that effective neuroprotection can be achieved in PID patients. Selection of agents that target mechanisms of demonstrated pathogenetic significance in PD patients will improve the likelihood that this success in vitro and in vivo will translate into similar success in PD patients. Evidence has accumulated that mitochondrial dysfunction and oxidative stress may play key roles in the pathogenesis of PD. Mitochondrial complex I activity is impaired in the substantia nigra in PD compared to age-matched controls. The ability of complex I inhibitors (MPTP and rotenone) to reproduce many features of PD when systemically administered in animals indicates that complex I dysfunction may play a causal role in PD. Levels of markers of oxidative damage to lipids, proteins, and DNA are elevated in the substantia nigra in PD (as well as in MPTP-treated animals). Thus, complex I dysfunction resulting in oxidative stress may play a key role in the pathogenesis of PD. The parallel work on the role of alpha synuclein in PD also now is revealing a strong connection to mitochondrial mechanisms. Inhibition of complex I or exposure to oxidative stress promotes alpha synuclein aggregation. Conversely, overexpression of mutant or wild-type alpha synuclein induces mitochondrial dysfunction and oxidative stress, and expression of mutant alpha synuclein enhances susceptibility to oxidative stress. In vitro and in vivo models of PD have demonstrated successful neuroprotection with strategies to enhance energy metabolism, block free radical damage, or enhance endogenous antioxidant mechanisms. The clinical trial now being planned provides a unique opportunity to determine if similar strategies can yield clinically meaningful neuroprotection in PD.

[unreadable] DESCRIPTION (provided by applicant): Recent data from studies of transgenic mice lend support to the mitochondrial theory of aging, which proposes that oxidative damage to mitochondrial DNA gives rise to mutations that accumulate with age, resulting in mitochondrial dysfunction and contributing to age-related disorders. In the human brain, we have found that oxidatively-induced somatic mitochondrial DNA (mtDNA) mutations accumulate with aging to reach high levels. Mitochondrial complex I dysfunction plays a key role in Parkinson's disease (PD), and indirect data implicates mtDNA mutations as the cause of this mitochondrial dysfunction. Yet, we and others have been unable to identify clearly pathogenic inherited mtDNA mutations in most PD patients. Together, these observations raise the possibility that the accumulation of somatic mtDNA mutations in the brain plays a key role in the pathogenesis of PD. However, little is known regarding the cell types in the brain that accumulate these mutations, or whether or not the accumulation of these mutations plays a role in mitochondrial dysfunction and neurodegeneration. We hypothesize that somatic mtDNA mutations accumulate with aging in single neurons and glia, that the levels of somatic mtDNA mutations in neurons, and possibly in astrocytes, are greater in PD compared to age-matched controls, and that these mutations contribute to mitochondrial complex I dysfunction. We will use laser capture microdissection (LCM) to isolate single neurons (with and without Lewy bodies), astrocytes, and microglia from the substantia nigra and control regions of young and old human control subjects, and from early and late stage PD patients. We have developed and validated a highly sensitive cloning-sequencing strategy that we will use to analyze these cells for levels and patterns of somatic mtDNA mutations, and will assess the relationship between these mutations and mitochondrial complex I dysfunction. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a common age-related neurodegenerative disorder that leads to progressive disability. Oxidative stress and mitochondrial dysfunction play key roles in the pathogenesis of PD. The proposed studies of somatic mtDNA mutations in single cells in the human brain will provide insights into the relationship between aging, oxidative injury to mitochondrial DNA and mitochondrial dysfunction, and might help to identify new treatment strategies to delay or prevent the disability of PD. