Dissertations / Theses on the topic 'Mitochondrial defects'

Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.
Doctor of Philosophy

Mestrado em Biotecnologia - Biotecnologia Molecular
The aim of this work is the study of phenotypic changes and mitochondrial morphology in Saccharomyces cerevisiae cells with specific mutations in genes involved in the ubiquitin-proteasome pathway. The protein turnover is important because it ensures organelles viability such as mitochondria, indispensable for cell survival. The COP9 complex is paralogous to the proteasome lid and eukaryotic translational initiator factor 3 (eIF3) complexes. The CSN5 subunit of the COP9 signalosome is responsible for the E3 ligase Cdc53/Cul1 activity through the removal of the ubiquitin-like protein, Rub1. Deletion of the Csn5 gene is lethal in high eukaryotes but not in yeast, this observation allow us to study the effects of this mutation in this organism (strain Δcsn5) together with other mutants or double mutants: rpn11-m1, Δrub1, rpn11-m1/Δcsn5 rpn11-m1/ Δrub1. Mutants and wildtype (W303-1A) were characterised regarding growth in different carbon sources and temperature as well as response to stress or DNA damage causing agents (methyl methanesulfonate and canavanin). The morphological results allowed us to investigate authophagy, and in particular mitophagy, through fluorescence microscopy (GFP-Atg8 and GFP-Atg32) and Western Blot analysis. We found a relation between deubiquitination undertaken by Rpn11 protein, from the 19S proteasome subunit, and the activation of rubylation/derubylation cycles by the CSN5 subunit of the CSN complex (COP9 signalosome). In fact, the rpn11-m1/ Δrub1 shows a semi-lethal phenotype and mitophagy in exponential phase in glucose rich medium. Also the Δcsn5 strain shows early mitophagy together with phenotypic changes, such as big vacuoles. In addition, it has been established a possible relationship between the CSN complex and the resilience to damage in the DNA caused by the methylating agent, methyl methanesulfonate (MMS).
O objectivo deste trabalho centrou-se no estudo das alterações fenotípicas e ao nível da morfologia mitocondrial em células de levedura Saccharomyces cerevisiae com mutações específicas em genes envolvidos na via de degradação proteica ubiquitinaproteasoma. O turnover proteíco é muito importante pois garante a viabilidade dos vários organelos celulares, de entre os quais, a mitochondria, cuja função principal é a produção de energia na forma de ATP. A subunidade Csn5 do COP9 signalosome, complexo com elevada similaridade com a lid proteasomal e com o factor 3 de iniciação translacional em eucariotas (eIF3), é responsável pela actividade da E3 ligase Cdc53/Cul1 através da remoção da proteina similar à ubiquitina, Rub1. A delação do gene que codifica para a subunidade Csn5 é letal em eucariotas superiores mas não em levedura o que nos permite estudar os seus efeitos juntamente com outros mutantes: rpn11-m1, Δrub1, rpn11-m1/Δcsn5 rpn11-m1/ Δrub1. Mutantes e wild-type (W303-1A) foram caracterizados a nível de crescimento em diferentes fontes de carbono e a diferentes temperaturas, assim como à resposta a factores causadores de dano ao nível do DNA e síntese proteica (sulfonato de metil metano e canavanina) juntamente com uma análise do potencial de membrana mitochondrial, autofagia/mitofagia através de microscopia de fluorescencia (GFP-Atg8 e GFP-Atg32) e Western Blot. Os resultados obtidos indicam que existe uma relação entre a acção de deubiquitinação da proteina Rpn11, da subunidade 19S do proteasome, e a activação dos ciclos de rubilação/ derubilação pela subunidade Csn5 do complex CSN (COP9 signalosome), sendo que o mutante rpn11-m1/Δrub1 apresenta um fenótipo semi-letal com instabilidade ao nível do DNA e alterações mitocôndriais que levam a um mitofagia em fase exponencial em meio rico em glucose. Por sua vez, o mutante rpn11-m1/Δcsn5 também revela mitofagia prematura em conjunto com alterações fenotípicas, como o aumento da dimensão celular (grande vacúolo), que ja é também evidente no mutante Δcsn5. Foi ainda estabelecida uma possível relação entre o complex CSN e a capacidade de resistência aos danos causados no DNA pelo agente metilante MMS.

Mitochondrial function is critical for the survival of eukaryotes. Hence, mitochondrial dysfunctions are involved in numerous human diseases. An essential process for a normal mitochondrial function is mitochondrial gene expression which is tightly regulated in response to various physiological changes. The accurate control of mitochondrial gene expression is essential in order to provide the appropriate oxidative phosphorylation capacity for diverse metabolic demands. Recent findings in the basic mitochondrial replication and transcription regulation helped advance our understanding of organelle function and basic pathogenetic mechanisms of mitochondrial DNA mutations associated with oxidative phosphorylation defects. Mitochondrial transcription is regulated by the mitochondrial transcription termination factor (mTERF) both at the initiation and termination levels. A protein family containing highly conserved mTERF motifs has been identified recently and its members named generically as "terfins." In this work, one of these factors, mTERFD3, has been characterized in vitro and in vivo. The mTERFD3 protein is highly conserved throughout evolution. It is a mitochondrial protein localized to the matrix and is abundantly expressed in high energy demand tissues. We found that it contains 4 putative leucine zippers and is able to form dimers in vitro. We showed that mTERFD3 binds mtDNA at the transcription initiation site in the mtDNA regulatory region. These findings suggest that mTERFD3 may be involved in regulating mitochondrial gene expression at the transcriptional initiation level. In order to study the functional significance of mTERFD3 in vivo we developed a mouse deficient in mTERFD3 using a gene trapping strategy. The KO mice had a normal lifespan but showed decreased weight gain and decreased fat content in females. Fibroblasts isolated from KO mice displayed decreased growth rate when compared with WT in respiratory media, and had decreased complex IV activity. Consistent with the above findings, we found that muscle, one of the tissues with high energy demands, showed abnormal mitochondrial function, displaying features characteristic of mitochondrial myopathy such as decreased muscle strength and endurance. Muscle mitochondria of the KO mice showed a significant decrease in the complex II +III and complex IV activity. The decrease in OXPHOS complexes activity was associated with increased citrate synthase activity, suggesting mitochondrial proliferation, a feature typical for mitochondrial disorders. Another important finding was a decrease in the muscle mitochondrial transcripts in the KO animals associated with decreased steady state levels of OXPHOS subunits. Together these data suggest that mTERFD3 is a mitochondrial protein involved in the regulation of mtDNA transcription. mTERFD3 KO is not embryonic lethal suggesting that it is involved in the fine tuning of mitochondrial transcription. We conclude that mTERFD3 is a mitochondrial protein that modulates oxidative phosphorylation function, probably by directed interactions with the mtDNA regulatory region. This work shows the importance of mTERFD3, an mTERF family member, in the mitochondrial gene expression regulation.

