Dissertations / Theses on the topic 'Mitochondrial biogenesis and quality control'

Le complexe III de la chaine respiratoire mitochondriale (OXPHOS III) chez S. cerevisiae est assemblé à partir de dix sous-unités structurales codées par le génome soit nucléaire, soit mitochondrial et fait intervenir une douzaine de protéines extrinsèques au complexe. Nous avons étudié l’une d’entre elle, Bcs1, une ATPase oligomérique conservée de la famille des protéines AAA (ATPases Associated with diverse cellular Activities), qui contrôle la dernière étape de l’assemblage du complexe III. Chez l’Homme, des mutations dans l’orthologue de BCS1, BCS1L, sont associées à différentes maladies. Nous avons montré que des mutations dans les résidus conservés du domaine AAA de Bcs1 peuvent être compensées par des mutations dans les sous-unités de l’ATP synthase mitochondriale (OXPHOS V). Ces mutations compensatrices diminuent toutes l’activité d’hydrolyse de l’ATP de l’enzyme et nous avons proposé que la biogenèse du complexe III puisse être modulée selon l’état énergétique mitochondrial par Bcs1 via sa dépendance à l’ATP. Nous avons aussi identifié des mutations compensatrices dans d’autres gènes et le cas particulier de la délétion du RRF1, facteur général du recyclage des ribosomes mitochondriaux, a été étudié. Nous avons montré que l’absence de Rrf1 a un effet différent sur la stabilité et la traduction des divers ARNm mitochondriaux. Nos résultats suggèrent une coopération entre les facteurs généraux et les facteurs spécifiques de la traduction mitochondriale dans le contrôle de l’expression des sous-unités des complexes OXPHOS traduites dans la mitochondrie<br>OXPHOS complexes are multi-subunit complexes embedded in the inner mitochondrial membrane. We have studied the assembly factor Bcs1 that is a membrane-bound AAA-ATPase, required for the assembly of complex III. Mutations in the human gene BCS1L are responsible for various mild to lethal pathologies. Extragenic compensatory mutations able to restore the assembly of complex III in yeast bcs1 mutants were found in different genes not directly connected to the complex, revealing new networks of protein interactions. Mutations in catalytic subunits of ATP synthase were identified and thoroughly characterized. This work has allowed us to propose a novel regulatory loop via the ATP-dependent activity of Bcs1 protein, connecting the production of mitochondrial complex III and the activity of the ATP synthase. Moreover, these results hold promise for the development of therapies, targeting the mitochondrial adenine nucleotide pool, in treatment of BCS1-based disorders. We also show that the absence of RRF1, a mitochondrial ribosome recycling factor, is able to compensate defects of bcs1 mutants. Deletion of RRF1 has a differential impact on the stability and translation of mitochondrial mRNAs. Our results suggest cooperation between general and specific translation factors in controlling the expression of mtDNA-encoded subunits of the OXPHOS complexes

Mitochondria are the powerhouses of eukaryotic cells and they must remain healthy in order to generate sufficient ATP for cellular function. Dysfunctional mitochondria pose a grave threat to high-energy demanding tissues and are associated with an array of human diseases. Mitochondria exist in a dynamic organelle network that is essential for their intracellular distribution and quality control. A damaged mitochondrion must first be exiled from the network by mitochondrial fission and next be neutralized by a process termed mitophagy. A number of mitophagy pathways exist to specifically target damaged or redundant mitochondria for engulfment by double-membrane autophagosomes in order to deliver them to the acidic lysosome for degradation. This dissertation explores the regulation and molecular mechanisms of the PINK1/Parkin mitophagy pathway. Mutated in several forms of Parkinson's disease, the PINK1 kinase and Parkin E3-ubiquitin ligase govern the selective degradation of dysfunctional mitochondria and they have been demonstrated to play key neuroprotective roles in vitro and in vivo. Here, the role of mitochondrial bioenergetics in regulating mitophagy is investigated. By employing a range of biochemical and imaging techniques in a cell-based model of Parkin-mediated mitophagy, the following data demonstrate how cells dependent on mitochondrial respiration can avoid mitophagy via intricate control of mitochondrial dynamics. In order to maintain the energy supply, respiring cells can resist mitophagy by preserving an interconnected mitochondrial network via inhibition of Drp1 and impaired OMA1-dependent OPA1 cleavage. This dissertation also questions the importance of close contact between the mitochondria and endoplasmic reticulum (ER) for the progression of Parkin-mediated mitophagy. A focused siRNA screen of ER-mitochondrial communication factors highlights a novel role for ER-mitochondrial Ca2+ signa ling during Parkin-mediated mitophagy. Together, the data presented in this dissertation place mitochondrial bioenergetic demand and Ca2+ flux as key players in the regulation of mitophagy. Further research will be required to identify whether these two regulatory arms are linked and will strengthen the therapeutic potential for positively modulating mitochondrial homeostasis in order to promote cell protection.

