Parkinson's disease (PD) is a slow-progressive neurodegenerative disorder affecting 5-6 million people around the globe. The disease is manifested by the rapid deterioration of dopaminergic cells in the substantia nigra portion of the brain; however, the pathological mechanism of selective dopaminergic neuronal death is unknown. A reduction in levels of 3,4-dihydroxyphenylacetaldehyde (DOPAL) is biologically critical as this aldehyde has been shown to be toxic to dopaminergic cells and is a highly reactive electrophile. Investigating neuronal protein targets is essential in determining the cause of toxicity. An essential protein-GAPDH (e.g., glyceraldehyde-3-phosphate dehydrogenase) is an abundantly expressed enzyme known for its glycolytic activity, and recent research has implicated its role in oxidative stress-mediated neuronal death. This work positively shows GAPDH as a target for DOPAL modification, and, for the first time, DOPAL is identified as a potent inhibitor for GAPDH enzymatic activity. LC-MS and other chemical probes (ie. thiol and amine modifiers) show that DOPAL modifies specific –Lys, -Arg, and –Cys residues in the cofactor binding-domain of GAPDH. The enzyme inhibition is also time and DOPAL dose-dependent. DOPAL has a unique structure, containing two reactive functional groups: an aldehyde and catechol ring. In-house syntheses of DOPAL analogues, containing the catechol group and lacking the aldehyde, and vice versa have been tested on GAPDH and do not inhibit or modify GAPDH. Therefore, both the catechol and aldehyde groups of DOPAL are specific to binding with GAPDH and are necessary to achieve modification and enzyme inhibition. In addition to finding a novel enzyme inhibited and modified by DOPAL, this work has also confirmed linking DOPAL levels to a fungicide associated with PD risk. This benzimidazole fungicide, benomyl was shown to inhibit ALDH2 in the SH-SY5Y neuroblastoma cell line via an increase in DOPAL and a decrease in DOPAC. The ratios of DOPAL and DOPAC, the product of ALDH, were measured by HPLC-ECD, and found that benomyl does inhibit ALDH2 in this dopaminergic cell model. The cytotoxicity of benomyl, DA, DOPAL and the combination of DA or DOPAL with benomyl was assessed by MTT assay. Surprisingly, the only toxic combination was the combination of DA or DOPAL with benomyl. In fact, this toxicity appears to be synergistic, as none of the single treatments are significantly toxic to the cells. This synergistic effect also affects GAPDH aggregation. The cell morphology is also drastically different in the presence of the combined treatments, compared to individual treatment of DA, DOPAL or benomyl; cells start to ebb and show apoptotic-like features at just 2h. A second class of pesticides, named chlorpyrifos and chlorpyrifos-oxon were tested for toxicity in PC6-3These compounds were toxic to these cells due to DOPAL accumulation reaching high levels in the 100 µM range. Exposure to environmental toxins such as pesticides and fungicides has long been linked to PD risk, but only recently to DOPAL levels. This work provides a novel mechanism by which fungicide exposure may stimulate PD pathogenesis.

Abstract not available

<p>Organophosphorus pesticides (OPs) are neuroactive compounds that can affect the development of the nervous system and behavior. Using zebrafish, we compared the effectiveness of three different OPs, chlorpyrifos (CPF), diazinon (DZN) and parathion (PA) on survival, AChE inhibition and larval motility. We conclude that at equimolar concentrations CPF is the most effective OP of the three tested. 300nM CPF inhibited AChE when measured at 5 days post fertilization (dpf) by 80%, which correlated to a 35% decrease in larval motility when measured at 6 dpf. </p><p>We isolated the cDNA of <italic>zfkcc2</italic>, which encodes for an important cation-chloride cotransporter responsible for establishing a chloride gradient that allows for the neurotransmitter GABA to become inhibitory for a subset of neurons. By isolating the cDNA of <italic>zfkcc2</italic>, we were able to characterize its expression pattern up to 4 dpf. Using qRT-PCR, we showed that the expression of <italic>zfkcc2</italic> rises from 2 to 96 hours post fertilization (hpf). The expression of <italic>zfkcc2</italic> is found at 1 dpf in the forebrain, midbrain and hindbrain in a distinct pattern. By 3 dpf, zfkcc2 expression is found throughout most of the developing zebrafish brain. We were then able to characterize the expression pattern of zfnkcc1, which encodes for a cation-chloride cotransporter that works opposite to <italic>zfkcc2</italic> and establishes a chloride gradient allowing GABA to be excitatory during development. Expression of <italic>zfnkcc1</italic> is found at 1 dpf throughout most of the developing zebrafish head including in some non-neural tissue, but appears concentrated to the otic vesicle at 3 dpf. Using double in situ hybridization, we investigated the nature of the developmental switch from zfnkcc1 to zfkcc2 by looking at regions where there was an interaction between both genes' expression patterns in the developing zebrafish nervous system. </p><p>We preliminarily examined how affecting nicotinic signaling with exposures to nicotinic agonists/antagonists can affect the expression of <italic>zfnkcc2</italic> and <italic>zfkcc2</italic>. While there were no apparent changes in the expression pattern from any of the exposures at 2 dpf, we found that by 3 dpf nicotine and CPF exposures resulted in an incomplete expression pattern for <italic>zfkcc2</italic> that seemed to be delayed. However, the expression of <italic>zfnkcc1</italic> at the otic vesicle was unaffected by exposure. </p><p>Our results suggest that disrupting nicotinic signaling during development can affect the expression of an important cation-chloride cotransporter, <italic>zfkcc2</italic>, that plays a major role in establishing the necessary chloride gradient for the proper functioning of GABA during development. Future studies should focus on investigating the nature of the developmental switch between <italic>zfnkcc1</italic> and <italic>zfkcc2</italic> to see if the possibility of their regulation being coupled is a target for exposures.</p> / Dissertation

