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.

Retinal degenerative diseases cause varying degrees of irreversible vision loss in millions of people worldwide. Common to all retinal degenerative diseases is the malfunction or demise of photoreceptor cells or its supportive cells, retinal pigment epithelium cell in the retina. A considerable part of these diseases were resulted from the inherited mutations of essential genes expressed in these retinal cells. The understanding of pathologic mechanism as well as developing of therapeutic treatment for these diseases were discussed in this study. A cutting-edge therapeutic genome editing technology is studied in the first part of study. This technology was invented to treat retinitis pigmentosa via engineered nucleases, which has great clinical potential for autosomal dominant genetic disorders that were previously irreparable by conventional gene therapy interventions. Though customizable gene editing tools can be engineered to target specific mutation sites, however it is too daunting for diseases like retinitis pigmentosa, a progressive retinal degenerative condition associated with more than 150 mutations in the rhodopsin gene alone. Here in this study, we present an “ablate-and-replace” combination strategy that 1) destroys expression of the endogenous gene by CRISPR/Cas9 in a mutation-independent manner, and 2) enables expression of wild-type protein through exogenous cDNA. As proof of concept, we show that our CRISPR-based therapeutic machinery efficiently ablates mRho in vivo, and when combined with gene replacement therapy, ameliorates rod photoreceptor degeneration and improves visual function in two genetically distinct autosomal dominant retinitis pigmentosa animal models. This mutation-independent, ablate-and-replace strategy represents the first electrophysiological recovery by a CRISPR-mediated therapy in an autosomal dominant disorder and it offers a clinically relevant, universal strategy to overcome allelic heterogeneity in debilitating inherited conditions. For the second part of the study, gene editing technology was used to study the pathogenesis of Doyne honey comb dystrophy, another Mendelian disease with extensive similarities to age-related macular degeneration. This monogenic disorder is caused by a unique point mutation on an extracellular matrix protein EFEMP1, expressed by retinal pigment epithelium cell. To precisely gauge the physiological effect resulted from this mutation, CRISPR-mediated gene correction was used to create isogeneic cell pairs from patient donated tissue-derived stem cells. These stem cells were differentiated into retinal pigment epithelium cell before analysis. We found unfolded protein response and immune response were not involved in the pathogenesis, which contradicts existing theories. Via proteomics analysis, we found expression level of a cholesterol catabolic enzyme was affected by the EFEMP1 mutation while those proteins controlling the cholesterol transport remains constant. This result provides supportive evidence to explain the aberrant intracellular accumulation of cholesterol found in patient retinal pigment epithelium cells. This imbalance in lipid homeostasis also suggests Doyne honey comb dystrophy is a retinal pigment epithelium cell-autonomous disease.