La mitophagie, la dégradation sélective des mitochondries par autophagie, est impliquée dans l’élimination des mitochondries endommagées ou superflues et requiert des régulateurs et protéines spécifiques. Chez la levure, Atg32, localisée dans la membrane externe mitochondriale, interagit avec Atg8, et permet le recrutement des mitochondries et leur séquestration à l’intérieur des autophagosomes. Atg8 est conjuguée à de la phosphatidyléthanolamine et est ainsi ancrée aux membranes du phagophore et des autophagosomes. Chez la levure, plusieurs voies de synthèse de PE existent mais leur contribution dans l’autophagie et la mitophagie est inconnue. Dans le premier chapitre, nous avons étudié la contribution des différentes enzymes de synthèse de PE, dans l’induction de l’autophagie et la mitophagie et nous avons démontré que Psd1, la phosphatidylsérine décarboxylase mitochondriale, est impliquée dans la mitophagie seulement en condition de carence azotée alors que Psd2, localisée dans les membranes vacuolaires, endosomales et de l’appareil de Golgi, est nécessaire en phase stationnaire de croissance. Dans le second chapitre, la relation entre Atg32, la mitophagie et le protéasome a été étudiée. Nous avons démontré que l’activité du promoteur d’ATG32 et la quantité de protéine Atg32 exprimée sont inversement régulées. En phase stationnaire de croissance, l’inhibition du protéasome empêche la diminution de l’expression d’Atg32 et la mitophagie est stimulée. Nos données montrent ainsi que la quantité d’Atg32 est reliée à l’activité du protéasome et que cette protéine pourrait être ubiquitinylée. Dans le troisième chapitre, nous nous sommes intéressés au rôle potentiel de Dep1, un composant du complexe nucléaire Rpd3 d’histones déacétylases, dans la mitophagie. Dans nos conditions, Dep1 semble être mitochondriale et elle est impliquée dans la régulation de la mitophagie. BRMS1L (Breast Cancer Metastasis suppressor 1-like) est l’homologue de Dep1 chez les mammifères. Cette protéine possède un rôle anti-métastatique dans des lignées de cancer du sein. Nous avons trouvé que l’expression de BRMS1L augmente en présence de stimuli pro-mitophagie<br>Mitophagy, the selective degradation of mitochondria by autophagy, is implicated in the clearance of superfluous or damaged mitochondria and requires specific proteins and regulators. In yeast, Atg32, an outer mitochondrial membrane protein, interacts with Atg8, promoting mitochondria recruitment to the phagophore and their sequestration within autophagosomes. Atg8 is anchored to the phagophore and autophagosome membranes thanks to phosphatidylethanolamine (PE). In yeast, several PE synthesis pathways have been characterized, but their contribution to autophagy and mitophagy is unknown. In the first chapter, we investigated the contribution of the different enzymes responsible for PE synthesis in autophagy and mitophagy and we demonstrated that Psd1, the mitochondrial phosphatidylserine decarboxylase, is involved in mitophagy induction only in nitrogen starvation, whereas Psd2, located in vacuole/Golgi apparatus/endosome membranes, is required preferentially for mitophagy induction in stationary phase of growth. In the second chapter, we were interested in the relationship between Atg32, mitophagy and the proteasome. We demonstrated that ATG32 promoter activity and protein expression are inversely regulated. During stationary phase of growth, proteasome inhibition abolishes the decrease in Atg32 expression and mitophagy is enhanced. Our data indicate that Atg32 protein is regulated by the proteasome activity and could be ubiquitinated. In the third chapter, we investigated the involvement of Dep1, a member of the nuclear Rpd3L histone deacetylase complex, in mitophagy. In our conditions, Dep1 seems to be located in mitochondria and is a novel effector of mitophagy both in nitrogen starvation and stationary phase of growth. BRMS1L (Breast Cancer Metastasis suppressor 1-like) is the mammalian homolog of Dep1 and has been described in breast cancer metastasis suppression. We found that BRMS1L protein expression increases upon pro-mitophagy stimuli

Thesis (M.A.)--Boston University<br>DJ-1 is a cytosolic sensor for oxidative damage which acts on the Mitochondria. It works to curb the negative effects of high membrane potential in mitochondria, but the mechanism of action is still uncertain. This study measured DJ-1’s potential in enchancing mitochondrial quality control in the context of pancreatic B-cells treated with a palmitate and glucose media to promote glucolipotoxicity (GLT). DJ-1 was proven capable of reversing GLT induced changed in mitochondrial morphology in the arenas of Feret’s diameter, aspect ratio, and form factor. We also showed that the mitochondrial membrane potential did not vary with the presence or absence of DJ-1. In addition, DJ-1 was shown capable of limiting the upward boundary of GLT induced increase in mitochondrial membrane potential. Furthermore, an experiment using INS1 cells with GFP-LC3 showed that DJ-1 can decrease the average number of autophagosomes in the cell.

