Jason R. Cannon, PhD - US grants

DESCRIPTION (provided by applicant) This proposal is for a pathway to independence award. The candidate will learn new techniques and create a divergent research focus from the mentor laboratory. The candidate's primary goal is to become an independent investigator and make major scientific contributions to the neurodegenerative disease research field. A detailed Career Development Plan that includes coursework, learning new techniques, scientific meeting attendance, and specific feedback from an advisory committee has been constructed to help the candidate achieve this goal. The research focus of this proposal is on gene-environment interactions in Parkinson's disease (PD). New approaches to modeling such interactions are a major focus of this proposal. The causes of most PD cases are unknown, about 10% are inherited. The causes of the remaining "sporadic", about 90% are unknown. Epidemiological evidence has repeatedly suggested that environmental exposures increase the risk for PD. However, no single toxicant has been identified as a causative agent. Most cases may arise from both environmental and genetic factors. However, such interactions are poorly understood. Research to date on gene-environment interactions has typically utilized toxicant models that have little relevance to human health. Indeed, current PD models have major etiological limitations. Toxicant models typically use large acute doses by unrepresentative routes of exposure and genetic models often use complete life-span knockout of a gene or massive transgene expression. The candidate hypothesizes that: "early-stage" environmental PD modeling is best suited to study gene-environment interactions. He proposes to overcome current barriers by: Aim 1) Creating both "early" and "late" stage PD models using relevant environmental toxicants. Here, toxicants recently linked to PD will be used to create new rodent models that reproduce the key features of both early and late-stage PD and utilize environmentally relevant toxicants. This aim will serve as a "screen" to identify the optimal toxicant model to advance to later aims. The toxicant producing the best model will move forward to later aims. Aim 2) "Real-world" exposure modeling: This aim will utilize toxicant exposure through dosing regimens that bear relevance to human health. Aim 3) Creating new gene-environment interaction models: Here, transgenic rats expressing mutations known to cause PD in humans will be exposed to the optimal toxicant from aim 1. Thus a new gene-environment rodent PD model will be created that utilizes an environmental toxicant linked to PD and expresses a mutation known to cause human PD. Brain toxicant levels will be determined and correlated with pathological observations (Aims 1-3). Aim 4) Testing gene therapy approaches in these models: In vivo modulation of PD genes will be tested as a potential therapeutic approach in newly created toxicant models. This project is expected to produce major advances in gene-environment interaction modeling. Newly created models will be used to identify pathogenic pathways and test new therapeutic approaches. Public Health Relevance: Parkinson's disease (PD) affects roughly 1 million Americans and the causes are largely unknown, although much data suggests that environmental exposures play a role. The proposed work will focus on the development of new models using compounds that have been linked to PD. These newly developed models will be used to test interactions between environmental and genetic factors and also to test potential treatments.

DESCRIPTION (provided by applicant): The causes of most Parkinson's disease (PD) cases are unknown (~90% are sporadic), while ~10 % are due to purely inherited factors. Environmental factors have long been suspected, but no toxicant has been convincingly identified. Numerous diverse classes of compounds, including pesticides and solvents have been linked to PD. This proposal tests the hypothesis that: exposure to the heterocyclic amine, 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP), a known carcinogen in rodents, replicates key features of human Parkinson's disease (PD). Currently, there are little preliminary data on the neurological effects of chronic PhIP. However, four key factors have led to proposing a role for PhIP in PD-relevant neurodegeneration. 1) A preliminary study from members of our group showing PD-relevant motor deficits in mice 2) Published dopaminergic effects of structurally related heterocylic amines in rats 3) Potential for high-level human consumption. PhIP is the most abundant amino-imidazoazaarene isolated from the crust of cooked meat (up to 15ug/kg uncooked meat) and 4) PhIP and PhIP metabolites cross the blood-brain barrier and, therefore, may have direct effects on distinct neuronal nuclei. The major goals of this proposal are to: 1) Determine if chronic administration of PhIP in the rat reproduces the key features of PD and 2) Identify potential mechanisms of toxicity using in vitro approaches. In this proposal, the following aims will be carried out: Aim 1. To determine if chronic PhIP administration replicates the key features of PD. Aim 2. To identify mechanisms of neurotoxicity of PhIP using a primary midbrain culture system. Success of this proposal would lead to at least three major advances in PD research. 1. Identification of a possible causative factor. PhIP is a common toxicant produced in meat preparation and may be consumed in single high doses and chronically vs. most rarely encountered dopaminergic toxicants. Success of this proposal would likely prompt epidemiological studies. Further, alterations in meat preparation are available, reducing PhIP formation and a potential PD-relevant exposure. 2. Development of a new PD model. There are numerous PD models, each with advantages/ disadvantages. None have adequately considered dietary factors. Current models have also failed to predict clinical trial successes and a novel model may prove to be more successful. 3. New PD mechanisms. If PhIP exposure reproduces the key PD features, mechanistic studies would be expected to identify novel pathogenic pathways that may be therapeutic targets. In summary, using both in vivo and in vitro systems we will carefully characterize neurodegeneration after PhIP exposure and preliminarily identify mechanisms of toxicity relevant to effects on the nigrostriatal dopamine system.

