published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

University of Macau / Institute of Chinese Medical Sciences

Mitochondria are essential organelles that provide the cell with energy and are involved in many housekeeping processes. Maintaining a healthy population of mitochondria is vital for the proper functioning of cells and the presence of dysfunctional mitochondria may lead to cellular damage and cell death. Neurons are particularly susceptible to the consequences of mitochondrial damage as they have high energy needs and are post-mitotic. The clearance of damaged mitochondria by autophagy, or mitophagy, has emerged as an important quality control mechanism. The Parkinson's disease related proteins phosphatase and tensin homolog-induced putative kinase 1 (PINK1) and Parkin have been identified as important regulators of mitophagy in mammalian cells, directly linking mitophagy to neurodegeneration. The role of these two proteins in this mitophagy is further explored in the first part of this dissertation. We propose a model whereby a cleavage product of PINK1 in the cytosol binds Parkin and prevents its translocation to mitochondria, which is regarded as the initiating step in Parkin/PINK1 mitophagy. Upon the occurrence of mitochondrial damage, however, full-length PINK1 accumulates on the mitochondrial outer membrane (MOM) and recruits Parkin, marking the damaged mitochondria for mitophagy. In the second part, we assess mitophagy in a cellular model based on disease caused by mutations in mitochondrial DNA (mtDNA). We find that the mere presence of damaged mitochondria in the cell does not activate mitophagy. Rather, this process is a complex interplay between mitochondrial membrane potential, levels of PINK1/Parkin and the activation of general macroautophagy. The final part of this dissertation describes the development and validation of a new method to study mitophagy. MitophaGFP, a red-green tandem fluorescent protein targeted to the MOM, changes color from yellow to red once mitochondria enter lysosomes, the final step of the mitophagy process. This new probe allows us to quantitatively and qualitatively assess mitophagy and fulfills a need in the mitophagy field. The work described in this dissertation contributes to elucidate the mechanisms underlying mitophagy regulation in mammalian cells. Its findings can serve as a basis to further explore the importance of mitophagy as a quality control mechanism and the role of its defect in neurodegeneration.

A defining characteristic of neurodegenerative disease is the accumulation of mutant or misfolded proteins within neurons. Selective macroautophagy of aggregates, or aggrephagy, is a lysosome-mediated protein degradation pathway implicated in the turnover of disease-relevant accumulated proteins, but its specific function in vivo in the mammalian nervous system is poorly understood. The large PI3P-binding protein Alfy (Autophagy-linked FYVE protein) is an adaptor required for selective macroautophagy of aggregated proteins in cellular model systems. We sought to address Alfy-mediated aggrephagy in the mammalian brain in mouse models of Huntington’s disease (HD). HD is a neurodegenerative disorder caused by autosomal dominant inheritance of an expanded CAG repeat within the IT15, or huntingtin (htt) gene. The mutation causes an expansion of a polyglutamine (polyQ) tract in the protein Huntingtin (Htt), which results in psychiatric, cognitive, and motor symptomology. A pathological hallmark of HD is the accumulation of intracellular deposits of mutant Htt and ubiquitin. The exact relevance of these deposits remains unclear, but their elimination, hypothesized to occur via macroautophagy, correlates with behavioral improvements in mouse models of HD. The selective mechanisms of this phenomenon are largely unexplored in vivo. We have created two mouse models to address the role of Alfy-mediated selective macroautophagy in mammalian HD brain. First, we created tamoxifen-inducible Alfy knockout mice (Alfy iKO) and crossed them with a redesigned inducible HD mouse (HD103Q) that uses a tetracycline-regulated system to control reversible expression of mutant exon-1 Htt. Western blot, in situ, and PCR analysis confirm that Alfy can be eliminated from brain in adult Alfy iKO mice. A timecourse of Htt aggregation and clearance reveals that HD103Q mice accumulate huntingtin deposits, which clear in a linear manner upon transgene suppression over the course of four months. The loss of Alfy significantly impedes the removal of these deposits. Second, an Alfy knockout mouse was created using gene-trap technology, and mice hemizygous for Alfy knockout were crossed with BACHD mice expressing full-length human mutant Htt. We find that 50% Alfy depletion in the BACHD leads to increased insoluble Htt aggregate deposition along with accelerated decline in motor behavioral performance. Furthermore, inducible knockout of Alfy alone has a severe and age-dependent motor behavioral phenotype. This work reveals an in vivo role for Alfy in turnover of mutant Htt deposits, suggests that the accumulation of detergent-insoluble mutant Htt species contributes to behavioral pathogenesis, and supports an important function for Alfy at the intersection of HD and aging.