Thesis (Ph. D.)--University of Oregon, 2004.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 113-119). Also available for download via the World Wide Web; free to University of Oregon users.

[EN] Post-transcriptional modification of the wobble uridine (U34) of a tRNA set is an evolutionary conserved process, produced by homologous proteins from the MnmA/MTU1, MnmE/GTPBP3 and MnmG/MTO1 families. Mutations in the human genes MTU1 and GTPBP3 or MTO1 produce acute infantile liver failure, and hypertrophic cardiomyopathy and lactic acidosis, respectively, which usually cause lethality in the first months of life. It is assumed that the primary cause of these diseases is the lack of the modifications introduced by the MTU1 protein in position 2 (tiol) and GTPBP3 and MTO1 proteins (taurinomethylation) in position 5 at U34 in a subgroup of mt-tRNAs. Nevertheless, the molecular mechanisms underlying these diseases (and other diseases associated with such modifications) are not clear. The reason why the typical defects of oxidative phosphorylation (due to impaired mitochondrial translation) produce such wide range of phenotypes is still unknown. Our hypothesis sustains that the mitochondria-nucleus retrograde signaling pathways triggered by the hypomodification at position 2 and 5 of U34 are different, and that each nuclear response is modulated by the genetic and epigenetic programs of cells and organisms. In this work, we have used the nematode Caenorhabditis elegans as a model organism to study the effects of inactivating the homologue proteins to MTU1, GTPBP3 and MTO1, which we have named as MTTU-1, MTCU-1 and MTCU-2, respectively. We have proved that these nuclear encoded proteins are located in mitochondria and are involved in U34 modification of mt-tRNAs. The mtcu-1 and mtcu-2 mutants show a reduction in fertility, while the mttu-1 mutant shows a reduction in fertility and a lengthening of the reproductive cycle (both phenotypes are thermosensitive). The phenotypes exhibited by the mttu-1, mtcu-1 and mtcu-2 mutants support our hypothesis, in which the mttu-1 single mutation, on the one hand, and the mtcu-1 and mtcu-2 single mutations, on the other hand, trigger different retrograde signaling pathways which produce specific nucear expression. Thus, a nuclear dependent phenotypic trait (as transcription or mt-tRNAs stability) and the expression of nuclear genes as ucp-4, hsp-6, hsp-60 and other genes involved in mitochondrial metabolism show a differential pattern in both group of mutants. hsp-6 and hsp-60 genes (UPRmt markers) are downregulated in mttu-1 single mutant, which could be related to fertility and reproductive cycle thermosensitivity. The three single mutants exhibit reduced expression of glycolysis and ß-oxidation genes (usually more drastic in the mttu-1 mutant), an induction of a glutaminolysis marker, and an induction of the ucp-4 gene, which encodes a transporter of the succinate to the mitochondria. Due to all three single mutants display a mild OXPHOS dysfunction, we propose that the observed changes in the expression of genes involved in the mitochondrial metabolism reveal a TCA cycle reprogramming aimed to compensate the reduction of acetil-CoA (coming from glycolysis and fatty acid oxidation) though the activation of anaplerotic pathways characterized by the succinate import to mitochondria by UCP-4 and the incorporation of 2-oxoglurate from glutaminolysis. We also analyze the effects of the simultanous suppression of modifications at positions 2 and 5 of U34 in C. elegans. The double mutant mtcu-2;mttu-1 displayed a severe OXPHOS dysfunction and a 5-fold higher AMP/ATP ratio, which was associated with embryonic lethality, developmental arrest in primary larval stages, penetrant sterility in adults and extended lifespan. This lifespan extension is modulated by signaling pathways which depend on AMPK (specifically on AAK-1 catalitic subunit) and steroid hormones, through DAF-9 and DAF-12 proteins. This work shows the important gene reprogramming related to mitochondrial metabolism in response to U34 hypomodification of mt-tRNAs, and shows new connexions between signaling pathways that extend lifespan.
[ES] La modificación post-transcripcional de la uridina de tambaleo (U34) de ciertos tRNAs es un proceso conservado evolutivamente, realizado por proteínas homólogas de las familias MnmA/MTU1, MnmE/GTPBP3 y MnmG/MTO1, y biológicamente relevante. De hecho, mutaciones en los genes humanos MTU1 y GTPBP3 o MTO1 causan fallo hepático infantil agudo y cardiomiopatía hipertrófica infantil, respectivamente, que producen letalidad durante los primeros meses de vida. Se asume que la causa primaria de estas enfermedades es la ausencia de las modificaciones introducidas por la proteína MTU1 en la posición 2 (tiol) y las proteínas GTPBP3 y MTO1 (taurinometil) en la posición 5 de la U34 en un grupo de mt-tRNAs. Se desconocen los mecanismos subyacentes y las razones por las que el déficit de OXPHOS resultante en todos los casos (atribuido a alteraciones de la traducción mitocondrial de proteínas) produce fenotipos tan diversos. Nuestra hipótesis es que la señalización retrógrada mitocondria-núcleo promovida por la hipomodificación de los mt-tRNAs en 2 ó 5 de la U34 es diferente y la respuesta nuclear viene modulada por el programa genético y epigenético de células y organismos. Hemos utilizado el nematodo C. elegans como modelo para estudiar los efectos producidos por la inactivación de las proteínas homólogas de MTU1, GTPBP3 y MTO1 a las que hemos denominado MTTU-1, MTCU-1 y MTCU-2. Hemos comprobado que estas proteínas, codificadas por el núcleo, son de localización mitocondrial y están implicadas en la modificación de la U34 de los mt-tRNAs. Los mutantes mtcu-1 y mtcu-2 presentan una reducción en su fertilidad y, en el caso del mutante simple mttu-1, fenotipos asociados a termosensibilidad. Los fenotipos exhibidos por los mutantes mttu-1, mtcu-1 y mtcu-2 sustentan la hipótesis de que la mutación mttu-1, y las mutaciones mtcu-1 y mtcu-2 promueven señales retrógradas diferentes que producen patrones de expresión nuclear específicos. Así, un rasgo fenotípico dependiente de genes nucleares (como lo es la transcripción y/o estabilidad de los mt-tRNAs) y la expresión de genes nucleares como ucp-4, hsp-6, hsp-60 y otros implicados en el metabolismo mitocondrial muestran un patrón diferente en los dos grupos de mutantes. Los genes hsp-6 y hsp-60 (marcadores de la UPRmt) están regulados a la baja en el mutante mttu-1. Los tres mutantes simples exhiben una reducción en la expresión de genes de la glicólisis y de la ß-oxidación de los ácidos grasos, una inducción en un marcador de glutaminolisis y una inducción en el gen ucp-4 (mayor en mttu-1) implicado en el transporte de succinato a la mitocondria. Dado que los tres mutantes simples presentan una disfunción OXPHOS relativamente suave, proponemos que los cambios de expresión en genes que modulan el metabolismo mitocondrial revelan una reprogramación del ciclo del TCA que compensa la disminución en el aporte de acetil-CoA procedente de glicólisis y oxidación de ácidos grasos con la activación de rutas anapleróticas del ciclo del TCA (importe de succinato a la mitocondria por UCP-4 y aporte de ¿-cetoglutarato procedente de la glutaminolisis). También analizamos los efectos de la anulación simultánea de las modificaciones en las posiciones 2 y 5 de la U34. El doble mutante mttu-1;mtcu-2 presenta una disfunción OXPHOS severa, con una ratio AMP/ATP 5 veces superior al control, que resulta en letalidad embrionaria, detención del desarrollo en estadios larvarios tempranos y esterilidad completa en los adultos que presentan, por otra parte, una longevidad unas dos veces superior a la cepa control. Este incremento de la longevidad está modulado por rutas de señalización que dependen de la subunidad catalítica AAK-1 (AMPK), y de hormonas esteroideas (proteínas DAF-9 y DAF-12). El trabajo muestra la importante reprogramación de genes relacionados con el metabolismo mitocondrial en respuesta a la hipomodificación de la U34 de los mt-tRNAs y
[CAT] La modificació post-transcripcional de la uridina de balanceig (U34) de certs tRNAs és un procés conservat evolutivament realitzat per proteïnes homòlogues a les de les famílies MnmA/MTU1, MnmE/GTPBP3 i MnmG/MTO1 i biològicament relevant. De fet, mutacions en els gens humans MTU1 i GTPBP3 o MTO1 causen fallada hepàtica infantil aguda i cardiomiopatia hipertròfica infantil amb acidosis làctica, respectivament, que produïxen letalitat durant els primers mesos de vida. S'assumix que la causa primària d'aquestes malalties és l'absència de les modificacions introduïdes per la proteïna MTU1 a la posició 2 (tiol) i per les proteïnes GTPBP3 i MTO1 (taurinometil) a la posició 5 de la U34 en un grup de mt-tRNAs. Es desconeixen els mecanismes subjacents en estes malalties i les raons per les quals el dèficit de la OXPHOS resultant en tots els casos (atribuït a alteracions de la traducció mitocondrial de proteïnes) produïx fenotips tan diversos. La nostra hipòtesi és que la senyalització retrògrada mitocondria-nucli promoguda per la hipomodificació dels mt-tRNAs en 2 o 5 de la U34 és diferent i la resposta nuclear en cada cas es dependent del programa genètic i epigenètic de cèl¿lules i organismes. Hem utilitzat el nematode C. elegans com a organisme model per a estudiar els efectes produïts per la inactivació de les proteïnes homòlogues de MTU1, GTPBP3 i MTO1 a les que hem denominat MTTU-1, MTCU-1 i MTCU-2. Hem comprovat que aquestes proteïnes, codificades pel nucli, són de localització mitocondrial i estan implicades en la modificació de la U34 dels mt-tRNAs. Els mutants mtcu-1 i mtcu-2 presenten una reducció en la seua fertilitat i, en el cas del mutant mttu-1, fenotipus associats a termosensibilitat. Els fenotipus exhibits pels mutants mttu-1, mtcu-1 i mtcu-2 sustenten la hipòtesi que la mutació mttu-1, i les mutacions mtcu-1 i mtcu-2 promouen senyals retrògrads diferents que produïxen patrons d'expressió nuclears específics. Així, un tret fenotípic dependent de gens nuclears (com ho és la transcripció i/o l'estabilitat dels mt-tRNAs) i l'expressió de gens nuclears com ucp-4, hsp-6, hsp-60 i altres implicats en el metabolisme mitocondrial mostren un patró diferent en els dos grups de mutants. Els gens hsp-6 i hsp-60 (marcadors de la UPRmt) estan regulats a la baixa en el mutant mttu-1. Els tres mutants simples exhibixen una reducció en l'expressió de gens de la glicòlisi i de la ß-oxidació dels àcids grassos, una inducció en un marcador de glutaminolisi i una inducció en el gen ucp-4 (major en el mutant mttu-1) implicat en el transport de succinat a la mitocondria. Atés que els tres mutants simples presenten una disfunció OXPHOS relativament suau, proposem que els canvis d'expressió en gens que modulen el metabolisme mitocondrial revelen una reprogramació del cicle del TCA que compensa la disminució en l'aportació d'acetil-CoA procedent de la glicòlisi i de l'oxidació d'àcids grassos amb l'activació de rutes anaplerótiques del cicle del TCA (importació de succinat a la mitocondria per UCP-4 i aportació de ¿-cetoglutarat de la glutaminolisi). També s'analitzen els efectes de l'anul¿lació simultània de les modificacions en 2 i 5 de la U34. El doble mutant mttu-1;mtcu-2 presenta una disfunció OXPHOS severa, amb una ràtio AMP/ATP 5 vegades superior al control, que resulta en letalitat embrionària, detenció del desenvolupament en estadis larvaris primerencs, esterilitat completa en els adults i una longevitat unes 2 vegades superior al control. Aquest increment de la longevitat està modulat per rutes de senyalització que depenen de la subunitat catalítica AAK-1 (AMPK), i d'hormones esteroidees (a través de les proteïnes DAF-9 i DAF-12). En resum, aquest treball mostra per primera vegada a nivell d'un animal model la important reprogramació de gens relacionats amb el metabolisme mitocondrial en resposta a la hipomodificació de la U34 dels mt-tRNAs i
Navarro González, MDC. (2016). Caenorhabditis elegans as a research tool to study mitochondrial diseases associated with defects in tRNA modification [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/61978
TESIS