Predatory shallow-water tropical marine snails within the genus Conus are estimated to consist of up to 700 species. These carnivorous mollusks have devised efficient venom harpoon-like radular teeth that allow them to predominantly incapacitate polychaete annelids (vermivores), in some cases fish (piscivores), or other mollusks (molluscivores) as an envenomation survival strategy for feeding, defense, and competitor deterrence. The venom of each Conus species contains a distinctive assortment of over 50 diversified disulfide-rich conotoxins with varied pharmacological specificities that selectively inhibit the function of ion channels (Ca2+, Na+, K+) or nicotinic acetylcholine receptors (nAChRs) involved in the animal neurotransmission. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing an exquisite selectivity to distinguish between channels / receptors and even particular their subtypes. Novel conotoxins detected in the molecular neurobiological approach, providing chemists and pharmacologists a vast library (>50,000 individual toxins) of conotoxins have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. In this article, we present the natural history of the Conus biology as well as the nomenclature, classification, structure, neurotoxicological mechanisms, post-translational modification, and pharmaceutical applications of conotoxins. In addition, we also set up the bioinformatic database and search engine about hitherto-identified name and distribution of Conus species and neuropharmacological mechanism, accession number, sequence, and 3D structure of conotoxins and provide researchers advantageous tools for further investigation.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

The growth of a neuronal dendritic tree depends on the neuron’s internal state and the environment within which it is situated. Different types of neuron develop dendritic trees with specific characteristics, such as the average number of terminal branches and the average length of terminal and intermediate segments. A key aspect of the growth process is the construction of the microtubule cytoskeleton within the dendritic tree. Neurite elongation requires assembly of microtubules from free tubulin at the growth cone. The stability of microtubule bundles is an important factor in determining how likely it is for a growth cone to split to form new daughter branches. Microtubule assembly rates and bundle stability are controlled by microtubule-associated proteins, principally MAP2 in dendrites. Extending previous work (Hely et al, J. Theor. Biol. 210:375-384, 2001) I have developed a mathematical model of neurite outgrowth in which elongation and branching rates are determined by the phosphorylation state of MAP2 at the tips of each terminal branch. Tubulin and MAP2 are produced in the cell body and transported along the neurite by a combination of diffusion and active transport. Microtubule (dis)assembly at neurite tips is a function of tubulin concentration. The rate of assembly depends on the amount of unphosphorylated MAP2 bound to the microtubules and linking them together. Phosphorylation of MAP2 destroys its linking capability and destabilises the microtubule bundles. Each terminal has a probability of branching that depends on the phosphorylation of MAP2 which, in turn, is a function of calcium concentration. Results from this model show that changes in the (de)phosphorylation rates of MAP2 affect the topology of the final dendritic tree. Higher phosphorylation promotes branching and results in trees with many short terminal branches and relatively long intermediate segments. Reducing phosphorylation promotes elongation and inhibits branching.

Exposure to methyl mercury (MeHg) is a global concern. Increased chronic exposure to MeHg among fish and marine mammal consuming populations will increase the risk of prenatal exposure and as a result, the risk of infant brain damage and neurotoxcity. It is therefore important to understand the role of environmental factors, such as nutrition, in determining susceptibility to MeHg toxicity. Three nutrients (selenium (Se), vitamin C and vitamin E) were selected for examination of their interactions with the mechanisms of McHg cytotoxicity in vitro. Two hybrid neural cell lines (M03.13 and NSC-34) were evaluated for their usefulness in the study of MeHg cytotoxicity. Sixteen toxic endpoints were selected for investigation of growth, viability, structure and biochemistry. Both cell lines responded to MeHg exposure in a dose dependent manner for the majority of endpoints suggesting that both MO3.13 and NSC-34 cells undergo structural and biochemical changes during exposure to McHg, but that MO3.13 cells are more sensitive to DNA, mitochondria) membrane damage and glutathione (GSH) depletion and that NSC-34 cells are more sensitive to protein damage and apoptosis. Se exposure lessened the MeHg-induced decrease in DNA and GSH concentrations in both cell lines. In NSC-34 cells, Se also increased F-actin concentrations and prevented an increase in caspase-3 activity. Se may alter the mechanism of cell death by preventing McHg disruption of DNA replication thus maintaining the production and function of peptides (GSH) and protein (polymerized actin) that aid in MeHg detoxification and neural function. In NSC-34 cells, vitamin C prevented the induction of caspase-3 activity and lessened DNA damage and GSH depletion. Vitamin E lessened GSH depletion and lessened G-actin depletion. Both vitamin C and E improved GSH status, but vitamin C also delayed McHg damage of DNA and prevented early signs of apoptosis suggesting these two vitamins interfere with MeHg metabolism by diffe

Methylmercury (MeHg) is an environmental contaminant mainly present in fish and seafood. The long-term consumption of these fish and seafoods could pose a health risk to pregnant women and their children. Animal studies were conducted to assess the effects of MeHg exposure on reproduction and offspring development as well as the potential benefits of nutrient supplementation. Adult female rats were treated by gavage with MeHg at dose levels of 0.5, 1.0 or 2.0 mg/kg/day for 4 weeks prior to mating and throughout pregnancy, and then were allowed to deliver. In a second study, adult female rats were treated with MeHg at 1.25 mg/kg/day for the same duration, and they were fed diets containing an extra 1 ppm selenium (Se), or 225 IU/kg vitamin E, or both of these two nutrients, 4 weeks prior to MeHg dosing, and then throughout McHg treatment. (Abstract shortened by UMI.)

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 203-226) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 149-179). Also available in print.