Kwong Man Kwong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 83-99). / TABLE OF CONTENTS --- p.i / ACKNOWLEDGEMENTS --- p.vi / LIST OF FIGURES --- p.vii / LIST OF ABBREVIATIONS --- p.ix / Chapter I. --- ABSTRACT --- p.1 / Chapter II. --- INTRODUCTION --- p.4 / Chapter III. --- LITERATURE REVIEW --- p.6 / Chapter A. --- EXCITATORY AMINO ACIDS AND EXCITOTOXICITY --- p.6 / Chapter 1. --- GLUTAMATE RECEPTORS --- p.7 / Chapter 2. --- NMDA RECEPTOR --- p.9 / Chapter 3. --- EXCITOTOXICITY --- p.10 / Chapter a. --- ACUTE PHASE --- p.10 / Chapter b. --- DELAYED PHASE --- p.11 / Chapter c. --- MECHANISM --- p.11 / Chapter i) --- Inhibition of Na+，K+-ATPase --- p.12 / Chapter ii) --- Impaired Mitochondrial Calcium Buffering --- p.12 / Chapter iii) --- Perturbation of Cytoskeletal Organisation --- p.13 / Chapter iv) --- Phospholipase Activation --- p.14 / Chapter v) --- Endonuclease Activation --- p.15 / Chapter vi) --- Protein Kinase C Activation --- p.15 / Chapter vii) --- Xanthine Oxidase Activation --- p.16 / Chapter viii) --- Nitric Oxidase Synthase Activation --- p.16 / Chapter B. --- APOPTOSIS --- p.19 / Chapter 1. --- MORPHOLOGICAL CHANGES --- p.19 / Chapter 2. --- BIOCHEMICAL AND MOLECULAR CHANGES --- p.20 / Chapter 3. --- APOPTOTIC MEDIATORS --- p.21 / Chapter a. --- INTERLEUKIN-1β CONVERTING ENZYME (ICE) --- p.21 / Chapter b. --- ENDONUCLEASE --- p.22 / Chapter c. --- NITRIC OXIDE SYNTHASE (NOS) --- p.23 / Chapter d. --- POLY(ADP-RIBOSE) POLYMERASE (PARP) --- p.24 / Chapter e. --- CALPAINS --- p.25 / Chapter IV. --- NMDA INDUCED APOPTOSIS IN RAT RETINA --- p.27 / Chapter A. --- RATIONALE --- p.27 / Chapter B. --- MATERIALS AND METHODS --- p.31 / Chapter 1. --- NMDA INDUCED EXCITOTOXICITY --- p.31 / Chapter a. --- INTRAVITREAL INJECTIONS --- p.31 / Chapter b. --- RETINAL GANGLION CELL COUNTS (RGCC) --- p.32 / Chapter i) --- Flat Preparation of Rat Retina --- p.33 / Chapter ii) --- RGCC --- p.33 / Chapter d. --- INNER RETINAL THICKNESS (IRT) --- p.34 / Chapter i. --- Preparation of Epoxyl Specimens --- p.34 / Chapter ii. --- Measurement of IRT --- p.36 / Chapter 2. --- DOSE RESPONSE STUDY OF NMDA --- p.36 / Chapter 3. --- NMDA INDUCED APOPTOSIS IN RAT RETINA --- p.37 / Chapter a. --- GEL ELECTROPHORESIS OF RETINAL DNA --- p.37 / Chapter b. --- HISTOPATHOLOGICAL STUDIES --- p.39 / Chapter i) --- Light Microscopy --- p.39 / Chapter ii) --- Terminal Deoxynucleotidyl Transferase-mediated dUTP-biotin Nick End Labelling (TUNEL) --- p.40 / Chapter iii) --- Electron Microscopy (EM) --- p.41 / Chapter c. --- MORPHOMETRY --- p.41 / Chapter i) --- TUNEL Positive Nuclei --- p.41 / Chapter ii) --- RGCC and IRT --- p.42 / Chapter 4. --- STUDY OF ENZYME INHIBITORS --- p.42 / Chapter C. --- RESULTS --- p.43 / Chapter 1. --- EXCITOTOXICITY IN RAT RETINA --- p.43 / Chapter a. --- RGCC --- p.43 / Chapter b. --- IRT --- p.44 / Chapter 2. --- DOSE DEPENDENT TISSUE RESPONSES AND REGIONAL RESPONSES --- p.44 / Chapter a. --- RGCC --- p.44 / Chapter b. --- IRT --- p.45 / Chapter 3. --- NMDA INDUCED APOPTOSIS IN RAT RETINA --- p.45 / Chapter a. --- RETINAL DNA GEL ELECTROPHORESIS --- p.46 / Chapter b. --- HISTOPATHOLOGY AND TUNEL --- p.46 / Chapter c. --- MORPHOMETRY OF TUNEL AT THE RGCL AND INL --- p.47 / Chapter d. --- TISSUE RESPONSES AT 7 DAYS AFTER INJECTION --- p.48 / Chapter e. --- EM --- p.48 / Chapter i) --- RGCL --- p.48 / Chapter ii) --- INL --- p.48 / Chapter 4. --- ENZYME INHIBITOR STUDY IN NMDA INDUCED EXCITOTOXICITY --- p.49 / Chapter a. --- EFFECT OF VARIOUS ENZYME INHIBITORS ON RGCC --- p.49 / Chapter b. --- EFFECT OF VARIOUS ENZYME INHIBITORS ON IRT --- p.50 / Chapter D. --- DISCUSSION --- p.51 / Chapter 1. --- NMDA INDUCED EXCITOTOXICITY IN RAT RETINA --- p.51 / Chapter 2. --- DOSE DEPENDENT RESPONSES AND REGIONAL RESPONSES --- p.55 / Chapter 3. --- NMDA INDUCED APOPTOSIS IN RAT RETINA --- p.58 / Chapter 4. --- INHIBITOR STUDY --- p.62 / Chapter a. --- ICE --- p.63 / Chapter b. --- ENDONUCLEASE --- p.65 / Chapter c. --- NOS --- p.67 / Chapter d. --- PARP --- p.69 / Chapter e. --- CALPAIN --- p.70 / Chapter V. --- NMDA INDUCED APOPTOSIS IN RABBIT RETINA --- p.72 / Chapter A. --- RATIONALE --- p.72 / Chapter B. --- MATERIALS AND METHODS --- p.73 / Chapter 1. --- INTRAVITREAL INJECTION OF NMDA --- p.73 / Chapter 2. --- HISTOPATHOLOGY AND TUNEL --- p.74 / Chapter 3. --- MORPHOMETRY OF TUNEL --- p.74 / Chapter 4. --- TISSUE RESPONSES AT 7 DAYS AFTER INJECTION --- p.74 / Chapter a. --- RGCC --- p.74 / Chapter b. --- IRT --- p.74 / Chapter 5. --- EM --- p.75 / Chapter C. --- RESULTS --- p.76 / Chapter 1. --- HISTOPATHOLOGY AND TUNEL --- p.76 / Chapter 2. --- MORPHOMETRY OF TUNEL --- p.77 / Chapter 3. --- TISSUE RESPONSES AT 7 DAYS POST INJECTION --- p.78 / Chapter a. --- RGCC --- p.78 / Chapter b. --- IRT --- p.78 / Chapter 4. --- EM --- p.79 / Chapter a. --- RGCL --- p.79 / Chapter b. --- INL --- p.79 / Chapter B. --- DISCUSSION --- p.80 / Chapter VI. --- CONCLUSION --- p.82 / Chapter VII. --- REFERENCES --- p.83 / Chapter VIII. --- FIGURES --- p.100