Heart disease is a complex and heterogeneous disease. Notably, studies have demonstrated gender differences in the expression and types of cardiovascular disease, such as dilated cardiomyopathy (DCM), a major underlying cause of heart failure. Previously we showed that loss of A-Kinase Anchoring Protein 13 (Akap13), a unique proto-oncogene and estrogen receptor modulator, resulted in enlarged embryonic hearts, defective cardiac sarcomere formation, and embryonic lethality in mice. Data have also shown cAMP-dependent Protein Kinase A (PKA) to be involved in DCM pathophysiology. Given the established role of AKAP13 in cell signaling, its ability to bind and modulate ligand-activated nuclear hormone receptors and transcription factors, and its association with actin and other cytoskeletal components, we hypothesized that a functional AKAP13 protein was required for cardiomyocyte function in the adult heart; defective function of AKAP13 could promote DCM. To this end, we established an inducible, cardiac-specific Akap13 conditional knockout (Akap13cKO) mouse model using a Cre-lox recombination strategy with two separate Cre-recombinase expressing mouse models (α-MHC-MerCreMer and Tnnt2-rtTA; TetO-Cre). Cardiac functional examination of Akap13cKO mice revealed significant biventricular dilated cardiomyopathy with compensatory hypertrophic remodeling of the left ventricle and left atrial enlargement, decreased left and right ventricular systolic function, and abnormal left ventricular diastolic function. Of note, female Akap13cKO mice displayed a more pronounced cardiac phenotype and were more likely to die post-recombination.