? DESCRIPTION (provided by applicant): The causes of most Parkinson's disease (PD) cases are unknown, ~90% are 'sporadic', ~10% are attributed to inheritance. Environmental factors, including pesticides and solvents, have long been suspected, but no toxicant has been convincingly identified. Most cases are thought to arise from gene -environment interactions. A specific example is leucine-rich repeated kinase 2 (LRRK2), a large multi-domain protein with an unknown endogenous function. Numerous LRRK2 mutations cause PD. However, incomplete penetrance in humans and heightened sensitivity to dopaminergic (DA) neuron toxicants in animals expressing mutations suggest the importance of gene-environment interactions. Mounting data from the Cannon laboratory and others suggests that heterocyclic aromatic amines (HAAs) are neurotoxic and associated with neurodegenerative diseases, including PD. HAAs have been primarily investigated as carcinogens in laboratory animals and as potential human carcinogens. HAAs are formed during high temperature meat and poultry cooking. Thus, chronic HAA exposure through diet may be much more common and occur at higher levels than for many environmental toxicants. This proposal tests the hypothesis that: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most mass abundant HAA in cooked meats and poultry, exhibits selective dopaminergic neurotoxicity by a newly proposed mechanism of neurotoxic action through N-hydroxylation, a metabolite in common with PhIP's mediated genotoxicity. Our data suggests that mechanistic studies on PhIP neurotoxicity are critical to understanding a potential role in PD. The major goals of this proposal are to: 1) Characterize PhIP-mediated neurotoxicity in vivo; 2) Determine if PhIP exposure potentiates pathology in animals expressing mutated (G2019S) LRRK2 (the most common genetic cause of PD); 3) Identify key mechanism(s) of PhIP-mediated DA-selective neurotoxicity through examination of whether N-hydroxylation is a key pathogenic event, and by assessing the propensity of N-oxidized PhIP metabolites to broadly form adducts with major biomolecules. Modifications and adduct formation in specific PD-implicated proteins will also be examined. Success of this project will lead to several major advances. 1) Identification of a possible causative factor: PhIP is a common toxicant produced in cooked meats and may be consumed in higher doses than rarely encountered known DA toxicants; 2) Creation of a new gene-environment interaction model utilizing the most common PD causing-mutation and a compound to which humans are widely exposed; 3) New PD mechanisms: mechanistic studies would likely identify novel pathogenic pathways that may be therapeutic targets; 4) Prompt follow-up epidemiological and biomarker studies. In summary, using both in vivo and in vitro systems, we will carefully characterize PhIP-induced neurodegeneration and identify mechanisms of DA neurotoxicity. If PhIP exposure is proven to have a causative role in PD, then recommendations can be provided to the public because PhIP exposure can be mitigated by changes in cooking methods.

Parkinson's disease (PD) is a debilitating movement disorder (affecting ~5 million world?wide) resulting from selective death of dopamine (DA) neurons. To date, numerous rarely encountered exposures have been investigated as risk factors, but none have been clearly linked to PD. Further, the translation of therapeutics that are promising in animal studies to successful clinical trials has been very poor. These gaps in the field suggest serious weaknesses in the utilization of animal models in PD research. Most PD studies test hypotheses in single model systems. However, there are clear advantages with respect to increasing the strength of the findings and advancing the field through understanding species differences. This R21 aims to be highly responsive to PAR? 17?039 (Comparative Biology of Neurodegeneration) by testing PD?relevant neurodegeneration across three phylogenetically diverse animal model systems. In the spirit of an R21, the proposal utilizes high risk/high reward approaches, where novel risk factors will be tested to advance the understanding of the biology of PD. Per? and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants that have been investigated as developmental toxicants, with little information on long?term neurotoxicity. Our preliminary mechanistic and neuropathology data in nematode and amphibian models suggest that exposure to PFAS, especially perfluorooctane sulfonate (PFOS) induces selective PD?relevant, DAergic neurotoxicity. This project will address an important gap on how PFAS exposure leads to long?term neurological disease risk. We will test the hypothesis: that species?specific responses to PFOS?induced dopaminergic neurodegeneration will advance understanding of the biology of PD. Importantly, the hypothesis will be tested across 3 animal model systems, where concordance will strengthen findings, and discordance will identify biological aspects of species?specific sensitivity to environmentally?induced neurodegeneration. We will test our hypothesis through two aims: Aim 1. To identify species specific?PFOS doses that induce DAergic neurodegeneration. PFOS doses will be harmonized across systems to achieve brain levels that bear environmental relevance. Harmonization of internal dose levels to set external applied dosages for each model system will allow us to interrogate mechanistic hypothesis under comparable insults; Aim 2. Identify neurobiological underpinnings across species that contribute to differential sensitivity to PFOS?induced dopaminergic neurodegeneration. Here, we will identify species?specific differences in neurodegeneration that may underlie critical aspects of selective dopaminergic neurotoxicity induced by PFOS exposure. We will conduct comparative biology studies that are both phenotypic and mechanistic. Resultant data will be critical in determining: 1) Which species is best suited to PFOS neurodegeneration studies; 2) Identifying which pathogenic pathways directly correlate with neurodegeneration across species. These studies will mechanistically advance the field far beyond data from typical single?species studies.