Submitted by Natalia Vieira (natalia.vieira@famerp.br) on 2016-05-19T19:07:09Z No. of bitstreams: 1 sabrinamayaracezario_dissert.pdf: 2283086 bytes, checksum: 130b0d9b52a17f23980c982605a0dad9 (MD5) / Made available in DSpace on 2016-05-19T19:07:10Z (GMT). No. of bitstreams: 1 sabrinamayaracezario_dissert.pdf: 2283086 bytes, checksum: 130b0d9b52a17f23980c982605a0dad9 (MD5) Previous issue date: 2015-05-27 / Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP / Background - Age-related macular degeneration (AMD) is a complex disease. The identification of risk factors may contribute to the prognosis and treatment. Objectives – Evaluate the influence of genetic variants related to lipid metabolism, angiogenesis and inflammation and its relation with clinical and lipid profile and lifestyle beyond their gene expression in patients with AMD. Casuistic and Methods – We studied 333 individuals aged ≥50 years, 108 with AMD in exudative form (G1); 45 with AMD in dry form (G2), and 180 individuals without clinical and angiographic signs of the disease (G3). The polymorphisms of apolipoprotein E (APOE-rs429358/rs7412), triphosphate binding cassette sub -family A transport -member 4 (ABCA4-rs472908), complement factor H (CFH-rs1061170) and vascular endothelial growth factor (VEGF-rs3025039/rs1570360) were analyzed by PCR/RFLP (polymerase chain reaction/restriction fragments length polymorphism), while the respective gene expression in blood by PCR/RT (reverse transcription-PCR) and serum levels of apo E, ABCR, CFH, VEGF by ELISA (enzyme linked immuno sorbent assay). Clinical and lipid profile data in addition to lifestyle were obtained from medical records and questionnaire. Level of significance was accepted for P<0.05. Results – Systemic arterial hypertension (SAH) and smoking prevailed in patients with AMD exudative form (P<0.05). Genetic polymorphisms: APOE- rs429358/rs7412 - APOE*3/3 was noted in all groups, followed by APOE*3/4, as well APOE*4 (P>0.05). ABCA4-rs472908 - Genotype A/G was more frequent in G3 (68%) versus G2 (44%; P<0.0001), while A/A in G2 (36%) versus G1 (19%; P=0.04) and G3 (14%; P=0.003). The mutant genotype (G/G) prevailed in combination (G1+G2) versus G3 (P< 0.0001), and allele G in all groups (P>0.05). CFH-rs1061170 – The wild homozygous (TT) was in evidence in G3 (58%) versus G1 (39%, P=0.003); as well the homozygous mutant (CC) in G1 (27 %) versus G3 (14%; P=0.002) and allele T in G3 (0.72) versus G1 (0.56; P=0.0002). VEGF-rs3025039 – Genotypic and allelic distribution were similar between groups (P>0.05), highlighting the CC genotype and allele C. VEGF-rs1570360 – Mutant homozygote (A/A) prevailed in G2 (21%) versus G1 (5%; P=0.002) and G3 (8%; P=0.015) as well as the wild type allele (G) G1 (0.75) and G3 (0.71) versus G2 (0.57, P=0.004, P=0.020, respectively). Gene expression – Similar values between groups for all analyzed genes (P>0.05). Serum levels (median values in ng/mL) – ApoE– Increased level in G1 (270.6) versus G2 (196.5; P<0.0001) and G3 (242.8; P=0.035), and G3 versus G2 (P=0.0002). ABCR – High levels in G1 (0.30) versus G2 (0.25; P=0.003) and G3 (0.25, P<0.0001). CFH – Increase in G1 (1198.9) versus G2 (859.8; P=0.0069), both higher than in G3 (618.3; P<0.001, P=0.001, respectively). VEGF – Similar values between groups (P>0.05). Lipid