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Mitochondrial complex I activity is impaired in Parkinson's disease (PD), and inhibition of complex I with MPTP or rotenone reproduces many features of PD in animal models. The complex I defect can be transferred to cell lines expressing mitochondrial DNA (mtDNA) from PD patients, suggesting that mtDNA mutations account for the complex I defect. But despite attempts to identify them, the specific mutations that account for this defect remain unknown. Mitochondrial complex I dysfunction increases free radical production in the mitochondria, resulting in damage to macromolecules, with particularly high levels of potentially mutagenic damage to mtDNA. This damage to mtDNA accumulates with age and reaches especially high levels in PD. We hypothesize that this oxidative damage to mtDNA leads to the accumulation of somatic mtDNA mutations, ultimately contributing to the loss of dopaminergic terminals and potentially to cell death. Therefore, we predict that substantia nigra (SN) neurons will harbor high levels of somatic mtDNA mutations at early stages of PD. Consistent with this prediction, we present preliminary data indicating remarkably high levels of somatic mtDNA point mutations in SN neurons at very early stages of PD, whereas neurons with high levels of mutations are largely absent by end stage PD. Furthermore, we find that levels of the subset of mtDNA mutations predicted to result from oxidative stress are nearly 10-fold more prevalent in SN neurons from early PD compared to controls or to late PD neurons. These data are consistent with our hypothesis that somatic mtDNA mutations accumulate in SN neurons at early stages of PD, and that these mutations contribute to neuronal loss in PD. We further predict that experimental acceleration of the age-related accumulation of somatic mtDNA mutations will lead to similar changes in transgenic mice expressing a proofreading deficient mtDNA polymerase (POLG). We propose to use laser capture microdissection to analyze point mutations and large deletions in neurons and glia from human postmortem SN neurons and other brain regions in early PD, late PD, and controls. We further propose to conduct parallel experiments in transgenic mice expressing mutant POLG. Together, these studies have the potential to reveal a key mechanism in the pathogenesis of PD, and may lead to novel neuroprotective strategies.PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a common disorder that leads to progressive disability. Though many symptomatic treatments exist for PD, each has limitations, and a strategy to slow the progression of PD could have an enormous positive impact on the quality of life of PD patients. The proposed experiments will test the hypothesis that the accumulation of somatic mitochondrial DNA mutations in the brain contributes to the pathogenesis of PD, and may lead to novel strategies to slow the progression of PD. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Recent data from studies of transgenic mice lend support to the mitochondrial theory of aging, which proposes that oxidative damage to mitochondrial DNA gives rise to mutations that accumulate with age, resulting in mitochondrial dysfunction and contributing to age-related disorders. In the human brain, we have found that oxidatively-induced somatic mitochondrial DNA (mtDNA) mutations accumulate with aging to reach high levels. Mitochondrial complex I dysfunction plays a key role in Parkinson's disease (PD), and indirect data implicates mtDNA mutations as the cause of this mitochondrial dysfunction. Yet, we and others have been unable to identify clearly pathogenic inherited mtDNA mutations in most PD patients. Together, these observations raise the possibility that the accumulation of somatic mtDNA mutations in the brain plays a key role in the pathogenesis of PD. However, little is known regarding the cell types in the brain that accumulate these mutations, or whether or not the accumulation of these mutations plays a role in mitochondrial dysfunction and neurodegeneration. We hypothesize that somatic mtDNA mutations accumulate with aging in single neurons and glia, that the levels of somatic mtDNA mutations in neurons, and possibly in astrocytes, are greater in PD compared to age-matched controls, and that these mutations contribute to mitochondrial complex I dysfunction. We will use laser capture microdissection (LCM) to isolate single neurons (with and without Lewy bodies), astrocytes, and microglia from the substantia nigra and control regions of young and old human control subjects, and from early and late stage PD patients. We have developed and validated a highly sensitive cloning-sequencing strategy that we will use to analyze these cells for levels and patterns of somatic mtDNA mutations, and will assess the relationship between these mutations and mitochondrial complex I dysfunction. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is a common age-related neurodegenerative disorder that leads to progressive disability. Oxidative stress and mitochondrial dysfunction play key roles in the pathogenesis of PD. The proposed studies of somatic mtDNA mutations in single cells in the human brain will provide insights into the relationship between aging, oxidative injury to mitochondrial DNA and mitochondrial dysfunction, and might help to identify new treatment strategies to delay or prevent the disability of PD. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A groundbreaking set of recent studies revealed that homozygous "mutator" mice expressing a proofreading deficient mitochondrial polymerase gamma (Polg ) accumulate both mtDNA point mutations and large deletions in association with a premature aging phenotype, demonstrating that somatic mitochondrial DNA (mtDNA) mutations can contribute to an aging phenotype. Large mtDNA deletions rather than point mutations have been proposed to be the driving force behind the aging phenotype, in part based on the lack of an overt phenotype in heterozygous mutator mice despite high levels of mtDNA point mutations and normal levels of deletions. However, we strongly argue in favor of the alternative hypothesis that mtDNA point mutations drive the aging phenotype in the mutator mice, an argument supported by our preliminary data, and propose to further test this hypothesis with particular attention to the neurological phenotype in the heterozygous mice. Several studies have demonstrated a normal age-related loss of dopaminergic substantia nigra (SN) neurons, as well as a marked enhancement with aging in the vulnerability of these neurons to mitochondrial toxins. However, studies of these or other age-related changes in the brain are lacking in the Polg mutator mice. Elucidating the cause of age-related vulnerabilities in the brain represents a critical step in advancing our understanding of normal aging in the brain and potentially of age-related neurodegenerative diseases such as Parkinson's disease (PD). We hypothesize that somatic mtDNA point mutations are a major cause of these age-related vulnerabilities of dopaminergic neurons, and therefore we predict that both heterozygous and homozygous Polg mutator mice will show increased spontaneous age related loss of dopaminergic neurons in addition to an enhanced susceptibility to toxin-induced degeneration of dopaminergic SN neurons compared to wild-type littermate controls. The proposed studies will directly test these predictions, and thus will help to clarify the important issue of the role of mtDNA point mutations in the aging brain. PUBLIC HEALTH RELEVANCE: set of cells in the brain that produce dopamine degenerate in Parkinson's disease. These same cells show a greatly enhanced vulnerability with advancing age to death from exposure to certain toxins, potentially accounting for the dramatic rise with age in the incidence of Parkinson's disease. We propose a series of studies to investigate the hypothesis that acquired mitochondrial DNA mutations account for the age-related vulnerability of these brain cells to degeneration.

DESCRIPTION (provided by applicant): Disorders caused by maternally inherited pathogenic mitochondrial DNA (mtDNA) mutations can lead to a wide array of neurological, cardiac, and other disorders. Unfortunately, clearly effective clinical treatments for these often devastating disorders are lacking. An ideal strategy would eliminate the mutant mtDNA and replace it with wild type (WT) DNA. However, classic "gene therapy" approaches are difficult to apply to mtDNA mutations. On the other hand, mitochondria undergo frequent turnover (every few days), even in postmitotic cells, with only a subset of copies of the mitochondrial genome being replicated during this process, providing an opportunity to influence which mtDNA molecules are replicated. We now propose to test a novel strategy to promote the selective elimination of deleterious mtDNA mutations that can be applied to heteroplasmic mtDNA mutations. Heteroplasmy is a common feature of pathogenic mtDNA mutations, and refers to a mix of WT and mutant mtDNA within the same cells or tissue. Our hypothesis takes advantage of a natural cellular process known as "mitophagy" (mitochondrial degradation by autophagy), which is a mechanism for selectively eliminating dysfunctional mitochondria. We hypothesize that some mitochondria within a cell will harbor greater levels of a heteroplasmic mtDNA mutation than others. Those with greater levels of a deleterious mutation will tend to have relatively greater impairment of mitochondrial function. Therefore, we propose to test the novel hypothesize that stimulating mitophagy by inhibiting mTOR kinase activity in cells harboring a heteroplasmic pathogenic mtDNA mutation will drive selection against the mutant mtDNA, over time leading to a substantial reduction in the mutational burden and hence an improvement in mitochondrial function. We have a unique resource available for testing this hypothesis: multiple SH-SY5Y cybrid cell lines harboring different levels of a heteroplasmic G11778A complex I (CI) gene mutation associated with Leber's Heredity Optic Neuropathy (LHON), all prepared at the same time from members of a single family. Our preliminary data with these cell lines support our hypothesis. A second important resource in our laboratory is the "mutator" mouse that expresses a proofreading deficient