In addition to ATP generation, mitochondria are essential in various cellular processes ranging from biosynthetic pathways, apoptosis, cell cycle progression, and calcium buffering. Studies in living cells have now firmly established that mitochondria exist as a dynamic network sculpted by fission and fusion reactions, rather than separated, individual organelles. Not surprisingly, mutations in genes involved in mitochondrial dynamics and quality control lead to human diseases such as Charcot-Marie-Tooth disease type 2A, Optic atrophy, and autosomal recessive Parkinson disease. I have designed a high-throughput protocol to permit genome-wide screening for novel genes that are required for normal mitochondrial morphology. I have executed a genome-wide RNA interference screen and identified several novel genes required for mitochondrial dynamics in addition to known regulators of mitochondrial dynamics. A detailed high-throughput genome-wide screening protocol is presented. I have shown that TID1, a gene identified from the screen, has a dual-role in maintaining the integrity of mitochondrial DNA and preventing the aggregation of complex I subunits. My analysis of the mitochondrial role of TID1 supports the existence of a TID1- mediated stress response to ATP synthase inhibition. The genome screen also identified the novel gene ROMO1 as essential for normal mitochondrial morphology. I have shown that ROMO1 may have an additional role in maintaining mitochondrial spare respiratory capacity, possibly by affecting cellular substrate availability. Finally, in a collaborative effort, we have shown that homozygous mutations in the mitochondrial fusion gene MFN2 lead to multiple symmetric lipomatosis (MSL) associated with neuropathy. Mechanistically, this mutation reduces MFN2 homocomplex formation. Taken together, these results show the utility of genome-wide screening in identifying genes involved in mitochondrial quality control.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Despite recent advances, the cause for most PD cases remains unclear. The discovery of mutations in PINK1 (PTEN-induced putative kinase 1) reinforced the importance of mitochondrial impairment in PD. Mitochondria are essential organelles for energy generation in eukaryotic cells, whose compromise can eventually cause cell death. Multicellular organisms have evolved quality control mechanisms to ensure the viability of mitochondria and ultimately the cell. Molecular quality control through the mitochondrial chaperones and proteases acts to promote the proper folding of polypeptides and the degradation of misfolded or damaged proteins. When molecular quality control is overwhelmed, organellar quality control ensures mitochondrial recycling through a selective form of autophagy called mitophagy. PINK1 has been proposed to act in both molecular and organellar quality control, by modulating the activity of chaperones, namely HtrA2 and TRAP1, and acting on mitophagy through Parkin recruitment to damaged mitochondria. The work in this thesis provides evidence of a genetic interaction between Trap1, Pink1 and parkin in Drosophila melanogaster. Trap1 is essential to maintain mitochondrial and dopaminergic neuronal functions and is associated with resistance to stress. Importantly, neuronal expression of Trap1 is sufficient to rescue the Pink1 mutants. Moreover, the expression of Trap1 ameliorates parkin-mutant phenotypes and parkin expression suppresses Trap1-mutant phenotypes, suggesting that molecular and organellar quality control pathways act in parallel downstream from Pink1. p62 is an autophagy adaptor that acts in the PINK1/Parkin pathway, facilitating the aggregation and elimination of depolarised mitochondria through mitophagy. In this work it is shown that loss-of-function mutations in the Drosophila orthologue of p62, ref(2)P, result in a reduction in lifespan and age-dependent neurodegeneration. ref(2)P expression rescues the Pink1-mutant phenotypes and its presence is essential for the parkin-mediated rescue of Pink1 mutant flies.