profile – G3 showed the highest level of HDLc (median=71mg /dL), compared to G2 (60mg/dL), and G1 (45mg/dL; P=0.003, P=0.029, respectively). Conclusion – Genetic variants of ABCA4, VEGF and CFH, besides SAH, smoking and increased serum levels of apo E, ABCR and CFH are associated with AMD, whereas the expression of the respective genes not differentiate AMD exsudative and dry forms, in contrast CFH (homozygous wild-type) has a protective character, as well as serum levels of HDLc. / Introdução – Degeneração macular relacionada à idade (DMRI) é uma doença complexa. A identificação de fatores de risco poderá contribuir no seu prognóstico e tratamento. Objetivos – Avaliar a influência de variantes genéticas, relacionadas com o metabolismo de lipídios, angiogênese e inflamação e sua relação com perfil clínico e lipídico e hábitos de vida, além das respectivas expressões gênicas em pacientes com DMRI. Casuística e Métodos – Foram estudados 333 indivíduos com idade ≥50 anos, sendo 108 com DMRI na forma exsudativa (G1); 45 com DMRI na forma seca (G2) e 180 indivíduos sem sinais clínicos e angiográficos da doença (G3). Os polimorfismos de apolipoproteína E (APOE-rs429358/rs7412), triphosphate binding cassette transporte sub-family A-member 4 (ABCA4-rs472908), complemento do fator H (CFH-rs1061170) e fator de crescimento endotelial vascular (VEGF-rs3025039/rs1570360) foram analisados por PCR/RFLP (polymerase chain reaction/restriction fragments lengh polymorphism), enquanto as respectivas expressões gênicas no sangue por PCR/RT (reverse transcription-PCR) e níveis séricos de apo E, ABCR, CFH, VEGF por ELISA (enzyme linked immuno sorbent assay). Dados de perfil clínico e lipídico, além de hábitos de vida, foram obtidos em prontuário médico e questionário. Admitiu-se nível de significância para P<0,05. Resultados – Hipertensão arterial sistêmica (HAS) e tabagismo prevaleceram em pacientes com DMRI exsudativa (P<0,05). Polimorfismos genéticos: APOE-rs429358/rs7412–APOE*3/3 destacou-se em todos os grupos, seguido de APOE*3/4, assim como o alelo APOE*4 (P>0,05). ABCA4-rs472908–O genótipo A/G foi mais frequente em G3 (68%) versus G2 (44%; P<0,0001), enquanto A/A em G2 (36%) versus G1 (19%; P=0,043) e G3 (14%; P=0,003). O genótipo mutante (G/G) prevaleceu na combinação (G1+G2) versus G3 (P<0,0001), e o alelo G em todos os grupos (P>0,05). CFH-rs1061170–O homozigoto selvagem (TT) destacou-se em G3 (58%) versus G1 (39%, P=0,003); já o homozigoto mutante (CC) em G1 (27%) versus G3 (14%; P=0,002), e o alelo T em G3 (0,72) versus G1 (0,56; P=0,0002). VEGF-rs3025039–Distribuição genotípica e alélica semelhante entre os grupos (P>0,05), destacando-se o genótipo CC e alelo C. VEGF-rs1570360–Homozigoto mutante (A/A) prevaleceu em G2 (21%) versus G1 (5%; P=0,002) e G3 (8%; P=0,015), assim como o alelo selvagem (G) em G1 (0,75) e G3 (0,71) versus G2 (0,57; P=0,004; P=0,020, respectivamente). Expressão gênica–Valores semelhantes entre os grupos para todos os genes analisados (P>0,05). Níveis séricos (valores de mediana em ng/mL)–ApoE–Aumento em G1 (270,6) versus G2 (196,5; P<0,0001) e G3 (242,8; P=0,035), e G3 versus G2 (P=0,0002). ABCR–Níveis elevados em G1 (0,30) versus G2 (0,25; P=0,003) e G3 (0,25; P<0,0001). CFH–Aumento em G1 (1.198,9) versus G2 (859,8; P=0,0069), ambos com acréscimo em relação a G3 (618,3; P<0,001; P=0,001, respectivamente). VEGF–Valores semelhantes entre os grupos (P>0,05). Perfil lipídico–G3 mostrou valor mais elevado de HDLc (mediana=71mg/dL), comparado a G2 (60mg/dL) e G1 (45mg/dL) (P=0,003; P=0,029, respectivamente). Conclusão – Variantes genéticas de ABCA4, VEGF e CFH, além de HAS, tabagismo e nível sérico elevado de apoE, ABCR e CFH associam-se a DMRI, enquanto a expressão dos referidos genes não diferencia DMRI exsudativa e seca, em contrapartida CFH (homozigoto selvagem) tem caráter protetor, assim como nível sérico de HDLc.

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

Age-related macular degeneration (AMD) is the leading cause of vision loss in individuals aged 50 years and older and is estimated to affect as many as 11 million individuals in the United States. The purpose of this study was to examine the association between vitamin D and AMD disease progression. The life course epidemiology framework model was used to explore how vitamin D level as a risk factor may have an association to AMD disease through time. Data in the 2005-2008 National Health and Nutrition Examination Survey (NHANES) database were collected on vitamin D levels and identified stages of AMD level based on graded fundus eye exams from an available sample size of 5,604 participants. A quantitative cross-sectional study approach was used to address this gap in knowledge. A bivariate analysis was used to examine each independent variable (age, race/ethnicity, smoking status, and diabetes) to the dependent variable AMD from the 2005-2008 NHANES dataset. A multivariate logistic regression analysis was performed with AMD including each independent variable found to be significant. The findings from this study failed to suggest an association between vitamin D levels to AMD, with or without the covariates included in the model. There was not an association found between vitamin D level and presence of AMD. An association was found between age, smoking, and race to presence of AMD in each of the bivariate models. The findings from this study could be used for positive social change by encouraging medical and public health agencies to target screening programs at high-risk age, smoking, and race groups. There remains to be conflicting data in the literature. This study adds to the body of literature suggesting that higher levels of vitamin D are not necessarily beneficial as they pertains to AMD.

Neurodegenerative Erkrankungen des Menschen sind eines der Hauptfelder molekularer neurobiologischer Grundlagenforschung. Um generell molekulare, komplizierte Vorgänge in vivo untersuchen zu können, nutzt man seit geraumer Zeit Modellorganismen wie Caenorhabditis elegans oder Drosophila melanogaster. In der vorliegenden Arbeit wird die Drosophila-Neurodegenerationsmutante loe (löchrig) beschrieben, die als Modell für die Rolle des Cholesterinhaushalts im Bezug auf Neurodegeneration herangezogen werden kann. Die Fliegen dieser Mutante zeigen stark progressive, altersabhängige Degeneration von Neuronen, dabei unterlaufen diese Nervenzellen einen nekrotischenZelltod. Verantwortlich für diese Mutation ist die Insertion eines P-Elementes in einem Intron des Drosophila-g-5'-AMP-aktivierten Proteinkinase- (AMPK)-Gens. Die verschiedenen Spleißprodukte des loe Gens kodieren für die regulatorische g-Untereinheit des AMPK-Komplexes, der , aktiviert durch 5'AMP, energieintensive Prozesse negativ reguliert. Die Spleißform loeI ist durch die P-Element-Insertion betroffen, Anteile des P-Elementes werden in das loeI-Transkript hineingespleißt. Eine neuronale Expression von loeI im loe-Hintergrund führt zur Revertierung des loe-Phänotypes. Mit der Expression anderer Spleißformen kann dieser Effekt nicht erzielt werden. Das LOE I-Protein birgt in seinem N-Terminus eine Reihe möglicher Interaktionstellen mit anderen Proteinen, die den AMPK-Komplex in einen Kontext mit den Proteinen der APP (Amyloid Precursor Proteins) ?Familie stellen oder z. B. Interaktionen mit dem Cytoskelett herstellen können. Eine molekulare Interaktion mit NiPSNAP, einem Protein, dass vermutlich eine Rolle im Vesikelverkehr spielt, konnte nachgewiesen werden. Ein direktes humanes Homolog von LOE I ist nicht bekannt, wohlgleich es im Menschen drei AMPK-g-Untereinheiten gibt, von denen zwei ähnliche Funktionen übernehmen könnten wie LOE I. Die loe-Mutante interagiert genetisch mit der Mutante clb ? columbus, die einen Defekt im Gen der HMG-CoA-Reduktase trägt. Dieses Emzym ist das Schlüsselenzym der Cholesterinbiosynthese. Die Art der Interaktion belegt eine negative Regulierung der HMG-CoA-Reduktase durch die AMPK. So schwächt die clb-Mutation den neurodegenerativen loe-Phänotyp ab, eine Überexpression von clb verstärkt diesen. Eine Verminderung der Neurodegeneration kann auch mit Medikamenten erreicht werden: Statine, potente Hemmer der HMG-COA-Reduktase, reprimieren deutlich den loe-Phänotyp. In loe ist der Cholesterinester-Spiegel auf 40% abgesenkt. Eine weitere genetische Interaktion von loe konnte nachgewiesen werden: Die Mutante für das Drosophila-Homolog von APP (Appl) verstärkt den neurodegenerativen Phänotyp in loe stark, wogegen die Appl-Mutante selbst keine neurodegenerativen Defekte aufweist. Darüberhinaus zeigt die Doppelmutante Defekte, die keine der Einzelmutanten aufweist: Sterilität oder eine extrem kurze Lebensdauer von nur 