This dissertation, "A Study on Mitochondrial Uncoupling Protein 4 (UCP4) in Parkinsonian Models" by Chi-yuen, Andrew, Chu, 朱志遠, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled A study on mitochondrial uncoupling protein 4 (UCP4) in parkinsonian models submitted by Andrew, Chi-Yuen CHU for the degree of Doctor of Philosophy at The University of Hong Kong in August 2007 Parkinson's disease (PD) is characterized by nigrostriatal neuronal cell death. The etiology of the disease is unclear but recent research focuses on the following mechanisms, mitochondrial dysfunction, oxidative stress and excitotoxicity. In this study, the primary aim is to demonstrate that UCP4 is an uncoupling protein by showing that it posses the ability to protect cells from toxic insults. It does so by reducing oxidative stress, mitochondrial membrane potential (MMP), and consequently reduce ATP synthesis. To achieve the aims of this study, UCP4 protein was stably overexpressed in a human neuronal cell line, SH-SY5Y. UCP4 (36 kDa) was confirmed to be overexpressed predominantly in the mitochondria. Two specific antibodies against the N-terminus or C-terminus of UCP4 were raised. They were shown to cross-react specifically with overexpressed UCP4 protein. The identity of this protein was confirmed by Matrix Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry/ Peptide Mass Fingerprint (MALDI-TOF/PMF). iiiThe tissue distribution of UCP4 in rat brain and human post mortem brain slides was studied. In rat, UCP4 was widely expressed in the cortex, striatum and substantia nigra. In these regions, uniform expression was observed, staining was relatively homogenous. On the other hand, UCP4 was predominantly located in the Purkinje cells in cerebellum and selectively in pyramidal cells in the hippocampus. In human post mortem brain slides, positive staining was seen only in the cerebellum. Overexpression of UCP4 in SH-SY5Y cells conveyed a significant survival advantage via reduced oxidative stress, which is in accord with the hypothesis of partial uncoupling, when cells were exposed to either MPP or dopamine. However, contrary to the concept of simple uncoupling, MMP was not lower in the cells overexpressing UCP4 when compared with vector transfected control cells, after MPP exposure. A similar result was seen when cellular ATP content was assayed. Overexpression of UCP4 in SH-SY5Y cells resulted in an elevated ATP level compared with that in vector control cells. A similar result was seen when both types of cells were treated with 2 mM ADP, (a substrate for complex V) driving ATP synthesis. No mitochondrial proliferation observed. The expression of other isoforms of uncoupling proteins, (UCP2, UCP5) did not change in cells overexpressing UCP4 when challenged with MPP, in contrast to the increase seen in vector control cells. At the same time, the proliferation of cells overexpressed UCP4 was higher than that in vector control cells, both in non-differentiated and differentiated states. Therefore, the elevated ATP level in cells overexpressing UCP4 was neither due to the proliferation of mitochondria, nor due to the effects of other UCPs, (namely UCP2 and UCP5) but due to the overexpression of UCP4 itself. iv In conclusion, overexpression of UCP4 attenuated the toxicity induced by MPP and dopamine, which indicated a neuroprotective role of UCP4. The mechanisms of the neuroprotective effects of UCP4 observed are not wholly in accord with the mild uncoupling hypo