mtDNA polymerase 3 (Polg) leading to accumulation with age of heteroplasmic somatic mtDNA mutations in association with a premature aging phenotype. Our preliminary data demonstrate substantial metabolic, behavioral, and neurochemical deficits in these mice. We now hypothesize that enhancing mitophagy in the Polg mutator mice will attenuate the accumulation of somatic mtDNA mutations and ameliorate the deficits in these mice. Ultimately, clinical applications of this strategy have the potential to be of benefit to patients with classic mitochondrial disorders associated with heteroplasmic mtDNA mutations, to families harboring Polg mutations associated with familial parkinsonism and other disorders, and potentially for age-related neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Mitochondrial genetic disorders can be disabling but clearly effective treatments are lacking. Ideally, one would want to use a strategy that would eliminate the mutant mitochondrial DNA and replace it with normal DNA. We now propose to test a strategy of enhancing the natural process by which cells eliminate dysfunctional mitochondria, which we hypothesize will drive selection against mutant mitochondrial DNA in favor of normal DNA in cells harboring a mix of mutant and normal DNA.

DESCRIPTION (provided by applicant): Physical exercise is inversely related to the risk of Parkinson's disease (PD), and in rodents can protect against mitochondrial dysfunction and neuronal loss induced by MPTP. Recently, exercise also has been shown to have a dramatic protective effect in Polg mutator mice expressing a proofreading deficient form of the mitochondrial DNA (mtDNA) polymerase ¿ (Polg). In these mice, there is an accelerated accumulation of somatic mtDNA mutations, leading to a premature aging phenotype. Exercise in these mice normalizes muscle mitochondrial function and significantly extends lifespan. Perhaps more surprisingly, physical exercise also improves brain mitochondrial function and completely prevents brain atrophy. The mechanisms of these protective effects in the brain are unknown. In skeletal muscle, exercise increases levels of mRNA of PGC-1¿, a transcriptional coactivator that upregulates mitochondrial biogenesis and antioxidant defenses. Exercise also may increase PGC-1¿ activity through posttranslational mechanisms. In muscle, exercise reduces levels of RIP140, a suppressor of PGC-1¿ activity, and induces SIRT1 dependent deacetylation of PGC-1¿, thereby promoting its activation. We hypothesize that the protective effects of exercise on brain mitochondrial function exercise may result from similar mechanisms that account for this effect in muscle. If correct, then these mechanisms may account for the association of exercise with a reduced risk of PD. This potential link between exercise and increased PGC-1¿ activity is particularly exciting in light of recent data implicating reduced brai PGC-1¿ activity in the pathogenesis of PD. Thus, increasing PGC-1¿ in brain is a promising potential neuroprotective strategy. The Polg mutator mice represent a valuable model for studying the protective effects of exercise on the brain. In addition to brain atrophy and impaired mitochondrial function, we have preliminary data indicating that the Polg mutator mice have significant behavioral (motor) deficits as well as loss of striatal tyrosine hydroxylase (TH) immunostaining intensity and reduced dopamine (DA) and dopamine metabolites, indicating that mitochondrial dysfunction caused by somatic mtDNA mutation accumulation can cause nigral-striatal pathology. These data raise the possibility that the high levels of somatic mtDNA mutations that we and others have identified in SN neurons in PD may contribute to nigral-striatal dysfunction in PD. Thus, if our hypothesis proves to be correct, then the proposed studies on the impact of exercise on somatic mtDNA mutations and PGC-1¿ activity in the brain may be of relevance to PD. The main goal of this project is to investigate potential mechanisms of the protective effect of physical exercise in the brain of Polg mutator mice, including the impact on somatic mtDNA mutation levels and on regulation of PGC-1¿ levels and activity in the brain. This targeted approach will be complemented by an unbiased metabolomics approach that may reveal a role for novel pathways linking exercise to protective effects in the brain. PUBLIC HEALTH RELEVANCE: Physical exercise improves mitochondrial function and has robust protective effects in the brain, but the mechanisms of these effects are unknown. Understanding these mechanisms provide insights into novel neuroprotective strategies of relevance to aging and age-related neurodegenerative diseases such as Parkinson's disease. We now propose to investigate the mechanisms of protection in the brain by exercise through studies of a mouse model of premature aging.