Darrerament s’han produït avanços importants que han contribuït al coneixement dels mecanismes de disfunció cel·lular i mort en la malaltia de Parkinson (MP) i en la malaltia de Huntington (MH). Ambdues malalties són trastorns del moviment que es caracteritzen per la pèrdua específica de neurones dels ganglis basals, les neurones dopaminèrgiques de la substància nigra (SN), en el cas de la MP i les neurones espinoses de l’estriat, en el cas de la MH. Malgrat les diferències, ambdues comparteixen processos patològics comuns com la presència de proteïnes malplegades, l’estrés oxidatiu i disfunció mitocondrial. La mitocòndria és la font d’energia principal en les cèl·lules eucariotes, però també és un orgànul dinàmic relacionat amb una gran quantitat de processos cel·lulars. La disrupció de la homeòstasis mitocondrial i la subseqüent disfunció mitocondrial juguen un paper important en la patofisiologia de les malalties neurodegeneratives. El manteniment de la integritat mitocondrial a través de diferents mecanismes de control és crític per a la superviviència neuronal. Aquesta tesi es centra en l’estudi dels mecanismes de control de qualitat mitocondrial en la MP i la MH, per tal d’entendre millor els mecanismes que duen a la mort cel·lular. En el primer capítol, he estudiat el transport de proteïnes a la mitocòndria en models in vitro i in vivo de la MP. In vitro, la inhibició del complexe I produeix una alteració del transport de proteïnes a la mitocòndria així com una disminució dels nivells de proteïnes OXPHOS, acumulació de proteïnes agregades i disminució dels nivells de chaperones mitocondrials. Per tal de restablir el transport de proteïnes mitocondrials es van sobreexpressar dos components clau del sistema de translocases: la translocasa de la membrana externa 20 (TOM20) i la translocasa de la membrana interna 23 (TIM23). La sobreexpressió in vitro de TOM20 i TIM23 va restaurar el transport de proteïnes mitocondrials i va alleugerar la disfunció mitocondrial i la mort cel·lular. La inhibició del complexe I en ratolins també dóna lloc a una alteració del transport de proteïnes mitocondrials i produeix neurodegeneració del sistema dopaminèrgic. La sobreexpressió de TIM23 va restaurar parcialment el transport de proteïnes i va protegir lleugerament les neurones dopaminèrgiques de la SN. En canvi, la sobreexpressió de TOM20 va ser incapaç de millorar el transport de proteïnes mitocondrials i, fins i tot, va exacerbar la mort cel·lular. Aquests resultats posen de relleu el paper de la disfunció del transport de proteïnes mitocondrials, en particular de dos dels seus components, en la patogènesis de la MP i suggereixen la necessitat de futurs estudis es centrin en altres elements d’aquest sistema. En el segon capítol, he estudiat el paper de la proteïna huntingtina en la mitofàgia i com la seva mutació, que dóna lloc a una expansió de glutamines, pot afectar a aquesta funció. Per a tal fi, he treballat en un model in vitro de cèl·lules estriatals ST-Q7 (control) i ST-Q111 (mutant). En condicions fisiològiques, la mitofàgia induïda no es troba mitjançada pel reclutament de parkin als mitocondris despolaritzats. La huntingtina mutada afecta la mitofàgia induïda a través de l’alteració de la seva funció de scaffold en diferents passos del procés de mitofàgia: (i) activació d’ULK1 a través de l’alliberament de mTORC1, (ii) formació del complexe Beclin 1-Vps15,(iii) interacció dels adaptadors de mitofàgia OPTN i NDP52 amb huntingtina i, (iv) amb LC3. Com a resultat, els mitocondris de les cèl·lules ST-Q111 estan més danyats i tenen una respiració mitocondrial deficient. Aquests resultats demostren la presència d’una alteració en la mitofàgia com un mecanisme lligat a la MH. En conclusió, el descobriment de noves dianes mitocondrials en la MP i MH emfatitza el paper important que juga el control de qualitat mitocondrial en la neurodegeneració.