Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 167-184). Also available via the Internet.

Parkinson's disease (PD) is a progressive neurodegenerative disorder with evolving layers of complexity. In the present thesis we replicated the PD neurodegenerative pattern using a mouse model characterized by systemic injections of the neurotoxin 1-methyl-4-phenyl1,2,3,6-tethrahydropyridine (MPTP). Firstly, we investigated the role of a pro-inflammatory mediator called high mobility group box 1 (HMGB1), in-vivo and in post-mortem human tissue of PD patients. Our study shows increased protein levels of HMGB1 in substantia nigra of PD patients and MPTP treated mice. Inhibition of HMGB1, using the antagonist glycyrrhizin, and a HMGB1 neutralising antibody, has shown neuroprotection against MPTP neurotoxicity as well as prevented nuclear translocation of the protein in dopaminergic neurons. Secondly, we profiled the expression of two HMGB1 cognate receptors: TLR2 and TLR4. Levels of the receptors were upregulated in mouse ventral midbrain following the acute and sub-acute MPTP regimen. However, TLR4 knock out mice were not protected against MPTP-induced dopaminergic toxicity. Thirdly, we developed a novel method of brain tissue dissociation and isolation of immune cells viable for downstream flow cytometric analyses. The technique allowed us to investigate the effects of low grade chronic systemic inflammation within the CNS of mice. The method has proven to be sensitive enough for the qualitative and quantitative measurement of immune cells isolated from brain parenchyma. Lastly, an extensive behavioural characterization on the three different MPTP mouse models was performed in order to identify translational behavioural paradigms in relation to the nigrostriatal lesion induced by the neurotoxin MPTP. Major gait and balance impairments were identified in acute and chronic MPTP mouse models. The findings on HMGB1, TLR2 and TLR4 proteins are the overarching element between the identification of the behavioural endpoints in the MPTP models and the newly developed method for the isolation of immune cells from the mouse brain parenchyma.

Thesis (Ph.D.)--Texas Christian University, 2007. / Title from dissertation title page (viewed Apr. 29, 2008). Includes abstract. Includes bibliographical references.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references (leaves 111-122). Also available via the Internet.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 209-226). Also available via the Internet.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 58-64). Also available via the Internet.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 98-115).

1-Methyl-4-(1-methylpyrrol-2-yl)-1 ,2,3,6-tetrahydropyridine (TMMP) is a neurotoxic analog of the parkinsonian inducing agent MPTP. TMMP and its putative metabolites 1-methyl-4-(1-methylpyrrol-2-yl)-2,3-dihydropyridinium (MMDP+) and 1-methyl-4-(1-methylpyrrol-2-yl)pyridinium (MMP+) were synthesized and fully characterized. Substrate/inactivation properties of TMMP and its analog N-propargyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine with MAO-B were investigated. Kinetic data was obtained, including Km and Vmax for TMMP as an MAO-B substrate, and KI and kinact values for N-propargyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine. The metabolic studies of TMMP and MMDP+ were conducted with an HPLC diode array assay. Both in-vivo and in-vitro metabolic studies showed that TMMP is oxidized to its dihydropyridinium species (MMDP+) in a reaction catalyzed by MAO-B. MMDP+ undergoes autoxidation to form the pyridinium species (MMP+), the mechanism of this conversion is not clear. In-vitro studies show that MAO-B is not responsible for this conversion and the oxidation of MMDP+ to MMP+ is likely to be enzyme catalyzed. Toxicity investigations include dopamine depletion studies of TMMP and MMDP+, mitochondrial respiration and microdialysis studies of MMDP+ and MMP+. The above studies show that TMMP is an MPTP-type neurotoxin. / Master of Science

The cyclic allylamine I-methyl-4-phenyl-l ,2,3,6-tetrahydropyridine (MPTP) is a potent and specific neurotoxin that causes a parkinsonian like syndrome in humans and subhuman primates. Research has revealed that MPTP is bioactivated in a reaction catalyzed by flavin containing monoamine oxidase B (MAO B) to yield the dihydropyridinium species MPDP+ which undergoes further oxidation to the ultimate toxin, the I-methyl-4-phenylpyridinium species MPP+. The research summarized in this thesis describes a potential model reaction for the MAO catalyzed conversion of MPTP to MPDP+ and the synthesis and biological evaluation of MPTP analogs bearing a heteroatom at C-4 of the tetrahydropyridine ring. The model for the enzyme catalyzed oxidation of MPTP to MPDP+ is based on the anhydride mediated conversion of MPTP Noxide to MPDP+. This reaction pathway was visualized to mimic a reaction sequence in which an FAD-MPTP adduct cleaves to yield MPDP+ and FADH2. Attempts were made to assess the isotope effect associated with this reaction and to compare that value with the corresponding values for the MAO-B catalyzed reaction [D(V max/Km) = 7-9], the cytochrome P-450 catalyzed reaction [D(V max/Km) = 1.04] and the electrochemical oxidation (D k = 1.35). Unfortunately experimental difficulties prevented a complete analysis of the problem. Specialized equipment will be required to obtain accurate isotope effect measurements. The second study concerns the preparation of the MPTP analogs 4-chloro-, 4-cyano-, and 4-( 4-fluorophenoxy)-1,2,3,6-tetrahydropyridine as potential MAO B substrates that could generate neurotoxic pyridinium metabolites. Results obtained with MAO B have revealed that the dihydropyridinium intermediate formed from 4-( 4-fluorophenoxy)-1 ,2,3, 6-tetrahydropyridine undergoes spontaneous hydrolysis to generate 4-fluorophenol and 1- methyl-4-pyridone. The significance of this finding with respect to neurotoxic mechanisms and design are discussed. / Master of Science