Wu, Chi Chung. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 86-96). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Chapter 1. --- INTRODUCTION / Chapter 1.1. --- Neurodegenerative disorders 一 a brief overview --- p.1 / Chapter 1.2. --- Polyglutamine diseases --- p.1 / Chapter 1.3. --- Polyglutamine protein conformers and toxicity --- p.5 / Chapter 1.4. --- in vivo modeling of polyglutamine diseases in Drosophila / Chapter 1.4.1. --- GAL4/UAS transgene expression system in Drosophila --- p.13 / Chapter 1.4.2. --- Temporal control of transgene expression systemin Drosophila --- p.15 / Chapter 1.4.3. --- Drosophila as a model to study polyglutamine diseases --- p.16 / Chapter 1.5. --- in vitro polyglutamine diseases models --- p.19 / Chapter 1.6. --- Aim of study --- p.23 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1. --- Drosophila culture and manipulation / Chapter 2.1.1. --- Drosophila culture --- p.25 / Chapter 2.1.2. --- Pseudopupil assay of adult retinal degeneration --- p.25 / Chapter 2.2. --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 2.2.1. --- Protein extraction from adult Drosophila heads --- p.26 / Chapter 2.2.2. --- Preparation of SDS-polyacrylamide gel and electrophoresis --- p.27 / Chapter 2.2.3. --- Western blotting --- p.28 / Chapter 2.2.4. --- Immunodetection --- p.29 / Chapter 2.3. --- Solubilization of SDS-insoluble protein --- p.31 / Chapter 2.4. --- Filter retardation assay --- p.31 / Chapter 2.5. --- Immunoprecipitation --- p.32 / Chapter 2.6. --- Nucleocytoplasmic fractionation --- p.33 / Chapter 2.7. --- PCR cloning / Chapter 2.7.1 . --- Drosophila DNA preparation --- p.34 / Chapter 2.7.2. --- Construction of pGEX4T3-MJDflQ27/81 expression plasmid --- p.34 / Chapter 2.8. --- in vitro aggregation assay / Chapter 2.8.1. --- Expression and purification of GST-MJDAQ27/81 protein --- p.36 / Chapter 2.8.2. --- in vitro aggregation --- p.37 / Chapter 2.8.3. --- Native slot-blot --- p.38 / Chapter 2.9. --- Reagents and buffers / Chapter 2.9.1. --- Reagents for Drosophila culture --- p.39 / Chapter 2.9.2. --- Reagents for SDS-PAGE --- p.39 / Chapter 2.9.3. --- Reagents for filter retardation assay --- p.42 / Chapter 2.9.4. --- Reagents for immunoprecipitation --- p.43 / Chapter 2.9.5. --- Reagents for nucleocytoplasmic fractionation --- p.43 / Chapter 2.9.6. --- Reagents for PCR cloning --- p.44 / Chapter 2.9.7. --- Reagents for in vitro aggregation assay --- p.46 / Chapter 3. --- Establishment of a GAL80ts-mediated transgenic Drosophila model of Machado-Joseph Disease (MJD) / Chapter 3.1. --- Introduction --- p.48 / Chapter 3.2. --- Results / Chapter 3.2.1. --- GAL80ts-mediated expression of expanded full-length MJD protein caused progressive neuronal degenerationin Drosophila --- p.49 / Chapter 3.2.2. --- Detection of SDS-insoluble expanded full-length MJD protein and its correlation with neuronal degeneration / Chapter 3.2.2.1. --- Progressive neuronal degeneration is not mediated by progressive accumulation of expanded full-length MJD protein --- p.51 / Chapter 3.2.2.2. --- SDS-soluble expanded full-length MJD protein does not correlate with progressive neuronal degeneration --- p.53 / Chapter 3.2.2.3. --- Progressive accumulation of SDS-insoluble expanded full-length MJD protein correlate with progressive neuronal degeneration --- p.55 / Chapter 3.3. --- Discussion --- p.57 / Chapter 4. --- Detection of conformational changes of expanded full-length MJD protein and its association with neuronal degeneration / Chapter 4.1. --- Introduction --- p.60 / Chapter 4.2. --- Results / Chapter 4.2.1. --- Expanded full-length MJD protein underwent conformational changes from monomer to fibrils and such conformational changes correlated with neuronal degeneration --- p.61 / Chapter 4.2.2. --- Mechanistic studies of how conformational changes of expanded full-length MJD protein triggers neuronal degeneration / Chapter 4.2.2.1. --- Expanded full-length MJD protein gradually accumulated in the nucleus during the course of neurodegeneration --- p.62 / Chapter 4.2.2.2. --- Fibrillar expanded full-length MJD protein caused transcriptional dysregulation of endogenous Hsp70 gene --- p.66 / Chapter 4.2.3. --- Consolidation of the role of fibrillar expanded full-length MJD protein in neuronal degeneration --- p.67 / Chapter 4.3. --- Discussion --- p.72 / Chapter 5. --- Attempts to generate new conformation-specific antibody against recombinant expanded full-length MJD proteins / Chapter 5.1. --- Introduction --- p.75 / Chapter 5.2. --- Results / Chapter 5.2.1. --- Recombinant expanded full-length MJD protein underwent conformational changes during in vitro aggregation --- p.75 / Chapter 5.3. --- Discussion --- p.77 / Chapter 6. --- GENERAL DISCUSSION --- p.81 / Chapter 7. --- CONCLUSION --- p.84 / Chapter 8. --- REFERENCES --- p.86

