Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmaceutical Sciences

First Advisor

Aloke K. Dutta

Second Advisor

David M. Thomas

Abstract

PROGRESS TOWARDS UNDERSTANDING OF MECHANISMS OF ACTION OF POTENT MULTIFUNCTIONAL DISEASE MODIFYING

THERAPEUTICS FOR PARKINSON'S DISEASE.

by

MRUDANG MANOJKUMAR SHAH

December 2013

Advisor: Dr. Aloke Dutta

Major: Pharmaceutical Sciences

Degree: Doctor of Philosophy

Our long term goal is to design and develop potent multifunctional disease modifying therapeutics for Parkinson's disease. The objective of my dissertation was to understand the mechanisms of action of some potent small molecules (synthesized in our lab) as a disease modifying Parkinson's disease therapeutic. The objective was achieved by pursuing the following two specific aims:

1. Investigating anti-oxidant and neuroprotective effects of a lead molecule (D-512) generated in our lab.

2. Assessing the ability of some of our potential lead compounds (D-240, D-436, and D-520) to prevent the aggregation of α-synuclein.

Oxidative stress is one of the major factors implicated in the pathogenesis and progression of Parkinson's disease. To investigate the neuroprotective effects of D-512 in Parkinson's disease, we determined the ability of D-512 to rescue against 6-hydroxydopamine induced cell death in dopaminergic cell line PC12 under two different protocols. Once it was established that D-512 is able to prevent/rescue the cell death induced by 6-hydroxydopamine in PC12 cells, various other assays to determine its ability to confer neuroprotection were performed.

As Parkinson's disease patients have elevated levels of oxidative stress and subsequent damage of their glutathione system, excessive lipid peroxidation, and DNA damage, we evaluated D-512's ability to protect against 6-hydroxydopamine induced changes in glutathione levels and DNA damage. We also evaluated the ability of D-512 to protect against the lipid peroxidation induced by sodium nitroprusside. In addition, we also evaluated the possible molecular mechanism by which D-512 may be able to confer neuroprotection against 6-hydroxydopamine induced cell death.

α-synuclein aggregation has been shown to be present in post-mortem brains of Parkinson's disease patients and SNCA gene mutations as well as gene duplication and triplication results in familial form of Parkinson's disease. To assess the ability of some of our lead compounds against α-synuclein aggregation, we developed a cell-free system assay. We simulated different Parkinson's disease associated factors to generate different types of α-synuclein aggregates. We also developed various assays to determine the type of aggregates formed following our experiments. After optimizing assays to generate α-synuclein oligomers and fibrils, we evaluated the ability of some standard compounds (ascorbic acid and rifampicin) and some of our lead compounds (D-436 and D-520) to prevent α-synuclein aggregation in presence of dopamine. We employed numerous assays to verify the morphology and structural characteristics of the aggregates formed under various conditions.

α-synuclein aggregates have been known to transfer from one neuron to another by their ability to form pores in biological membranes. Therefore, we developed an in-vitro assay to determine the extracellular toxicity of α-synuclein aggregates generated under high concentration of α-synuclein and α-synuclein in presence of dopamine by cell-free system assay. Afterwards, we evaluated the ability of a standard compound (rifampicin) and our lead compounds (D-240, D-436, and D-520) to prevent α-synuclein aggregation and subsequent cytotoxicity of extracellular α-synuclein aggregates. We also evaluated the ability of our compounds to prevent α-synuclein aggregation and the morphological changes induced in presence of our lead compounds.

INVESTIGATING THE METHAMPHETAMINE-INDUCED

STRIATAL MICROGLIA ACTIVATION

by

MRUDANG MANOJKUMAR SHAH

December 2013

Advisor: Dr. David Thomas

Major: Pharmaceutical Sciences

Degree: Doctor of Philosophy

Methamphetamine is a highly abused, addictive and neurotoxic drug which causes striatal-specific activation of microglia cells. Our long term goal is to identify the potential molecular targets of methamphetamine induced neurotoxicity and addiction. The objective of my dissertation was to develop a cell-culture model of methamphetamine neurotoxicity and determine proteomic expression changes in microglia isolated from striatum of methamphetamine treated animals. I achieved my objective by pursuing following two specific aims:

1. Identifying the proteomic expression changes of cultured microglia cells after exposure to methamphetamine or similar insult.

2. Optimization of a protocol to isolate highly purified fraction of microglia from various regions of brain and identification of protein expression changes in striatal microglia isolated from control and methamphetamine treated animals.

The addictive property of methamphetamine is because of its ability to cause release of dopamine from striatum, which cause euphoria. Dopamine quinones have been shown to cause significant changes in gene expression profile of culture microglia cells (BV2 cells). We determined to evaluate the effect of different concentrations of methamphetamine and lipopolysaccharide (gram negative endotoxin which causes strong immune response) on cultured microglia cells alone and cultured microglia cells which have access to various soluble factors secreted by cultured dopaminergic cell line (MN9D cells). We also verified the differences in protein expression profile of BV2 cells alone or BV2 cells co-cultured with dopaminergic neuronal cell line to determine if either of the cell-culture systems can be used as a model to determine methamphetamine-induced neurotoxicity in striatal microglia.

As striatum is a region where all dopaminergic nerve terminals of nigrostriatal tract end and methamphetamine has been shown to cause significant alterations in dopaminergic neuronal system, we determined to isolate microglia from striatum. We compared and optimized various protocols for microglial isolation to achieve method to isolate highly pure fraction of microglia cells. Afterwards, we also isolated microglia from striatum of untreated animals and animals treated with methamphetamine to determine protein expression changes between them. We analyzed the differences in protein expression between isolated microglia to understand the molecular mechanisms or pathways which are altered or affected by methamphetamine which might be responsible for addictive and neurotoxic properties of methamphetamine.

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