Valproic Acid Promotes Acetylation of Superoxide Dismutase-2 During Neurogenesis. Valproic acid (VPA) is a known developmental toxicant associated with a high prevalence of neural tube defects (NTD). The mechanism of VPA-induced NTD is unclear, but oxidative stress may be implicated. To understand how embryotoxic oxidative stress may occur, we measured superoxide dismutase (SOD) activity following VPA treatment in the embryonic pluripotent P19 mouse carcinoma cell line. In undifferentiated P19 cultures treated with VPA (5 mM), dichlorofluorescein fluorescence increased 15% compared to untreated controls over 20 min, indicating a modest, yet statistically significant increase in ROS generation. Undifferentiated P19 cells were treated with VPA for 6 h, after which total SOD and mitochondrial SOD (SOD2) activities were measured. VPA treatment decreased total SOD activity by approximately 20% but SOD2 activity was undetectable; but this was not a consequence of changes to SOD (SOD1 or SOD2) protein concentrations. Interestingly, glutathione redox state increased from -262 mV to -245 mV after a 6 h treatment with VPA, indicating significant oxidation of the cellular redox environment. Measurement of mitochondrial superoxide levels showed an increase following VPA treatments. While it is unlikely that VPA works directly as an oxidant, these data suggest that VPA may promote oxidative stress through an alternative means, such as via the inhibition of SOD activity and thus, allow for an increase in ROS. Importantly, VPA is a known deacetylase inhibitor, and SOD2 function is regulated by acetylation. As such, we evaluated the acetylation state of SOD2 to determine potential disruption via acetylation. Treated undifferentiated P19 cells showed a significant increase in SOD2 acetylation. However, in fully differentiated P19-derived neurons, cells showed no such SOD2 acetylation. Additionally, pretreatment with dithiole-3-thione (D3T), a Nrf2 activator of the antioxidant response, attenuated VPA-induced mitochondrial ROS production and SOD2 acetylation and improved SOD2 activity, suggesting Nrf2 as a potential means to reduce VPA-mediated oxidative stress. To evaluate the effects in the embryo proper, gestational day 8 mouse embryos were treated with VPA in culture for 6 h. Similar to P19 cells, VPA-treated neurulating embryos showed significant SOD2 acetylation and a concomitant decrease in total SOD activity. These data support a similar consequence of VPA-induced oxidative stress in embryos as is demonstrated in our cellular model. Since no SOD2 acetylation is observed in differentiated neurons and VPAinduced SOD2 acetylation occurs more prevalently in undifferentiated/differentiating cells, these data purport means by which VPA preferentially induces oxidative stress in developing systems.

Despite the importance of muscle in the development of type 2 diabetes, few studies have investigated the effect of polychlorinated biphenyls (PCBs) on muscle energy metabolism. Previous results from our lab suggested that PCB126 exposure induced an indirect negative effect on muscle mitochondrial function. Since PCBs are stored in adipose tissue, we hypothesized that PCB126 alters adipokine secretion which in turn affects muscle metabolism. Objectives. Study the adipose-to-muscle communication in PCB126-induced metabolic defects. Methods. Communication between adipocytes and myotubes was reproduced by exposing C2C12 or mouse primary myotubes to the conditioned medium (CM) of 3T3L1 adipocytes exposed to environmentally relevant PCB126 levels. Results. PCB126 significantly increased adipokine secretion and decreased mitochondrial function, glucose uptake and glycolysis in insulin resistant (IR) but not in insulin sensitive 3T3L1. However, exposure of myotubes to CM of IR 3T3L1 only decreased glucose uptake and insulin sensitivity, without altering myotubes glycolysis or mitochondrial function. Conclusion. Our results suggest that the increased adipokine secretion by adipocytes could explain the decreased muscle glucose uptake and insulin sensitivity when exposed to PCB126.

Mitochondria are the main sites for adenosine triphosphate (ATP) generation within most cells. Structural and functional alterations of mitochondria due to genetic abnormalities of mitochondria can cause respiratory chain dysfunction. In this study, the important role of mitochondria in energy metabolism was determined by comparing the effect of mitochondrial DNA (mtDNA) mutations on growth patterns and oxidative phosphorylation (OXPHOS) enzyme activities of six isolated clones (B5, B12, D4, D9, E1 and E8); as well as the effect of ATP supplement to culture using the slowest growing clone. The isolated clones had shown distinct growth pattern and morphology. The difference in proliferation rates among the clones was ascertained by the doubling times (B5=26.4h. B12=43.2h. D4=25.7h. D9=33.6h. E1=26.9h and E8=28.8h). The clone's slow growth rate was likely the result of mitochondrial mutations in the 16S rRNA gene, ND1, ND4, ND6 and COX III. Five heteroplasmic mutations were found in clone B12 (G2480T, C2513G, A2520T, C9527T and C14263G), one heteroplasmic mutation in clone D9 (A4137G) and one homoplasmic mutation in clone D4 (C11496). The mutations in clone B12 appeared to be deleterious to the cell by disrupting mitochondrial OXPHOS activities and reducing energy output. Additionally, extracellular ATP supplement to OXPHOS deficient clone B12 facilitated cell growth and enhances the gene expression. Increased expression of mtDNA-encoded respiratory chain complexes observed in clone B12 compared to clone D4 may reflect mitochondrial genomic adaptation to perturbations in cellular energy requirements. The stimulation of mitochondrial biogenesis may be a cellular response in compensation for defects in OXPHOS associated with mtDNA mutations. My data support the hypothesis that the variability in functional manifestations of mtDNA is attributed to the nature of the mutation, number of mutation and the gene specifically affected. These results will help to further our understanding of the relationship between mitochondrial mutation and cellular function.