<br>In the past years, several important advances have expanded our understanding of the pathways that lead to cell dysfunction and death in Parkinson’s disease (PD) and Huntington’s disease (HD). Both diseases are movement disorders characterized by the loss of a specific subset of neurons within the basal ganglia, dopaminergic neurons in the substantia nigra pars compacta (SNpc), in the case of PD, and medium spiny neurons in the striatum, in the case of HD,. Despite distinct clinical and pathological features, these two neurodegenerative disorders share critical underlying pathogenic mechanisms such as the presence of misfolded and/or aggregated proteins, oxidative stress and mitochondrial anomalies. Mitochondria are the prime energy source in most eukaryotic cells, but these highly dynamic organelles are also involved in a multitude of cellular events. Disruption of mitochondrial homeostasis and the subsequent mitochondrial dysfunction plays a key role in the pathophysiology of neurodegenerative diseases. Therefore, maintenance of mitochondrial integrity through different surveillance mechanisms is critical for neuronal survival. In this thesis I have studied in depth some mitochondrial quality control mechanisms in the context of PD and HD, in order to broaden the knowledge about the pathomechanisms leading to cell death. In the first chapter I have studied mitochondrial protein import in in vitro and in vivo models of PD. In vitro, complex I inhibition, a characteristic pathological hallmark in PD, impaired mitochondrial protein import. This was associated with OXPHOS protein downregulation, accumulation of aggregated proteins inside mitochondria and downregulation of mitochondrial chaperones. Therefore, we aimed to reestablish the mitochondrial protein import by overexpressing two key components of the system: translocase of the outer membrane 20 (TOM20) and translocase of the inner membrane 23 (TIM23). Overexpression of TOM20 and TIM23 in vitro restored protein import into mitochondria and ameliorated mitochondrial dysfunction and cell death. Complex I inhibition also impaired mitochondrial protein import and led to dopaminergic neurodegeneration in vivo. Overexpression of TIM23 partially rescued protein import into mitochondria and slightly protected dopaminergic neurons in the SNpc. On the contrary, TOM20 overexpression did not rescue protein import into mitochondria and exacerbated neurodegeneration in both SNpc and striatum. These results highlight mitochondrial protein import dysfunction and the distinct role of two of their components in the pathogenesis of PD and suggest the need for future studies to target other elements in the system. In the second chapter, I have studied the role of huntingtin in mitophagy and how the polyglutamine expansion present in mutant huntingtin can affect its function. For such, I worked with differentiated striatal ST-Q7 (as control) and ST-Q111 (as mutant) cells, expressing full length huntingtin. In these conditions, induced mitophagy was not mediated by Parkin recruitment into depolarized mitochondria. Mutant huntingtin impaired induced mitophagy by altering wildtype huntingtin scaffolding activity at different steps of mitophagy process: (i) ULK1 activation through its release from the mTORC1, (ii) Beclin1-Vps15 complex formation, (iii) interaction of the mitophagy adapters OPTN and NDP52 with huntingtin and (iv) with LC3. As a result, mitochondria from ST-Q111 cells exhibited increased damage and altered mitochondrial respiration. These results uncover impaired mitophagy as a potential pathological mechanism linked with HD. In conclusion, we have discovered new mitochondrial targets for PD and HD emphasizing the important role that mitochondrial quality control plays in neurodegeneration

Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis it is released to the cytoplasm where it participates in cell death. While confined in the mitochondria it has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. We used the yeast two-hybrid system to dissect Omi/HtrA2's pathway by identifying novel interactors and substrates. Our studies revealed a novel function of Omi/HtrA2 in the regulation of a Lys-63 deubiquitinating (DUB) complex. In addition, we found the mechanism by which Omi/HtrA2 protease participates in mitophagy by directly regulating the protein level of Mulan E3 ubiquitin ligase, especially during mitochondrial stress. Abro1 is a scaffold protein of the DUB complex known as BRISC (BRCC36 isopeptidase complex). In addition, Abro1 is involved in a cytoprotective pathway and is regulated by Omi/HtrA2. Three specific interactors of Abro1 protein were identified, ATF4, ATF5 and JunD, all members of the activating protein 1 (AP-1) family. We focused our studies on ATF4 since, like Abro1, it is ubiquitously expressed and is important in cell cycle regulation and survival. Abro1's interaction with ATF4 was specific and occurred only when cells were stressed. The significance of this interaction was the translocation of Abro1 from the cytoplasm to the cell nucleus. These results establish a new cytoprotective function of cytoplasmic Omi/HtrA2 as a regulator of the BRISC DUB complex. Furthermore, we have recently identified the mitochondrial Mulan E3 ubiquitin ligase as a substrate of Omi/HtrA2 protease. Mulan, along with MARCH5/MITOL and RNF185, are the only three mitochondrial E3 ubiquitin ligases identified thus far. The function of Mulan has been linked to cell growth, cell death, and autophagy/mitophagy. To investigate Mulan's function and its control by Omi/HtrA2, E2 conjugating enzymes that form a complex with Mulan E3 ligase were identified. Four specific interacting E2s were isolated, namely Ube2E2, Ube2E3, Ube2G2, and Ube2L3. To identify substrates for each unique Mulan-E2 complex, fusion baits were used in a modified yeast two-hybrid screen. Our results suggest that Mulan participates in various pathways, depending on the nature of its E2 conjugating enzyme partner. One of the interactors isolated against the Mulan-Ube2E3 bait was the GABARAP (GABAA receptor-associated protein), a member of the Atg8 family. We characterized this interaction both in vitro and in vivo and its potential role in mitophagy. Our studies defined a new pathway by which Mulan participates in mitophagy by recruiting GABARAP to the mitochondria.<br>Ph.D.<br>Doctorate<br>Molecular Biology and Microbiology<br>Medicine<br>Biomedical Sciences

Central in oxygenic photosynthesis, the cytochrome b6f complex, couples electron transfer to proton translocation across the thylakoid membrane via its quinol:plastocyanin oxidoreductase activity, contributing to ATP formation. Cytochrome b6f complex differs from its respiratory homolog, the bc1 complex, by the presence of an additional heme, heme ci located within the quinone reduction site Qi and attached by a unique thioether bond. Mutants lacking heme ci show low accumulation of partially functional b6f complex and, hence, cannot grow phototrophically. This grounded a screen for suppressor mutations that would restore higher accumulation of b6f complexes whose function, even if compromised, would sustain phototrophic growth.The genetic suppressor approach undertook in Chlamydomonas reinhardtii during this PhD thesis led to the isolation and characterisation of the ftsh1-1 protease mutant (mutation R420C which should affect ATP hydrolysis). The mutant ftsh1-1 proved to be a versatile tool for the functional study of otherwise degraded proteins. The combination of genetic, biochemical, physiological and biophysical experiments demonstrated notably that: (i) a QiKO mutant, whose b6f complexes are devoid of both bh and ci hemes, can grow phototrophically despite a broken Q-cycle, (ii) the absence of covalently bound heme ci, in the Rccb2 mutant, triggers photosensivity enhanced in the presence of O2 supporting a role for heme ci in oxygen rich environment, (iii) FtsH is involved in the maintenance of the main photosynthetic complexes.