Abstract not available

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Neurodegeneration is a collective term for the progressive loss of structure, function or even death of neurons. This includes diseases like Alzheimer’s disease, frontotemporal dementia, Parkinson’s disease and motor neuron disease. Recent researches have shown great interest in the role of tau proteins, which have versatile functions including microtubule stabilization and signal relay in the central nervous system. Retina and optic nerve, being part of the central nervous system, can also be affected by similar processes. In neurodegenerative diseases visual disturbances including difficulties in reading and finding object, depth perception, perceiving structure from motion, color recognition and impairment in spatial contrast sensitivity have all been observed. Some of these defects may be attributed to changes at ocular level. The effect of tau mutation was investigated in this study utilizing a transgenic P301L tau mice model. Morphometric analysis has been utilized to quatify the neurodegenerative changes, including the thickness of inner nuclear layer(INL), density of retinal ganglion cells(RGCs) and size of RGCs. Retinal sections stained by hematoxylin and eosin(H&E) were analyzed. Comparisons were made between the P301L tau mice and the control mice in addition to comparisons between different age groups. The study found that there was a significant decrease of thickness of INL of P301L tau mice when compared with control mice. The effect was more pronounced in the peripheral area and the effect increased with age. Regarding density of RGCs, P301L tau mice showed a similar age-related decline as control mice. And regarding the size of RGCs, the RGCs from P301L tau mice increased in size with age and the RGCs from control mice decreased in size with age. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

Major depression and Alzheimer’s disease (AD) are highly prevalent psychiatric disorders. Further investigation demonstrated that depression itself is a risk factor for AD, and several associated genetic mutations have been found Moreover, significant proportion of AD patients suffer also suffer from depression. These findings generated interests in finding the neurobiological linkages between depression and AD. The elucidation of pathophysiological mechanisms common in both disorders would be important, as the knowledge could provide additional insights regarding the pathogeneses of the disorders and possible interventions. The present study proposes that synaptic degeneration plays a central role in the pathogenesis of depression and AD. Using in vitro disease models, this study demonstrated abnormalities in pre-synaptic and cytoskeletal proteins, which leads to impaired synaptic function. Further investigation into the upstream events demonstrated the involvement of ubiquitin-mediated protein degradation mechanism and the preferential activation of the autophagic-lysosomal pathway. This study also investigated the neuroprotective properties of the antidepressants imipramine and escitalopram. Antidepressants have originally been thought to exert their therapeutic effects through monoaminergic system modulation. Interestingly, results in this study showed that these two agents were able to ameliorate the observed synaptic protein changes, thereby implicating other possible mechanism of action for antidepressants. In conclusion, this study provides evidence that similar synaptic pathologies exist between depression and AD, which could be responsible for the development of these two disorders. Furthermore, antidepressants may be exerting its effects through alleviating synaptic degeneration. / published_or_final_version / Psychiatry / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences