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Investigation of Pathology and Potential Drugs for the Neurodegenerative Diseases

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Tutor: ZhouFeiZuo
School: Central South University
Course: Chemical Engineering and Technology
Keywords: Alzheimerí»s disease (AD),Parkinsoní»s disease (PD),copper ion,dopamine,inhibitor,
CLC: R741
Type: PhD thesis
Year:  2013
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Abstract:
Neurodegenerative disease is one of the most common diseases in the elderly. Alzheimerí»s disease (AD) and Parkinsoní»s disease (PD) are the common forms of dementia. Most neurodegenerative diseases termed as the progressive loss of structures and functions of neurons, which eventually results in death of neurons. The metal-induced oxidative stress and the aggregation of the amyloidogenic proteins (e.g., Ž┬-amyloid in AD, alpha-synuclein in PD) have been implicated in the pathogenesis of neurodegenerative diseases. The mitochondria and neuronal membrane damage can be caused by the metal-induced oxidative stress, and the aggregates of the amyloidogenic proteins can induce the neuron death. Consequently, the mechanics of the metal-induced oxidative stress and the compounds which inhibit the aggregation process of the amyloidogenic proteins have been the areas under active pursuit. Based on these considerations, we have carried out the following studies:(1). Copper coexists with amyloid beta peptides (A(3) at a high concentration in the senile plaques of AD patients and has been linked to oxidative damage associated with AD pathology. However, the origin of copper and the driving force behind its accumulation are unknown. We designed a sensitive fluorescent probe, AŽ┬(1-16)(Y10W), by substituting the tyrosine residue at position10in the hydrophilic domain of AŽ┬(1-42) with tryptophan. Upon mixing Cu2+, AŽ┬(1-16)(Y10W), and aliquots of AŽ┬(1-42) taken from samples incubated for different lengths of time, we found that the Cu2+binding strength of aggregated AŽ┬(1-42) has been elevated by more than2orders of magnitude with respect to that of monomeric AŽ┬(1-42). Electron paramagnetic spectroscopic measurements revealed that the AŽ┬(1-42) aggregates, unlike their monomeric form, can seize copper from human serum albumin, an abundant copper-containing protein in brain and cerebrospinal fluid. The significantly elevated binding strength of the AŽ┬(1-42) aggregates can be rationalized by a Cu2+coordination sphere constituted by three histidines from two adjacent AŽ┬(1-42) molecules. Our work demonstrates that the copper binding affinity of AŽ┬(1-42) is dependent on its aggregation state and provides new insight into how and why senile plaques accumulate copper in vivo.(2). An RP-IPC-ESI-MS method was estabilished by coupling the reversed-phase ion-pairing chromatography (RP-IPC) on-line with the electrospray ionizationmass spectrometry (ESI-MS). Regeneration of the anion exchange column can be accomplished on-line by switching a four-way switch valve to interconnect the column to a regeneration solution. The IPC-ESI-MS method enabled us to separate and on-line detect four intermediates of the Fe3+-catalyzed dopamine oxidation. Among thses intermediates,6-hydroxydopamine, which is short-lived and highly neurotoxic, was detected and quantified. Together with the separation of other intermediates, gaining insight into the mechanism and kinetics of the Fe3+-catalyzed dopamine oxidation becomes possible.(3). The aggregation of amyloidogenic proteins/peptides has been closely linked to the neuropathology of several important neurological disorders. In AD, AŽ┬ peptides and their aggregation are believed to be at least partially responsible for the etiology of AD. The aggregate-inflicted cellular toxicity can be inhibited by short peptides whose sequences are homologous to segments of the AŽ┬(1-42) peptide responsible for Ž┬-sheet stacking (referred to as the Ž┬-sheet breaker peptides). Here, a water-soluble ferrocene (Fc)-tagged Ž┬-sheet breaker peptide, Fc-KLVFFK6, was used as an electrochemical probe for kinetic studies of the inhibition of the AŽ┬(1-42) fibrillation process and for determination of the optimal concentration of Ž┬-sheet breaker peptide for efficient inhibition. Our results demonstrate that Fc-KLVFFK6interacts with the AŽ┬ aggregates instantaneously in solution, and a substoichiometric amount of Fc-KLVFFK6is sufficient to inhibit the formation of the AŽ┬ oligomers and fibrils and to reduce the toxicity of AŽ┬(1-42). The interaction between Fc-KLVFFK6and AŽ┬(1-42) follows a pseudo-first-order reaction, with a rate constant of1.89í└0.05í┴10-4s-1. (4). The AŽ┬ aggregates are believed to be responsible for the neuropathology of AD. In this work, Fc is attached to the aggregating core of the AŽ┬ peptides, KLVFFAE. Inhibition of Fc-KLVFFAE aggregation by curcumin, a natural compound, was monitored by HPLC-electrochemical detection (HPLC-EC). The Fc oxidation current is dependent on the incubation condition and curcumin can retain Fc-KLVFFAE in its monomeric form. It is demonstrated that tagging Fc to KLVFFAE affords a cost-effective and electroactive mimicry of AŽ┬(1-42) and HPLC-EC is suitable for sensitive, reproducible, and facile screening of drugs for inhibiting the aggregation of AŽ┬ peptides.(5). The AŽ┬ peptide and its aggregates are believed to play an important role in the neuropathology of AD. The various AP aggregates, especially the P-sheet-containing oligomers and the fibrils, cause cell death. Small molecular compounds can inhibit the AŽ┬ aggregation process and reduce the AP aggregates-induced cytotoxicity. In this study, tabersonine, an indole alkaloid, was used to inhibit the aggregation process of AŽ┬(1-42). The results demonstrate that a sub-stoichiometric amount (12.5%) of tabersonine can effectively inhibit the formation of the AŽ┬ fibrils. Tabersonine can also disaggregate the mature AŽ┬ fibrils, reducing the AŽ┬(1-42) aggregates-induced cytotoxicity.
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