Mutações em genes da biologia dos telômeros, causando o seu encurtamento, são as bases moleculares de um grupo heterogêneo de doenças denominadas telomeropatias. O protótipo das telomeropatias é a disceratose congênita (DC), uma falência de medula óssea, caracterizada por sinais mucocutâneos e anemia aplástica (AA). Além da DC e AA, a fibrose pulmonar (FP) e a cirrose hepática (CH) também fazem parte do espectro das telomeropatias. Como pulmão e fígado são órgãos com baixa taxa proliferativa, suspeita-se que existe outros componentes celulares interagindo com os telômeros para o estabelecimento dessas doenças. Diferentes abordagens vêm estabelecendo uma relação entre e a biologia dos telômeros e as mitocôndrias. No entanto, ainda não se sabia sobre o funcionamento mitocondrial em células primárias de pacientes com telomeropatias. No nosso estudo, utilizamos fibroblastos dermais de indivíduos saudáveis (n=4) e de pacientes (n=6), diagnosticados com diferentes telomeropatias (AA, DC, FP e CH) e telômeros abaixo do 10º percentil (curtos para a idade). Ao avaliarmos parâmetros mitocondriais, observamos um fenótipo senescente, nas células dos pacientes, que refletiu num aumento na massa mitocondrial (85%), no número de copias de DNA mitocondrial, no consumo de oxigênio (71%) e na produção de superóxidos (74%), precursor das espécies reativas de oxigênio (EROs) mitocondriais. O superóxido levou a um aumento na expressão de antioxidantes, como a SOD1 e a UCP1. Dessa maneira, o estresse oxidativo gerado pelas EROs mitocondriais parece ter um papel fundamental na patogênese das telomeropatias. Além disso, sequenciamos amostras de sangue periférico de outros 72 pacientes com falência medular, com telômeros curtos e normais, para compararmos a taxa de variantes somáticas entre os grupos. Observamos que os pacientes com falência medular e telômeros curtos apresentaram maiores frequências de variantes somáticas. Supomos que o aumento da taxa de variantes somáticas possa ser consequência do desequilíbrio redox, observado nas células dos pacientes, causando danos no DNA das células-tronco hematopoéticas.Estudos como esse podem basear discussões sobre o uso de terapias antioxidantes em pacientes com telomeropatias
Mutations in telomere-related genes are the molecular basis of a phenotypically heterogeneous group of disorders that are collectively termed telomeropathies. The prototype of telomeropathies is the dyskeratosis congenita (DC), an inherited bone marrow failure characterized by mucocutaneous stigmata and aplastic anemia (AA). In murine telomerase knockout models, telomere shortening provokes mitochondrial deficiency and increases reactive oxygen species (ROS) production. However, the mitochondrial function in human telomeropathies has not been addressed. We evaluated mitochondrial parameters in fibroblasts from four healthy individuals (controls) and six patients with inherent bone marrow failure (DC and AA), carrying pathogenic variants in TERC, DKC1, RTEL1 and POT1 genes and, consequently, telomere shortening (<10th percentile). Patient fibroblasts displayed an 85% increment in mitochondrial mass, resulting in a 71% increase in oxygen consumption in the state of maximum respiration (ETS) compared to controls. As a consequence, mitochondrial ROS production was 74% higher in patients\' fibroblasts than in controls. Increased ROS level may explain the overexpression of SOD1 and UCP1 observed in patient cells. We further assessed the mitochondrial DNA (mtDNA) copy number in fibroblasts and peripheral blood of patients with telomere shortening. The mtDNA content was significantly higher in patients compared to controls. These findings indicate that mitochondria are affected in human telomere diseases and may play a role in disease development. Furthermore, overproduction of mitochondrial ROS could induce oxidative stress and result in somatic mutations in hematopoietic stem-cells, causing clonal disorders in patients with telomeropathies

Causé par un parasite du genre Plasmodium transmis par les moustiques, le paludisme est encore aujourd’hui un fléau causant des centaines de milliers de morts tous les ans. Le développement de nouvelles molécules antipaludiques et/ou l’amélioration des molécules déjà présentes sur le marché est une urgence sanitaire. Des molécules pouvant cibler tous les stades du cycle de vie du parasite seraient idéales. Ces molécules permettraient le traitement symptomatique et la prévention des rechutes mais aussi empêcheraient la transmission de la maladie. Dans ce travail, j’ai étudié le mécanisme d’action de deux molécules à activité antipaludique : la plasmodione et le proguanil. La plasmodione est en cours de développement et cible le parasite à la fois au stade sanguin et sous sa forme gamétocyte responsable de la transmission de l’homme au moustique. Le proguanil est une molécule déjà sur le marché depuis longtemps mais dont le mode d’action n’est pas encore compris bien qu’il soit un excellent partenaire synergique de l’antipaludique atovaquone, un inhibiteur du complexe bc1 de la chaine respiratoire mitochondriale. Pour cette étude, à l’aide du modèle levure, j’ai utilisé une approche génétique (construction, sélection et analyse de mutants) et biochimique (test d’activité enzymatique) afin de décrypter le mode d’action de ces molécules. Les résultats obtenus indiquent que la plasmodione est une ‘pro-drogue’ qui est transformée en métabolites actifs qui entrent ensuite dans un cycle de réactions d’oxydo-réduction (redox) produisant des espèces réactives de l’oxygène (ROS). Le stress oxydant qui en résulte provoque l’arrêt de croissance. Ces résultats sont en accord avec les données venant des études avec le parasite. J’ai montré que des flavoprotéines mitochondriales, principalement de la chaine respiratoire et du cycle de Krebs, jouent un rôle clé dans l’activité de la plasmodione. Ainsi les NADH déshydrogénases catalysent les réactions redox en utilisant les métabolites actifs de la plasmodione comme substrats aboutissant à une production de ROS. La succinate-déshydrogénase pourrait être impliquée dans la transformation de la plasmodione en métabolites actifs. Le fonctionnement de ces enzymes mitochondriales est donc requis pour que l’activité inhibitrice de la plasmodione soit observée. La mitochondrie est aussi un élément clé dans le mode d’action du proguanil. Dans une étude publiée précédemment, les auteurs avaient émis l’hypothèse que le proguanil ciblerait un système enzymatique mitochondrial participant au maintien du potentiel membranaire. Mais les résultats que j’ai obtenus ici ne semblent pas valider ce modèle. Ils conduisent plutôt à l’hypothèse que le proguanil s’accumulerait dans la mitochondrie, peut-être à de fortes concentrations. Cette accumulation affecterait des fonctions mitochondriales essentielles, résultant dans la mort de la cellule. De nombreuses questions restent ouvertes et plus de travail sera requis pour décrypter le mode d’action du proguanil
Malaria is caused by the Plasmodium parasite transmitted by mosquitoes. The disease remains today a major health issue with hundreds of thousands deaths every year. The development of new antimalarial compounds and/or the improvement of already available drugs is urgently needed. Compounds that could target all the stages of the life cycle of the parasite would be the best. Such drugs could be used in symptomatic treatment, for relapse prevention but also to block the transmission of the disease. In this work, I studied the mode of action of two antimalarial compounds, namely plasmodione and proguanil. Plasmodione is a new lead compound that targets the parasite both at the blood stage and at the gametocyte stage responsible for the transmission from human to mosquito. Proguanil has been in use for many years. Yet its mode of action is not understood although it is known to be an efficient synergistic partner of the antimalarial atovaquone that targets the mitochondrial respiratory chain bc1 complex. I used yeast as a model organism and an approach of genetics (construction, selection and analysis of mutants) and biochemistry (enzymatic activity tests) in order to decipher the mode of action of these compounds. The results indicate that plasmodione is a pro-drug transformed into active metabolites that then enter in a cycle of oxido-reductions (redox) producing reactive oxygen species (ROS). The resulting oxidative stress causes growth arrest. These data are in agreement with results obtained with the parasite. I showed that mitochondrial flavoproteins, mainly of the respiratory chain and Krebs cycle, play a key role in plasmodione activity. The NADH-dehydrogenases catalyse the redox reactions using the active plasmodione metabolites as substrates, leading to ROS production. The succinate-dehydrogenase is likely to be involved in the transformation of plasmodione in its active metabolites. Thus the functioning of the mitochondrial enzymes are required for the activity of plasmodione. The mitochondria is also a key element in the mode of action of proguanil. In a previously published report, the authors had hypothesized that proguanil would target a mitochondrial enzymatic system involved in the generation of the membrane potential. However the data I obtained do not seem to validate that hypothesis. They lead rather to the following hypothesis: proguanil would accumulate in the mitochondria, probably to high concentrations, which would affect essential mitochondrial functions leading to cell death. A number of questions remain open and more work are needed to uncover proguanil mode of action

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1.
També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut.
Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation.
We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.

Thesis (M. Sc.)--York University, 2001. Graduate Programme in Kinesiology and Health Science.
Typescript. Includes bibliographical references (leaves 84-88). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ66403.

碩士
長庚大學
生物醫學研究所
97
Mitochondrial complex I is a multi-subunit of inner mitochondrial membrane protein that serves as a gateway for electrons transferred from NADH to molecular oxygen through the RC (respiratory chain). In this study, short and long term effects of rotenone are investigated in RBA1. Short-term rotenone (500 nM) exposure enhanced Ca2+ stress-induced apoptosis upon ionomycin (5 µM) and arachidonic acid (50 µM) exposure but not oxidative stress-induced apoptosis upon H2O2 (10 mM) exposure which suggest its primary enhancement of Ca2+-inudced apoptosis. Rotenone alone (500 nM) induced greatly cCa2+ and mCa2+ stresses. At higher concentration (1 µM), rotenone diminished mitochondrial Ca2+ uptake possibly due to diminished △Ψm. In the presence of rotenone, ionomycin-induced cCa2+ and mCa2+ were further enhanced. Thus, rotenone enhanced ionomycin-induced cCa2+ and mCa2+ stresses. RT-RBA1 is RBA1 of long-term exposure to rotenone (20 nM 20 days). RT-RBA1 altered significantly mitochondrial dynamics for enhancing fission of mitochondria. At the same time, RT-RBA1 decrease mitochondrial mass and movement. Interestingly, vitamin E significantly prevented RT-RBA1 from enhancing apoptosis upon ionomycin exposure. Precise mitochondrial level of investigation showed that △Ψm was significantly depolarization for a reduced mCa2+ uptake during Ca2+ stress induced by ionomycin exposure. Nevertheless dose dependent mCa2+-induced ROS formation for an enhanced CL depletion was observed during ionomycin exposure in RT-RBA1. mCa2+-induced ROS formation upon Ca2+ stress is crucial for the enhanced depletion of cardiolipin in RT-RBA1. However, under Ca2+ free condition H2O2-induced CL depletion was found to be similar in RBA1 and RT-RBA1. RT-RBA1 significantly enhanced the opening of the mitochondrial permeability transition (MPT) and later apoptosis induced by ionomycin exposure. Finally, RT-RBA1 induced fission of mitochondria significantly reduced the propagation of mROS upon 561 nm laser irradiation possibly as a protective mechanism against oxidative stress. RT-RBA1, however, enhanced propagation of mCa2+ upon multiphoton 850 nm irradiation. Inhibition of fission in RT-RBA1 prevented mCa2+ propagation without altering the amount of mCa2+ upon 850 nm irradiation. It suggested fusion of mitochondria may protect against mitochondrial Ca2+. On the contrary, inhibition of fission enhanced cardiolipin depletion possibly mediated by an enhanced propagation of ROS. Thus, prevention of mCa2+-medated ROS formation may play a significant role in the future treatment of complex I defect associated pathological condition and neurodegeneration diseases including Parkinsonism and aging.

博士
國立成功大學
工程科學系碩博士班
100
Mitochondria are the energy production and metabolism centres of human and animal cells, which supply most of the energy for maintaining physiological functions and play an important role in the process of cell death. Meanwhile, cellular overproduction of reactive oxygen species and oxidative damage on biological molecules occur when mitochondrial functions decline, directly accelerating the alterations of mitochondrial DNA. Alterations of mitochondrial DNA have been reported to be strongly associated with mitochondrial dysfunction, mitochondria-related diseases, aging, and many important human diseases such as diabetes and cancers. Because it lacks an effective repair system, mitochondrial DNA suffers much higher oxidative damage and usually harbours more mutations than nuclear DNA. Molecular defects in mitochondrial DNA significantly contribute to a wide variety of mitochondrial diseases. Both qualitative and quantitative mitochondrial DNA defects affect clinical presentation and the severity of the diseases due to variations in the mitochondrial dysfunction profiles. As a result, the same mitochondrial DNA defect may present different severity in different organs or tissues and cumulatively contribute the overall clinical symptoms and treatment options. Therefore, quantification of different mitochondrial DNA defects is important for the diagnosis and treatment plan of mitochondria diseases. Traditional protocols for assess mitochondrial DNA involve micro-cloning, direct sequencing, real-time polymerase chain reaction processing and microarray detection; all of which are time-consuming and labor-intensive processes. Recently, rapid development in microfluidic devices fabricated by micro electro mechanical systems technology has made substantial impact on miniaturization of biomedical devices. The micro electro mechanical systems technology is able to integrate all functional micro components in one single chip with advantages such as compactness, high sensitivity and rapid analysis. In this study, an extraction module of mitochondrial DNA was first designed that integrated micropumps, a micromixer and a micro temperature sensor in three-dimensional format to automate the entire process. The experimental results showed that the proposed microchip has higher extraction efficiency for mtDNA. The extraction times for the microchip and a commercial kit of mtDNA extraction were 50 minutes and 300 minutes, respectively. A mitochondrial DNA deletion detection system integrated with a mtDNA extraction module, a micro polymerase chain reaction module and a micro capillary electrophoresis module has further been developed. The experimental results showed the PCR module could provide a comparable amplification yield when compared to a conventional instrument. The deletion rate of the mtDNA in the samples can be further quantified by measuring the percentage between the amplicon representing for the deletion and that for the total mtDNA. Furthermore, a mitochondrial DNA mutation detection system including an extraction module, a micro polymerase chain reaction module, and a mutation detection module capable of precise quantitative measurements was developed in this study. Experimental results showed that a new quantitative detection system can be utilized for the analysis of a point mutation in mtDNA. The DNA detection module can detect the mtDNA mutation using restriction enzyme digestion. Compared to traditional methods, the new chip system demonstrates excellent mutation detection limit for small starting specimen amount and capable of both qualitative and quantitative analysis. Thus the integrated microfluidic systems harbor a great potential for fully automatic high-throughput mitochondrial DNA detection to augment future clinical diagnosis and management of mitochondria diseases.

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Biology.
Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR19698

The WWOX gene has been identified as the gene that spans the FRA16D common chromosomal fragile site (CFS), which is a frequent site of DNA instability in cancer. Perturbation of the WWOX gene has been reported in various cancers, with low WWOX levels correlating with poorer prognosis. Individuals who inherit a non-functional copy of WWOX have also been found to be at greater risk of developing cancer. WWOX has been implicated in various cellular pathways, however the role of WWOX in tumourigenesis is not yet fully defined. There is therefore a need to determine the normal function(s) of WWOX and how perturbation of these roles is likely to contribute to cancer. A model was previously established to examine the cellular function of the Drosophila orthologue, Wwox and to identify novel functional interactors. Loss of Wwox in Drosophila was not found to result in any obvious cellular dysfunction that manifested as a phenotype. The aim of this study was to identify the types of cellular dysfunction brought about by other genes that could be modulated by Wwox. As Wwox has previously been implicated in metabolic processes, particularly aerobic metabolism and redox homeostasis, an RNA interference (RNAi) screen was performed to identify the types of metabolic stress that can be modulated by altered Wwox levels. Wwox was found to regulate cellular homeostasis in cells with mitochondrial dysfunction, with a requirement for the active site of its shortchain dehydrogenase/reductase (SDR) enzyme. Other genetic effectors of the mitochondrial dysfunction were also identified as candidates for further investigation into the pathway(s) in which Wwox participates. The contributions of Wwox to two other models of cellular dysfunction were also examined. Wwox was found to have a role in a Drosophila model of intrinsic tumour suppression. In addition, Wwox was also shown to affect cells with chromosomal instability (CIN), with loss of Wwox resulting in oxidative stress, DNA damage and subsequently apoptosis of CIN cells. This study has identified roles for Wwox in three different novel models of cellular dysfunction. These findings provide further insight into the tumourigenic potential of WWOX and could contribute to the ultimate aim of designing therapeutics for treatment of cancers with low WWOX levels.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Molecular and Biomedical Science, 2015.

AAbbssttrraacctt:: The mammalian organism is entirely dependent on ATP production by oxidative phosphorylation system (OXPHOS) on the inner mitochondrial membrane. OXPHOS is composed of respiratory chain complexes I-IV, ATP synthase and also include two electron transporters cytochrome c and coenzyme Q. Disorders of mitochondrial energy metabolism caused by OXPHOS defects are characterized by extreme heterogeneity of clinical symptoms, variability of tissues affected and the severity of the defect at the level of individual tissues. The mitochondrial disorders are not always clearly expressed at the level of available tissue or most easily available cultured fibroblasts and/or currently available methods are not capable to detect the defects on the fibroblasts level. The aim of this master thesis was to identify by biochemical methods, especially by high sensitive polarography, OXPHOS disorders in cultured fibroblasts. Cell lines from 10 patients with isolated (SURF21, SCO1 ND1, ND5) or combined defects of OXPHOS complexes whose biochemical defect was confirmed in muscle tissue as well as 14 patients with non- mitochondrial diseases (8 patients with Huntington disease, 6 patients with disorder of sulphur amino acids metabolism) were analysed. Furthermore impact of various cultivation conditions on OXPHOS...

The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....