DESCRIPTION (provided by applicant): A major goal of the neuroscience community is to develop treatment strategies that will slow or forestall the progression of chronic neurodegenerative diseases. Parkinson's disease (PD) is one of the most common adult neurodegenerative disorders, affecting over 1 million people in North America and the European Union, As a first step in identifying such therapies, the NINDS Exploratory Trials in Parkinson's disease (NET-PD) network successfully completed futility studies, which identified creatine as a potential agent to slow clinical decline in PD. The NET-PD network is now conducting a large, long-term, Phase 3 trial (known as LSI) comparing creatine to placebo. An additional futility study is underway (the FS-ZONE study) to examine the potential for pioglitazone as a disease modifying therapy in PD.

DESCRIPTION (provided by applicant): Mitochondrial dysfunction and oxidative stress play important roles in Parkinson's disease (PD). PGC-1alpha, a transcriptional coactivator, upregulates mitochondrial biogenesis and antioxidant defenses, and thus is an attractive target for neuroprotection in PD. Susceptibility to MPTP, a mitochondrial toxin, is increased in mice lacking PGC-1alpha, whereas overexpressing PGC-1alpha protects against an oxidative challenge in cell lines. Levels of expression of genes regulated by PGC-1alpha are low in substantia nigra (SN) neurons in early PD. Two important genetic causes of PD now have been linked to low PGC-1alpha. First, a recent study showed that loss of Parkin function leads to increased levels of novel protein called PARIS which transcriptionally inhibits expression of PGC-1alpha. And recently it was demonstrated that alpha-synuclein binds to the PGC-1alphha promoter and also suppresses its transcription. Together, these data strongly implicate a pathogenic role for low PGC-1alpha activity in PD, and raise the hope that correction of the PGC-1alpha deficit in PD will be neuroprotective. However, unexpectedly, we and others find that overexpressing PGC-1alpha at very high levels leads to reduced Bdnf and suppression of the dopaminergic phenotype. Our preliminary data suggest that this may result from suppression of Pitx3, a transcription factor that is critical for maintaining the dopaminergic phenotype and also for expression of Bdnf. Vulnerability to MPTP is increased by the very high levels of PGC-1alpha achieved using our AAV-PGC-1alpha vector. Thus, either low or very high levels of PGC-1alpha can be deleterious. Together, these data reveal that maintenance of PGC-1alpha activity levels within a therapeutic range is critical for the survival and function of dopaminergic neurons. We hypothesize that very high levels of PGC-1alpha lead to suppression of Pitx3, leading to loss of the dopaminergic phenotype and to enhanced vulnerability to MPTP due to loss of Bdnf. We further hypothesize that it will be possible to harness the neuroprotective potential of viral vector- mediated increases in PGC-1alpha activity while avoiding the potentially deleterious effects associated with very high levels of overexpression. We propose to test this by studying the impact in dopaminergic neurons on mitochondrial function, oxidative stress, the dopaminergic phenotype, and susceptibility to MPTP following more modest levels of upregulation of PGC-1alpha, or following co-expression of Pitx3 to prevent the deleterious effects of higher PGC-1alpha levels. The potential neuroprotective effects of Pitx3 on its own also will be studied. These experiments will test our hypothesis that suppression of Pitx3 mediates the PGC-1alpha-induced downregulation of Bdnf and of the dopaminergic phenotype. In addition, multiple gene therapy trials have been conducted in PD patients, and thus the proposed studies also will serve as initial tests of therapeutic strategies with translational potential.

