Document Type

Dissertation

Date of Degree

2012

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacy

First Advisor

Jonathan A. Doorn

Abstract

Parkinson's disease (PD) is a prevalent neurodegenerative disorder for which the greatest risk factor is age. Four to five percent of 85-year-olds suffer from this debilitating disease, which is characterized by the selective loss of dopaminergic neurons within the substantia nigra and the presence of protein aggregates known as Lewy bodies. While the etiology of this disease is still unknown, recent research implicates oxidative stress, activated microglia, and reactive dopamine (DA) metabolites to play a role in the initiation or progression of the disease. Activated microglia cause injury to dopaminergic neurons via a host of mechanisms, including reactive oxygen species production, release of cytokines, and phagocytic activity. Microglial activation has been detected in the brains of PD patients, but the source of this activation has not been elucidated. Previous research has shown electrophiles and endogenous neurotoxins to play a role in this microglial activation. The interaction between the neurotoxic metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and microglia has not been explored.

DOPAL is a highly reactive, bifunctional electrophile produced by oxidative deamination of DA by monoamine oxidase (MAO). DOPAL is oxidized in the major metabolism pathway to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH). DOPAL has previously been shown to be 100-fold more toxic than DA in vitro and in vivo. Potent inhibition of the rate-limiting enzyme in DA biosynthesis, tyrosine hydroxylase, by DOPAL has been well-established. DOPAL-mediated aggregation of Α-synuclein, the primary component of PD-hallmark Lewy bodies, has been suggested but was further explored in this work.

Results presented in this body of work include further determination of the aggregation of Α-synuclein by DOPAL, including evidence of covalent modification. The interaction of DOPAL with BV-2 microglia, an immortalized cell line, was addressed in depth through exploration of DOPAL catabolism, toxicity, and generation of an activational response. Metabolism of DOPAL to DOPAC was altered in activated microglia, with the production of DOPAC reduced by ~40%. Metabolism of DOPAL to DOPAC was also inhibited by both 4-hydroxynonenal and malondialdehyde, gold standards of lipid peroxidation. Both of these compounds were found to be significantly toxic to BV-2 cells at concentrations well below those considered toxic to dopaminergic cells. Alternatively, DOPAL and DA were found to be non-toxic to this cell line, while DOPAL was shown to be significantly toxic to dopaminergic cells at concentrations as low as 10 ΜM.

Significant activation of BV-2 microglia by DOPAL was observed at 10 ΜM and above by release of TNF-Α. Morphological changes, release of IL-6, and changes in expression of COX-2 also indicated activation by DOPAL but not DA or DOPAC. BV-2-conditioned media, generated by incubation with DA, DOPAL, or DOPAC, was added to MN9D cells, and toxicity was measured by the MTT assay. BV-2 conditioned media generated by DOPAL incubation produced the greatest toxicity for MN9D cells. These results implicate DOPAL in dopaminergic cell death through microglial activation.

Keywords

3,4-dihydroxyphenylacetaldehyde, alpha-synuclein, microglia, oxidative stress, Parkinson's disease

Pages

xvii, 111 pages

Bibliography

Includes bibliographical references (pages 101-111).

Copyright

Copyright 2012 Laurie Leigh Eckert

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