Over the past two decades, significant understanding of the pathogenesis of Parkinson's disease (PD) has been attributed to the discovery of genes, that when mutated, are responsible for familial forms of PD. Recently a novel autosomal dominant mutation (AD) causing PD was identified in receptor-mediated endocytosis-8 (RME-8). When mutated, symptoms of PD manifest with an onset ~ 70 years of age. RME-8 is a DnaJ domain containing protein that plays an important role in intercellular trafficking and recycling of retrograde cargo. Loss of function of RME-8 disrupts the endosome to Golgi transport resulting in cargo accumulation in the endosome and its re-routing to the lysosome for degradation. Studies have shown that VPS35 (another AD-PD gene, and part of the retromer that RME-8 interacts with) is involved in the mitochondrial quality control pathway implicated in PD. In addition, recent studies have shown that bec-1, a protein long studied as a regulator of autophagy (part of the mitochondrial quality control pathway), to also be involved in the retrograde trafficking co-localizing with RME-8. These findings suggest a possible new role of RME-8 in the mitochondrial quality control pathway. Here we investigated the possible role of RME-8 in the mitochondrial quality control pathway implicated in PD. Using loss of function approach by knocking-down RME-8 and gain of function approach by overexpressing the mutant form of RME-8 we investigated its role in two pathways involved in mitochondrial quality control: mitophagy and mitochondrial vesicle formation. Our results show that RME-8 is not involved in either pathways and thus the exact role of RME-8 in the pathogenesis of PD has to still be elucidated.<br>Des avancées significatives dans la compréhension de la pathologie propre à la maladie de Parkinson (MP) ont marqués les deux dernières décennies grâce, notamment, à la découverte de mutations génétiques responsables de formes familiales de la MP. Récemment, une mutation autosomale-dominante (AD) dans le gène RME-8 (receptor-mediated endocytosis-8) a été identifiée comme cause de la MP dont les manifestations cliniques associées à cette mutation apparaissent vers 70 ans. La protéine codée par RME-8, contient un domaine DnaJ qui joue un rôle important dans le trafic intracellulaire et le recyclage de cargos rétrogrades. La protéine RME-8 est exprimée dans plusieurs tissus et possède une forte affinité pour la chaperonne HSC70 (heat shock protein 70). RME-8 recrute HSC70 aux membranes couvertes de clathrine et interagit avec le complexe du retromère pour désassembler les triskelions de clathrine. La perte de fonction de RME-8 perturbe le transport de l'endosome au Golgi, ce qui entraîne l'accumulation du cargo dans l'endosome et sa redirection vers le lysosome. De plus, il a été démontré, que VPS35, fait partie du complexe du retromère et interagit avec RME-8, et que BEC-1 est impliquée dans le trafic rétrograde et que l'appauvrissement de RME-8 ou BEC-1 donne des phénotypes similaires. Puisque VPS35 et BEC1 jouent un rôle dans le contrôle de la qualité mitochnodriale, nous avons émis l'hypothèse que RME-8 est aussi impliquée dans ce processus. Ni l'ablation de RME-8 via l'ARN interférence ou sa surexpression n'a permis de montrer un rôle pour RME-8 dans la mitophagie ou la formation de vésicules mitochodriales. Nos données tendent à montrer que RME-8 n'est pas impliquées dans le contrôle de la qualité mitochondriale et que son rôle dans la pathogénèse de la MP demeure obscur.

Chez les eucaryotes, l’information génétique est transcrite en ARN messager qui subit plusieurs étapes de maturation et évènements d’assemblage avant d’être exporté hors du noyau. Ces modifications du transcrit sont effectuées par de nombreux facteurs protéiques recrutés au transcrit naissant, formant ainsi une particule ribonucléoprotéique (mRNP). La biogenèse du mRNP est étroitement liée avec la transcription et le contrôle qualité afin d’assurer l’efficacité et l’exactitude de la production de mRNPs matures. Des études récentes suggèrent que les membres du complexe THO-Sub2 pourraient être des facteurs cruciaux dans le couplage de la transcription, de la biogénèse du mRNP et de l’export. Dans notre groupe, nous avons mis en oeuvre un essai novateur pour étudier la biogénèse du mRNP et le contrôle qualité, basé sur l’expression du facteur Rho bactérien dans Saccharomyces cerevisiae. Rho interfère avec l’assemblage adéquat du mRNP et génère des transcrits aberrants qui sont dégradés par la machinerie de dégradation nucléaire. Dans cette étude, nous avons utilisé le système expérimental Rho pour mieux comprendre Rrp6 et l’implication de l’exosome dans la dégradation des transcrits liée au contrôle qualité, ainsi que pour mieux caractériser le rôle et la fonction du complexe THO-Sub2 dans le processus de biogénèse du mRNP. Les résultats obtenus révèlent une différence intéressante dans le comportement des membres du complexe THO sous l’action de Rho et dévoilent leur dépendance à la liaison à l’ARN, ce qui n’aurait pas pu être observé avec d’autres techniques expérimentales. Cela confirme le potentiel attendu du système expérimental basé sur Rho dans l’étude des facteurs protéiques impliqués dans la biogénèse et le contrôle qualité du