碩士
長庚大學
基礎醫學研究所
93
Pathogenic mutation of mitochondrial DNA (mtDNA) is often fatal to cells and can cause a large variety of human mitochondrial diseases. Precise mechanisms by which mtDNA mutation results in mitochondrial dysfunction and cell death, however, remain unclear. Two commonly seen mitochondrial DNA deletion and mutation are large scale mtDNA 4977 bp deletion (so called: “large-scale deletion” or “common deletion”, CD) and NADH dehydrogenase point mutation (ND) which each results in chronic progressive external ophthalmoplegia (CPEO) syndrome; Leber’s hereditary optic neuropathy (LHON); Dystonia and Parkinsonism, respectively. The precise mechanisms by which CD and ND mutation results in cellular and mitochondrial dysfunction remain unclear. This study, therefore, aims to investigate how particular mtDNA mutation alters mitochondrial function and activates subsequent apoptosis in established mitochondrial cybrids that harbor specific defect mtDNA from patients including CD and mitochondrial complex I defect. Functional visualization and comparison of individual live mitochondrion in intact mtDNA defect cybrids was studied by confocal laser scanning and time-lapse multi-photon imaging microscopy. Dynamic changes in mitochondrial reactive oxygen species (mROS) formation, mitochondrial membrane potentials (△Ψm) alteration and mitochondrial calcium([Ca2+]m) regulation was obtained for elucidating their relation to mitochondrial permeability transition pore opening, mitochondrial lethal proteins release and down streams apoptotic events activation. Both lines was conducted for experiments. Results of this study are devied into three parts: Part I: CD-augmented mitochondrial dysfunction and apoptosis upon stresses induced by H2O2 and Ca2+. CD dose-dependent enhances stress induced apoptosis (D cybrids > 50% >16% > W cybrids). Detail mechanistic investigation revealed that CD significantly enhanced mROS formation at resting which resulted in a more homogeneous ROS distribution in W cybrids and a much higher mROS gradient in D cybrids due to specific mROS formation. CD-augmented mROS formation was further enhanced upon H2O2 stress without threshold effect. In addition, this study reported for the first time that CD-induced mROS omits threshold effect indicating that small amount of CD (in this study 15%) is effective in causing pathological outcome of mROS formation and mitochondrial dysfunction. Similar results were found that CD significantly elevated resting [Ca2+]m in thread-like mitochondria as well as upon stresses induced by H2O2 and laser irradiation. CD also induced a significant depolarization in △Ψm that levels of resting △Ψm were found to be W cybrids > 16% > 50% > D cybrids (hyperpolarization) both at resting and upon stress. Finally, CD-induced augmentation of mitochondrial dysfunction upon H2O2 stress was protected by an angiotensin II antagonist, valsartan. Valsartan processes a potent inhibitory effect on mROS formation and △Ψm depolarization and down stream apoptosis induced by H2O2. Part II: ND-augmented mitochondrial dysfunction and apoptosis upon H2O2 stress. Similar to CD cybrids, although ND mutation did not cause morphological difference among three ND cybrids, ND5 and ND6 grew much faster than NDW (cell doubling time shifted from 37 hrs to 20 hrs). Intriguingly, although grew faster, ND mutation enhanced stress, including arachidonic acid (AA) and H2O2, -induced mitochondrial dysfunction and apoptosis. ND mutation also induced retardation in mitochondrial movement. Resting mitochondrial movement velocity (μm/min) decreased from 3.2 to around 2.7 (ND5) and 1.8 (ND6) and decreased further to 1.0 (NDW), 0.7 (ND5) and 0.5 (ND6). Mitochondrial membrane potential detected by JC-1 show not much difference among three cybrids. However, ND6 depolarized much faster than ND5 and then NDW cybrids. ND also enhanced mitochondrial ROS formation at resting as well as upon H2O2. Interesting, I observed heteroplasmic of mROS formation in ND5 cybrids including supersensitive ND5 (SS-ND5) and lowsensitive-ND5 (LS-ND5). Opening of the mitochondrial permeability transition pore (MPTP) was enhanced. ND mutation enhanced pore opening and triggered a faster release of mitochondrial lethal protein, Smac/DIABLO. Part III: Involvement of antioxidant, complex I, complex III, mitochondrial Ca2+ uniporter in H2O2-induced mitochondrial dysfunction in ND cybrids. Melatonin, a pineal gland secreted hormone, showed dose-dependent protective effect on H2O2-induced apoptosis. Mitochondrial electron transport chain complex inhibitor complex I, rotenone, reduced H2O2-induced mROS generation in all ND cybrids. Complex III inhibitor, antimycin A, however, enhanced H2O2-induced mROS generation in all ND cybrids. Antimycin A-enhanced mROS formation was partially reduced by rotenone. Finally, a mitochondrial Ca2+ uniporter inhibitor, Ru360, significantly reduced H2O2-induced mROS generation in all ND cybrids. The protective mechanisms indicate specific therapeutic approaches to the treatment of mitochondrial diseases associated with CD. The information acquired from this study will appreciably facilitate future therapeutic development for the prevention and treatment of mDNA mutation associated diseases.

碩士
長庚大學
生物醫學研究所
100
Mitochondrial complex II is a multi-subunit of inner mitochondrial membrane protein that serves as a gateway for electrons transferred from flavin adenine dinucleotide to molecular oxygen through the respiratory chain (RC). Recent evidences indicate that RC defects are closely associated by neurodegenerative diseases. For instance, Huntington disease (HD) is characterized with severe mitochondrial complex II inhibition. However, it remains largely unknown whether and how complex II dysfunction affects the regulation of mitochondrial and cellular stresses in astrocytes to lead to final pathology of HD. In this study, 3-nitropropionic acid (3-NP), an irreversible complex II inhibitor, was used to mimic HD. We investigated precisely the effects of 3-NP on dynamic change of mitochondrial membrane potential (ΔΨm), mitochondrial reactive oxygen species (mROS) formation, mitochondrial calcium (mCa2+) regulation and cardiolipin (CL) depletion in RBA-1 astrocytes. The results showed that high doses of 3-NP (10, 20 mM) induced cell death immediately and lower doses of 3-NP (2, 5 mM) dose-dependently induced ΔΨm depolarization and CL depletion. Interesting, 3-NP promoted depletion of CL, mitochondria fragmentation and inhibited 488nm laser irradiation induced oscillations of ΔΨm, suggesting its potential targeting on the transient mitochondrial permeability transition (MPT), a protective mode of MPT to crucially release overloaded mitochondria toxins such as mCa2+. Importantly, we observed that mCa2+ stress is superior to mROS stress during 3-NP-induced cell death in RBA-1 cells. Furthermore, we observed that long-term 3-NP treated RBA-1 were more sensitive to mCa2+ stress caused by ionomycin compared to wild type RBA-1 cells. These findings thus suggest that mCa2+-mediated CL dependent modulation of the t-MPT may play an important role in 3-NP-induced complex II inhibition associated apoptotic death of astrocytes and the pathogenesis of HD which may serve as a potential targeting for the treatment for HD and complex II defect associated diseases in the future.

碩士
國立清華大學
分子醫學研究所
106
NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) is a nuclear-encoded subunit of human mitochondrial complex I. It contains a binuclear iron sulfur cluster N1a, and may play a role in temporary storage of excess electrons to prevent free radical production. Defects of NDUFV2 have been associated with Alzheimer's disease and Parkinson's disease. Cell-penetrating peptide derived from HIV-1’s transactivator of transcription (TAT) has been successfully applied as a carrier to bring fusion proteins into cells by crossing plasma membranes without compromising the biological function of proteins. In this study, we tried to develop a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFV2 defects. Two fusion proteins (TAT-NDUFV2 and NDUFV2-TAT) were exogenously expressed and purified from E. coli for transduction of human cells. In addition, similar constructs were also generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFV2 and NDUFV2-TAT could be delivered into mitochondria and correctly assembled in complex I. Interestingly, the mitochondrial import of TAT-containing NDUFV2 was independent of mitochondrial membrane potential. To explore the therapeutic application of the developed system, a NDUFV2 knockdown cell line (IF4) generated in previous studies was applied for rescuing studies. Treating with TAT-NDUFV2 not only significantly improve the assembly of complex I in IF4 cells, but also partially rescue complex I functions both in the in-gel activity assay and the complex I enzymatic activity assay. In addition, the oxygen consumption rate and mitochondrial membrane potential of IF4 cells were also greatly increased. Similar results were also observed while IF4 cells were treated with NDUFV2-TAT. Our current findings suggest a great potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency and other mitochondrial disease.

Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...