Acquired (somatic) mitochondrial DNA (mtDNA) mutations accumulate with age and reach high levels in dopaminergic neurons at early pathological stages in Parkinson's disease (PD). POLG ?mutator? mice have an accelerated accumulation of somatic mtDNA mutations and develop a premature aging phenotype at levels of mutations comparable to levels found in neurons in PD patients, demonstrating that somatic mtDNA mutations can be functionally significant. Furthermore, some patients with POLG mutations develop parkinsonism associated with ?-synuclein (?Syn) pathology and loss of dopaminergic neurons in the substantia nigra (SN). Based on these and other data, we hypothesize a ?two-hit? hypothesis whereby somatic mtDNA mutations contribute to mitochondrial dysfunction, and thereby exacerbate vulnerability to ?Syn. This may help to explain the dramatic rise with age in the incidence of PD. The overall goal of this proposal is to assess the relationship between somatic mtDNA mutations and vulnerability to ?Syn toxicity. We will do this using 2 strategies. First, we we will assess the impact of increased levels of somatic mtDNA mutations on ?Syn toxicity by assessing heterozygous and homozygous POLG mutator mice that overexpress double-mutant (A30P and A53T) ?Syn in dopaminergic neurons. Double-mutant ?Syn mice (dMut?Syn) develop a slowly progressive phenotype including striatal dopamine deficiency with behavioral deficits and loss of dopaminergic SN neurons. We predict earlier and more severe behavioral deficits, mitochondrial dysfunction, impaired mitophagy, more severe loss of striatal dopamine and dopaminergic terminals, and increased SN dopaminergic neuronal loss compared to ?Syn overexpression in the SN of WT mice. Second, we will perform stereotaxic SN injections of AAV-?Syn (wild-type or A53T) or a control vector (GFP-degron) in wild-type and heterozygous and homozygous POLG mutator mice. We again predict that somatic mtDNA mutations will exacerbate the phenotype associated with increased ?Syn expression, including the progressive death of neurons normally seen following AAV-?Syn injections, resulting in earlier and more severe deficits. These studies may yield the first experimental evidence that somatic mtDNA mutations, at levels relevant to early stages in PD, influence vulnerability to ?Syn toxicity, a result that may in part explain the dramatic rise in the incidence of PD with age. These studies also will have characterized a novel mouse model of dopaminergic neuronal degeneration that combines two mechanisms of pathophysiological relevance to PD.

A large body of evidence implicates dysfunction of mitochondrial homeostasis as a key pathophysiological mechanism in Parkinson?s disease (PD). Maintenance of a pool of healthy functioning mitochondria requires a system for selectively degrading dysfunctional mitochondria (?mitophagy?). Autosomal recessive (AR) PD due to Parkin deficiency links directly to a defect in mitophagy. Mitochondrial dysfunction causes Parkin to translocate to the outer mitochondrial membrane where it interacts with PINK1 (another gene where mutations cause AR PD) to ubiquitinate mitochondrial proteins, thereby inducing fusion of mitochondria with autophagosomes, followed by autophagic degradation. Thus, loss of Parkin leads to the accumulation of dysfunctional mitochondria due to impaired mitophagy. We hypothesize that defective mitophagy also may exacerbate ?-synuclein (?Syn) toxicity. ?Syn induces mitochondrial complex I dysfunction, potentially by directly binding to TOM20 on the mitochondrial membrane and thereby interfering with mitochondrial protein import. Conversely, dysfunctional mitochondria produce increased reactive oxygen species (ROS), consistent with increased markers of oxidative damage in the PD brain. Furthermore, ROS can increase ?Syn accumulation. However, the role of mitophagy in clearing away dysfunctional mitochondria in the setting of ?Syn induced mitochondrial impairment is unknown. Most strategies to modulate mitophagy also alter autophagy in general, or impact other mechanisms, making it difficult to specifically study mitophagy. A target that allows a specific molecular manipulation of mitophagy is USP30. USP30 is a deubiquitinating enzyme (DUB) tethered to the outer mitochondrial membrane, where it directly removes ubiquitin that had been attached by Parkin, thereby counteracting Parkin?s ability to promote mitophagy. USP30 itself is a Parkin substrate, as Parkin normally ubiquitinates and inactivates USP30. Knock-down of USP30 by siRNA rescues mitophagy in Parkin-deficient cells and protects DA neurons in Parkin-deficient Drosophila and protects in an acute toxin model (paraquat). Thus, inhibition of USP30 is an attractive therapeutic strategy for restoring mitophagy to achieve neuroprotection in PD. However, thus far there are no data evaluating the impact of USP30 inhibition on mitophagy in vivo in mammals. Furthermore, the impact of USP30 inhibition has not been tested in a pathophysiologically relevant model of PD. We will address these two issues as outlined in this proposal by testing our main HYPOTHESIS that inhibiting USP30 can serve as an effective strategy to protect dopaminergic neurons against ?Syn toxicity in a progressive degenerative mammalian model of PD.