3-4 Tagen. Diese Interaktion ließ sich auf molekularer Ebene charakterisieren. Die proteolytische Prozessierung von APPL durch Sekretasen ist in loe alteriert. In der vorliegenden Arbeit konnte gezeigt werden, dass durch die loe-Mutation die b-Sekretase aus Vertebraten (BACE) und eine bisher noch nicht beschriebene endogene Sekretase aus Drosophila negativ beeiflusst werden. Ein AMPK-Komplex mit LOE I als g-Untereinheit scheint über den Cholesterinester-Spiegel die Aktivität einer speziellen Untergruppe der Sekretasen zu beeinflussen. Die Missfunktion dieser Sekretasen ist ein kritischer Punkt in der Pathogenese der Alzheimer-Krankheit. Die loe-Mutation wirft neues Licht auf die bekannten Verbindungen zwischen Cholesterin-Stoffwechsel, Vesikelverkehr und Prozessierung von APP(L). Mit den großen Möglichkeiten, die die Drosophila-Genetik bietet, stellt diese neue Mutante ein weiteres Werkzeug zur Charakterisierung von Therapie-Ansätzen für die Alzheimer-Kankheit dar. Die vorliegende Arbeit belegt um ein weiteres Mal, dass Drosophila ein potentes Modellsystem zur Untersuchung humaner, neurodegenerativer Erkrankungen wie Chorea Huntington, Parkinson oder der Alzheimer Krankheit ist. / Human neurodegenerative diseases are the main topic of molecular neurobiological basic research. To investigate detailed mechanisms in vivo one uses the tool of genetic model organisms like Caenorhabditis elegans or Drosophila melanogaster for quite a long while. This thesis describes the Drosophila neurodegenration mutant löchrig (loe), which can be used as a model for cholesterol metabolism in respect to neurodegeneration. Mutant loe flies show strong and progressive age-dependent degenration of neurons undergoing necrotic cell death. The P-element inserted in an intron of the gene coding for the Drosophila 5'-AMP activated protein kinase (AMPK) complex gamma subunit is responsible for the mutation in loe. The various splice forms of the loe gene code for different regulatory gamma subunits of this complex consisiting of three subunits. The splice form loeI is affected by the P-element insertion, parts of the P-element are spliced into the loeI transkript in the loe mutant. The neuronal expression of one copy of loeI in the mutant background revertes the neurodegenerative phenotype which can not be achieved by expression of one of the other splice forms. The LOE I protein contains in its N-terminus several putative interaction motifs and domaines. These could get a LOE I-containing AMPK complex in context with the APP (amyloid precursor protein) or the cytoskeletton. An interaction with NiPSNAP ? a protein with a putative function in the vesicular transport ? has been proved molecularly. A human homolog of LOE I is not yet known, although there are three different isoforms of a AMPK gamma subunit described in humans. The loe mutant interacts genetically with the columbus (clb) mutant, wich is affected in the gene of the HMG-CoA reductase, the key enzyme in cholesterol biosynthesis. This shown interaction verifies a negative regulation of the HMG-CoA reductase by the AMPK complex in Drosophila. Thus the clb mutation supresses the loe phenotype, an overexpression of clb enhances the neurodegeneration. A supression of the neurodegenerative phenotype can be also achieved by a statin treatment of loe flies. Statins are potent inhibitors of the HMG-CoA reductase. Another genetic interaction exists between loe and the Appl mutant. Appl d, the null mutant of the Drosophila APP homolog, enhances strongly the neurogenerative phenotype of loe, whereas the Appl mutant itself shows no neuronal defects. In addition the double mutant shows defects which none of the single mutants show: sterility of females and a dramatic shortened lifespan of only 3-4 days. This interaction could be characterized on the molecular level: The proteelytic processing of APPL by sectretases is altered in the loe mutant. Both the BACE sectretase from vertebrates and an so far uncharakterized endogenous sectretase in Drosophila are negatively influenced by the loe mutation. An AMPK complex containing LOE I as the gamma subunit seems to regulate the activity of a subgroup of the sectretases via the cholesterolester level. The misfunction of secretases is a crutial point in the pathogenesis of Alzheimer's disease. The loe mutation gives new insights in the already known links between cholesterol homeostasis, vesicular transport, and processing of APP(L). Together with the exstensive tools of Drosophila genetics this new mutant will supply new possibilities to characterize putative therapies to cure Alzheimer's disease. This thesis at another time presents Drosophila as an potent model system for the research on human neurodegenerative diseases like Huntington's disease, Parkinson or Alzheimer's disease.