Self-administration of 1-methyl-4-pheny]-1,2,3,6-tetrahydropyridine (MPTP) has resulted in irreversible symptoms of Parkinson's disease in several young drug abusers. It was found that this neurotoxicant selectively destroys neuronal cells in the substantia nigra of humans and other primates. Although the mechanism of action of MPTP is not fully understood, it is now generally believed that the crucial species for MPTP neurotoxicity is not MPTP itself, but rather some of its metabolites. MPDP⁺, an intermediate in the metabolism of the neurotoxin MPTP, was found to generate superoxide radical (⋅O₂⁻) during its autoxidation process. The generation of ⋅O₂⁻ was detected by their ability to reduce ferricytochrome c. Superoxide dismutase (SOD) inhibited this reduction in a dosedependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP⁺, but also on the pH of the system. Thus, the rate of autoxidation of MPDP⁺ and the sensitivity of this autoxidation to superoxide dismutase inhibitable ferricytochrome c reduction were both augmented as the pH was raised from 7.0 to 10.5. The rate constant (k_c) for the reaction of superoxide radical with ferricytochrome c to form ferrocytochrome c was found to be 3.48 x 10⁵ M⁻¹S⁻¹. The rate constant (k_MPDP⁺) for the reaction of MPDP⁺ with ferricytochrome c was found to be 4.86 M⁻¹S⁻¹. The generation of ⋅O₂⁻ was further confirmed by spin-trapping in combination with EPR techniques using 5, 5-dimethyl-1-pyrrolonine-N-oxide (DMPO) as the spin trapping agent. The rate of formation of spin adduct (DMPO-O₂⁻) was dependent not only on the concentrations of MPDP⁺ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP⁺, and of SOD to effectively compete with this reaction for the available ⋅O₂⁻, was used as a convenient competition reaction to quantitatively determine various kinetic parameters. Using this technique, the rate constant for scavenging of superoxide radicals by superoxide dismutase was found to be 7.56 x 10⁹ M⁻¹S⁻¹. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP⁺ was found to be 4.48 x 10⁻¹⁰ M⋅S⁻¹. The rate constant (k₁) for MPDP⁺ making superoxide radical was found to be 3.97 x 10⁻⁶ Sec⁻¹. The second order rate constant (k_DMPO) for DMPO trapping superoxide radicals was found to be 10.2 M⁻¹S⁻¹. The life time of superoxide radical at pH 10.0 was calculated to be 1.25 seconds. These data indicate that superoxide radicals are produced during spontaneous oxidation of MPDP⁺ and that EPR spin trapping techniques can be used to determine the rate constants and life time of free radicals generated in aqueous solution. Monoamine oxidase type B (MAO-B), an enzyme present in mitochondrial membranes, is known to metabolize MPTP to MPDP⁺, which then spontaneously oxidizes to MPP⁺. In the studies of MAO-B catalyzed oxidation of MPTP, the neurotoxicant was found to generate reactive oxygen species during its interaction with the enzyme. The kinetic parameters, K_m and V_max, for MAO-B catalyzed oxidation of MPTP to the corresponding species MPDP⁺ were found to be 0.194 mM and 0.335 µM/min, respectively. The generation of ⋅O₂⁻ and hydroxyl (⋅OH) radicals was detected as the DMPO spin adduct by spin trapping in combination with EPR techniques. Addition of Fe²⁺ (10 µM) to this system caused a 5-fold enhancement in EPR signal intensity of the DMPO-OH adduct. Catalase, a scavenger of hydrogen peroxide (H₂O₂), inhibited the DMPO-OH spin adduct formation in a dose-dependent manner, indicating that H₂O₂ is produced in the MAO-B catalyzed oxidation of MPTP. Ethanol, a well known scavenger of hydroxy] radical, rapidly produced an alpha-hydroxyethyl radical signal. SOD inhibited the formation of DMPO-O₂⁻ and DMPO-OH spin adducts in a dose-dependent fashion. These data suggest that ⋅O₂⁻ are produced during the oxidation of MPTP by MAO-B and that the generation of H₂O₂ and ⋅OH was secondary to the production of ⋅O₂⁻. MPTP and its metabolites, MPDP⁺ and MPP⁺, were found to inhibit the activity of acetylcholinesterase (AChE). The kinetic parameter, K_m for the substrate (acetylthiocholine), was found to be 0.216 mM and K_i values for MPTP, MPDP⁺ and MPP⁺ to inactivate AChE were found to be 2.14, 0.265 and 0.197 mM, respectively. The inactivation of AChE by these neurotoxicants was found to be dose-dependent. It was found that MPTP, MPDP⁺ and MPP⁺ are neither substrates of AChE nor the time-dependent inactivators. The studies of reaction kinetics indicate that the inactivation of ACHE by these inactivators is via a mixed-type inhibition. The dilution of the enzyme-inhibitor complex completely reversed the MPTP inhibition but only partially reversed the MPDP+ and MPP+ inhibition. These data indicate that MPTP and its metabolites can inactivate AChE and thereby increase ACh level in the basal ganglia of the brain, leading to potential cell dysfunction. These results suggest that once MPTP enters the basal ganglia of the brain, it can be catalyzed by MAO-B to generate a series of reactive species, including ⋅O₂⁻, H₂O₂ and ⋅OH, which are known to destroy cell membranes, enzymes and other important biological molecules. The nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may largely be due to the reactivity of these reactive oxygen species in combination with the inactivation of the AChE enzyme in the brain, leading to potential cell dysfunction. / Ph. D.