Phip-Induced Dopaminergic Neurotoxicity: In vitro and in vivo Mechanisms of Action
Parkinson’s disease (PD) is the second most common neurodegenerative disease with ~1% prevalence in the population over 60 years of age. PD patients manifest cardinal motor symptoms including bradykinesia, postural instability, resting tremor and rigidity, while non-motor symptoms arise decades earlier, and can be also debilitating. The neuropathology of the disease is characterized by loss of dopaminergic neurons in substantia nigra and subsequent depletion of dopamine in striatum in the midbrain. The hallmark molecular mechanisms underlying PD are aggregation of the protein alpha synuclein, oxidative stress, mitochondrial inhibition, disruption of dopamine homeostasis and neuroinflammation. Currently, there is no cure for this devastating disease whereas the treatments available only relieve the symptoms temporarily. ~10% of PD patients have been found to carry one or more mutations in the PD-relevant genes such as alpha synuclein and LRRK2. However, the rest is considered as sporadic comprising approximately nine million individuals worldwide. The role of environmental factors has been repeatedly emphasized in sporadic PD cases. Some of the PD-associated neurotoxins include pesticides, heavy metals, and accidental byproducts of illicit opioid drug synthesis. Although association of these compounds with the increased risk of PD has been confirmed in epidemiological studies, as well as cellular and animal models, the exposures only occur in minority of population such as in occupational settings. Thus, the search for neurotoxins that are exposed frequently in higher doses is still ongoing. Dietary factors represent such a group that receives far less attention compared to some of the PD-associated environmental toxins. Micro- and macronutrients (i.e. vitamins, polyphenols, fatty acids), and dietary habits (high calorie intake, Mediterranean diet) have been investigated as causative or protective factors of PD. However, the byproducts xx of cooking processes have not received much attention so far compared to raw dietary factors regarding neurodegeneration. Heterocyclic amines (HCAs) are formed during the cooking of meat at high temperatures, and were identified as potent mutagens three decades ago. Since then, they have been shown to induce tumor formation in several different types of tissues. HCAs have not been considered as dopaminergic neurotoxicants. However, structural similarities HCAs and PD-associated dopaminergic neurotoxins share, and limited number of studies showed their adverse effects in dopamine metabolism suggest their possible roles as contributors to dopaminergic neurotoxicity and PD-relevant mechanisms. Thus, we hypothesized that 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most HCAs found in cooked meat, induces selective PD-relevant dopaminergic neurotoxicity. In this dissertation, three specific aims are represented to test the role of PhIP as a dopaminergic neurotoxicant. First aim was to test PhIP-induced dopaminergic neurotoxicity in vitro using primary midbrain cultures. Second aim involved detailed and more in depth examination of in vitro mechanisms of PhIP-induced toxicity. The final goal in this thesis was to test whether PhIP exposure elicits motor deficits and neuropathology relevant to PD in vivo. Our studies showed that PhIP and its select phase I metabolite N-OH-PhIP cause selective dopaminergic neuronal loss, and neurite shortening in surviving dopaminergic neurons in primary midbrain cultures, while they did not affect other neuronal populations. Cell loss observed was found to be mediated by oxidative stress, and was alleviated in the presence of anti-oxidants such as blueberry extracts. In depth examination of mechanism of PhIP-induced dopaminergic neurotoxicity in vitro revealed that it inhibits proteasome degradation pathway, possibly leading to protein aggregation. Increase in free cytosolic dopamine levels was shown to enhance PhIP toxicity, suggesting dopamine metabolism as a contributor of the toxicity observed. Finally, PhIP induced DNA damage in astrocytes while sparing the neuronal populations. In vivo testing of PhIP neurotoxicity showed that acute PhIP exposure in wild type rats caused oxidative damage in dopaminergic neurons of substantia nigra while other dopaminergic xxi populations and neuronal cells nearby are less sensitive. Acute PhIP exposure also revealed decreases in striatal dopamine metabolites and turnover rate, suggesting possible inhibition of dopamine metabolism in the midbrain. These results suggest that acute PhIP exposure induces early mechanisms of PD without an overt lesion in the midbrain. Later, our studies testing subacute PhIP suggested systemic toxicity observed by decreased weight gain, and also subtle decreases in striatal dopamine turnover and nigral serotonin turnover in the absence of dopaminergic system lesion or motor deficits. Finally, we investigated gene-environment interaction using a transgenic line of rats that lacks an efflux transporter that results in increased accumulation of PhIP in brain. However, these animals did not show any exacerbated motor deficits or neuropathology compared to wild type rats. Overall, the findings presented in this dissertation, suggest the role of PhIP as a dopaminergic neurotoxicant in vitro and its contribution to early PD-relevant mechanisms including oxidative stress and disruption of dopamine metabolism in vivo. We believe the data generated in this thesis will pave the way for understanding how food consumed everyday might lead early neurodegeneration. Further studies are needed to test the chronic exposure in rodent models and to investigate gene-environment interactions considering differential metabolism of PhIP in rodents and humans.
Cannon, Purdue University.
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