Subsequent to an hypoxic-ischaemic (HI)-insult a multi-faceted complex cascade of events occurs that ultimately results in cellular and neurological impairments within cortical and sub-cortical central nervous system (CNS) regions. In the present studies a modified �Levine� rat-pup model of HI (left carotid artery ligation + 1 hour global hypoxia on post-natal day (PND) 26) was employed to assess the neuroprotective properties of clomethiazole (CMZ; a γ-aminobutyric acid (GABA)A receptor agonist). In this study, histological and electrophysiological paradigms were used to assess the long-term neuroprotective properties of CMZ (414mg/kg/day via mini-pumps). Key enzymes involved in inflammation, namely nitric oxide synthase (NOS) and arginase, were also examined to assess other potential CMZ mechanisms. Assessments were carried out 3- and 90-days post-HI, with extensive ipsilateral CNS lesions evident at a gross histological level, at both the early and long-term stages, with CMZ significantly decreasing the lesion size at 3- and 90-days (P<0.01; P<0.05). Evoked field potential analyses were used to assess hippocampal CA1 neuronal activity ex vivo. Electrophysiological measurements contralateral to the occlusion revealed impaired neuronal function following HI relative to short- and long-term controls (P<0.001, 3- and 14-days; P<0.01, 90-days), with CMZ providing near complete protection (P<0.001 at 3- and 14-days; P<0.01 at 90-days). Both inducible NOS (iNOS) and arginase activities were significantly increased at 3-days (P<0.01), with arginase activity remaining elevated at 90-days post-HI (P<0.05) ipsilaterally. CMZ suppressed the HI-induced increase in iNOS and arginase activities (P<0.001; P<0.05). These data provide evidence of long-term functional neuroprotection afforded by CMZ in a model of HI-induced neurodegeneration. In addition, under conditions of HI, functional deficits were not restricted to the ipsilateral hemisphere and were due, at least in part, to changes in the activity of NOS and arginase. Underlying mitochondrial dysfunction is eminently present in many neuropathological conditions. The full extent of mitochondrial dysfunction in cortical, hippocampal and cerebellar tissues was assessed following HI. Assessment of mitochondrial FAD-linked respiration at both 1- and 3-days post-HI revealed a significant decrease in activity from ipsilateral cortical and hippocampal regions (P<0.001). In addition, significant changes in respiratory function were also evident in contralateral regions and cerebellum, 3-days post-HI (P<0.05). Assessment of the mitochondrial electron transport chain (complexes I-V) and mitochondrial markers of integrity (citrate synthase) and oxidative stress (aconitase) confirmed ipsilateral mitochondrial impairment following HI. Complexes I, II-III, V and citrate synthase were also impaired, in contralateral regions and cerebellum, 3-days post-HI. CMZ treatment provided significant protection to all mitochondrial aspects of neuronal tissue assessed. This study provides evidence of the full extent of mitochondrial damage following an HI-insult and may contribute, in part, to the impairment seen contralaterally. In addition, protection afforded by CMZ extends to preservation of mitochondrial function and integrity. Cerebral ischaemia-induced angiogenesis has been shown within and around infarcted regions and may contribute to a more favourable neurological outcome. The level of angiogenesis was examined using platelet endothelial cell adhesion molecule-1 (PECAM-1 / CD31). CD31 immunolabelling 7-days post-HI revealed a significant increase in angiogenesis compared with non-intervention controls (P<0.001). Treatment with CMZ decreased the level of angiogenesis compared to HI + saline (P<0.001) back to non-intervention control levels. Conversely, N[omega]-nitro-L-arginine methyl ester (L-NAME) treatment (5mg/kg/day) exacerbated the ischaemic lesion (P<0.001) and resulted in a marked decrease in angiogenesis compared to non-intervention controls (P<0.001). The extent of cerebral infarction in these studies is dependent on the level of NOS activity with CMZ increasing total NOS levels compared to HI + saline, while L-NAME halted the HI-induce increase in total NOS activity (P<0.001). These results show for the first time, that angiogenesis may be used as an assessment of neurodegeneration / neuroprotection in pathologies of cerebral ischaemia and are directly correlated with changes in NOS activity. These studies have therefore shown that following HI, damage also occurs contralateral to the occlusion, and is not restricted to the ipsilateral hemisphere. In addition, the neuroprotective effects of CMZ have been shown to extend out to 90-days post-HI, whereby significant protection to CA1 neuronal activity was seen. These studies also provide in vivo evidence that CMZ may also afford neuroprotection via anti-inflammatory pathways, as evidenced by a decrease in iNOS and arginase activities. Furthermore, these studies have also show evidence that angiogenesis (CD31) can be used as a diagnostic tool to assess neuroprotection / neurodegeneration.

