Date of Award

5-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Neuroscience

Research Advisor

Mark S. LeDoux, M.D., Ph.D.

Committee

Joseph C. Callaway, Ph.D. Angela R. Cantrell, Ph.D. Duane D. Miller, Ph.D. Lawrence T. Reiter, Ph.D.

Keywords

HPLC-EC, LC-MS/MS, Neurodegeneration, Neurotoxins, Parkinson Disease, Tetrahydroisoquinolines

Abstract

The goal of this dissertation work was to (1) determine the distribution of several tetrahydroisoquinoline (TIQ) derivatives in rodent, normal human and Parkinson disease (PD) brain, (2) quantify the levels of these TIQ derivatives in common food sources in an effort to link specific food intake patterns with the development of PD and (3) examine the neurotoxicity of select TIQ derivatives in human dopaminergic cell culture. The TIQs are a family of monoamine alkaloids that share structural homology with 1-methyl-4-phenyl-1,2,3,6-tetrahyrdropyridine (MPTP), can be formed from dopamine or its oxidized metabolites and may be involved in the pathogenesis of monoaminergic cell death.

In our studies, we utilize enantiomeric-selective high-performance liquid chromatography with electrochemical detection (HPLC-EC) and liquid chromatography with tandem mass spectroscopy (LC-MS/MS) to determine the concentrations of TIQ derivatives in brain, as well as in common dietary plants in the United States. The dopaminergic SH-SY5Y human neuroblastoma cell line was utilized to document the neurotoxicity of N-methylated salsolinol derivatives.

Several TIQ derivatives were detected in all regions subjected to analysis. In general, salsolinols were present at higher concentrations than TIQ and its benzyl and methyl derivatives, especially in human brain. Moreover, salsolinols were concentrated in areas with increased dopamine synthesis and turnover such as the ventral midbrain and striatum, respectively. Significantly lower levels of (R)salsolinol, (S)salsolinol, N-methyl-(R)salsolinol andN-methyl-(S)salsolinol were found in the caudate nuclei of PD in comparison with normal human brain. N-methyl-norsalsolinol was detected in all regions of rodent and human brain subjected to analysis. In comparison to normal human controls, N-methyl-norsalsolinol levels were significantly lower in the substantia nigra and caudate nuclei from PD patients. N-methyl-norsalsolinol-immunoreactivity co-localized with a general neuronal marker and a monoaminergic cell marker. N-methyl-norsalsolinol-immunoreactivity was not observed in glia.

Our analysis of common foods revealed high concentrations of TIQ derivatives in banana, cherry, peach, grapefruit, avocado, button mushroom, leaf lettuce and celery. Enantiomeric salsolinol derivatives were detected as racemic mixtures, without evidence of stereoselective synthesis. Utilizing a lifetime food-item intake questionnaire, we identified trends for increased intake of several plants products in PD patients. Members of the rue (Rutaceae), banana (Musaceae), gourd (Cucurbitaceae), carrot (Apiaceae) and nightshade (Solanaceae) families were found to be highly consumed in both normal human and PD groups, and to concentrate several potentially neurotoxic TIQ derivatives.

We correlated indices of cellular energy production and cell viability in dopaminergic SH-SY5Y cells after exposure to N-methyl-norsalsolinol and N-methyl-(R/S)salsolinol. Both toxins induce dose-dependent decreases in cell survival with LC50 values of 0.305 mM and 0.377 mM after 24 hrs, respectively. These results suggest that cell death induced by these N-methylated salsolinols is due to impairment of cellular energy supply, caused in particular by inhibition of mitochondrial complex I. Their concentration in dopaminergic regions of human brain and relatively potent cytotoxicity among TIQs makes these compounds strong candidates for both an endogenous and exogenous link to cell death in PD.

DOI

10.21007/etd.cghs.2010.0067

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