Mitochondrial dysfunction and oxidative stress are hypothesized to play key roles in Parkinson's disease (PD)), motivating the development of therapies aimed at improving mitochondrial energy metabolism and blocking oxidative stress. Peroxisome-proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1?) fills this niche as a master regulator of mitochondrial biogenesis and antioxidant defenses. In support of a critical role of PGC-1?, dopaminergic (DA) neurons of knockout mice show increased vulnerability to degeneration in response to (MPTP). Pgc-1? and its targeted genes are underexpressed in DA neurons at early pathological stages in PD patients, suggesting a failure of this protective response. Two genetic causes of PD: increased ?- synuclein or loss of Parkin (PRKN) each has been shown to suppress PGC-1? expression. Furthermore, loss of PGC-1? increases vulnerability to ?-synuclein (?Syn) toxicity. Thus,increasing PGC-1? could be neuroprotective. Yet, we and others have found that viral-mediated Pgc-1? overexpression at high levels in the nigrostriatal system causes DA neurons to degenerate and increases their susceptibility to MPTP toxicity. Thus, either too much or too little PGC-1? is detrimental to DA neurons. A method to precisely control PGC-1? expression is essential before this can become a viable therapeutic strategy. We now hypothesize a novel post-translational regulatory mechanism to control PGC-1? protein levels, which arises from two observations. First was our unexpected observation that increasing CMA increases PGC-1? protein levels. Next was the recognition that FBXW7, an E3 ubiquitin ligase that tags PGC-1? for degradation by the ubiquitin proteasome system (UPS), harbors the perfect CMA consensus sequence, ?KFERQ?. These findings led us to hypothesize that CMA degrades FBXW7 and thereby reduces UPS-mediated degradation of PGC-1? protein: ?CMA ? ? FBXW7 ? ? PGC-1? . By contrast, inhibiting CMA should increase FBXW7, which would then ubiquitinate PGC-1? leading to its degradation by the UPS. Interestingly, FBXW7 has been identified as a target of PRKN, with PRKN loss leading to increased FBXW7 levels in Prkn-null mice. Furthermore, FBXW7 levels are increased in the brain of PD patients with PRKN mutations. Based on these and other preliminary data, we hypothesize that PGC-1? is regulated through a novel mechanism involving FBXW7 as a link between CMA and UPS. We have obtained additional preliminary data in support of this hypothesis, and will further test it by studying in vitro the mechanism by which manipulations of CMA alter PGC-1? levels, by testing our hypothesis that FBXW7 is a direct substrate for CMA, and by testing our hypothesis that decreasing FBXW7 levels specifically within dopaminergic neurons will be neuroprotective in vivo against ?Syn toxicity. These studies could significantly advance our understanding of mechanisms regulating PGC-1? activity, validate FBXW7 as a therapeutic target in PD, and demonstrate a novel mechanism for regulation of the UPS through CMA-mediated degradation of an E3 ubiquitin ligase.