Mitoquinone (MitoQ₁₀ mesylate) is a mitochondria-targeted antioxidant for the treatment of neurodegenerative diseases. As the oral bioavailability of mitoquinone is low in rat, it is necessary to better understand the mechanisms of its absorption in rat and in human. The aims of this thesis were 1) to investigate oral absorption mechanisms of mitoquinone in Caco-2 cell monolayers and in a rat intestinal tissue model; 2) to investigate the correlation between chemical structure and permeability of mitoquinone analogues in Caco-2 cell monolayers; and 3) to explore the hypothesis that active transport and/or drug metabolism contribute to the pharmacokinetics of oral mitoquinone in rat. In Caco-2 studies, transport of mitoquinone was polarized with the apparent permeability (P[app]) from basolateral (BL) to apical (AP) (P[appBL to AP]) being >2.5-fold the P[app] from AP to BL (P[appAP to BL]). The P[appBL to AP] value decreased by 26%, 31% and 61% by P-glycoprotein (P-gp) inhibitors verapamil 100 [mu]M, cyclosporine A (CsA) 10 [mu]M and CsA 30 [mu]M, respectively, whereas the P[appAP to BL] increased 71% by CsA 30 [mu]M. Some of the intracellular mitoquinone was reduced to mitoquinol and subsequently metabolized to glucuronide and sulfate conjugates. Apical effluxes of mitoquinol sulfate and mitoquinol glucuronide conjugates were significantly decreased by cyclosporine A 30 [mu]M and the breast cancer receptor protein (BCRP) inhibitor, reserpine 25 [mu]M, respectively. In the presence of 4% bovine serum albumin on the BL side, the P[appAP to BL] was 4.52 � 0.92 x 10⁶ cm/s. Based on a absorption-disposition model, F[a] value of mitoquinone in human is estimated to be 56%. A bellshaped relationship exists between the Caco-2 permeability of mitoquinone analogues and their lipophilicity. Permeability of mitoquinone analogues initially increases as lipophilicity increase, reaches a maximum, and then decreases due to significant cellular accumulation and active efflux. The physicochemical parameters of mitoquinone and its analogues (such as log P or polar surface area) alone do not predict their permeability across the cell membranes. The bidirectional transport of mitoquinone displays polarity across rat ileal mucosa. The P[app] from s to m (P[app s to m) of mitoquinone decreased and P[app m to s] increased but not significantly by P-gp inhibitor CsA 30 [mu]M. The tissue accumulation of mitoquinone was ~16% of the total amount of mitoquinone added. In addition, several phase I and one phase II metabolites generated by rat ileum tissue were detected. Results from pharmacokinetic studies indicate that mitoquinone was poorly (~24%) but rapidly absorbed and conjugated after oral administration. It was quickly excreted as unchanged drug and as its glucuronides (the major metabolites in rat) into intestine where it was reabsorbed. P-gp inhibition studies in rat indicate that inhibition of P-gp may increase the intestinal absorption of mitoquinone, but cannot change its oral bioavailability due to increased first-pass phase II metabolism and decreased enterohepatic recycling. In conclusion, mitoquinone is poorly absorbed in rat but may be well absorbed in human. The barrier functions of intracellular metabolism and the action of P-gp to oral absorption of mitoquinone in human may be less significant, whereas P-gp play an important role in the absorption and disposition of mitoquinone in rat in vivo. These results, together with those from its analogues, demonstrate that the actual absorption profile of a compound depends on its intrinsic membrane permeability, transporter affinity, metabolizing enzyme affinity and plasma protein binding affinity.