mRNP<br>In eukaryotes, the genetic information is transcribed into messenger RNA which undergoes various processing and assembly events prior to its export from the nucleus. These transcript modifications are performed by numerous protein factors recruited to the nascent transcript, thus making a messenger ribonucleoprotein particle (mRNP). mRNP biogenesis is tightly interconnected with both transcription and quality control to ensure efficiency and accuracy in production of mature mRNPs. Recent findings suggest that members of THO-Sub2 complex might be crucial factors in coupling transcription, mRNP biogenesis and export. In our group, we have implemented an innovative assay to study mRNP biogenesis and quality control, based on the expression of the bacterial factor Rho in Saccharomyces cerevisiae. Rho interferes with proper mRNP assembly and generates aberrant transcripts degraded by the nuclear degradation machinery. In this study, we use Rho experimental system to expand our findings on Rrp6 and exosome involvement in quality control degradation of transcripts, as well as to better characterize the role and function of THO-Sub2 complex in the process of mRNP biogenesis. Obtained results reveal an interesting difference in behavior of THO complex members upon Rho action and disclose their dependence on binding to the RNA, which could not be observed by other experimental techniques. This substantiates the expected potential of Rho-based experimental system in the study of protein factors involved in mRNP biogenesis and quality control

Mitochondria are dynamic; they alter their shape through fission, fusion and budding of vesicles. Mitochondrial vesicles serve as a quality control mechanism enabling these organelles to rid themselves of damaged lipids and proteins. Dysregulation in mitochondrial dynamics and quality control have been linked to Parkinson’s Disease, making the identification of molecules requisite for these processes a priority. We identified the endocytic protein, Sorting nexin 9 (Snx9) through a genome wide siRNA screen for genes which substantially alter mitochondrial morphology and therefore are important for its maintenance. In this work, the role of Snx9 in mitochondrial morphology is examined. Ultrastructural imaging of mitochondria within cells silenced for Snx9 revealed unbudded vesicles along a hyperfused mitochondrial reticulum suggesting a role for Snx9 in the release of these vesicles. The vesicular profiles contained concentric membranous whorls enriched for neutral lipids. Localization studies suggest the Parkinson’s disease genes, Parkin and Vps35 localize to the unbudded profiles.

Autosomal dominant spinocerebellar ataxias (SCA) are a heterogeneous group of neurological disorders characterized by cerebellar dysfunction. We recently showed that AFG3L2 mutations cause dominant ataxia SCA28. AFG3L2 and its partner protein paraplegin, which causes recessive spastic paraparesis SPG7, are components of the m-AAA complex, involved in mitochondrial protein quality control. Since yeast functional studies showed that paraplegin coexpression can modulate AFG3L2 mutations, we investigated the possible coinheritance of AFG3L2 and SPG7 mutations in patients with spinocerebellar syndromes. We identified 3 probands with heterozygous mutations in both the AFG3L2 and the SPG7 genes. Two ataxic patients carry an AFG3L2 mutation affecting highly conserved amino acids located in the ATPase or in the proteolytic domains of the protein along with the parapleginA510V. The third proband carries a de novo AFG3L2 mutation in the highly conserved SRH region of the ATPase domain along with the inherited deletion of SPG7 exons 4-6. The clinical presentation of this patient is characterized by early onset optic atrophy and a L-dopa-responsive spastic-ataxic syndrome with extrapyramidal signs. A muscle biopsy revealed an isolated complex I deficiency. Moreover, evaluation of substrates processing in patient’s fibroblasts showed abnormal processing pattern of OPA1. In conclusion, our data indicate that the presence of a loss-of-function mutation in paraplegin may act as a disease modifier for heterozygous AFG3L2 mutations. Concurrent mutations in both components of the mitochondrial m-AAA complex may result in a complex phenotype, thus expanding the clinical spectrum of AFG3L2-associated mutations. Moreover, biochemical and cell biology studies revealed a crucial role of the m-AAA complex in the processing of OPA1 and the maintenance of mitochondrial morphology.

O conhecimento das implicações da variabilidade genética em populações de inimigos naturais, principalmente parasitoides, é de vital importância para a otimização de programas de controle biológico. Desta forma, o presente trabalho teve por objetivo determinar como a variabilidade genética influencia diferentes parâmetros biológicos de Trichogramma pretiosum Riley, 1879 em experimentos de laboratório e de campo. Para que este objetivo fosse atingido, foram avaliados: 1) o efeito da seleção de isolinhagens de T. pretiosum em condições de laboratório, marcadas por meio do DNA mitocondrial, no subsequente desempenho de campo; 2) como a UR afeta a capacidade de voo de isolinhagens, marcadas por meio do DNA mitocondrial, de espécimes de T. pretiosum oriundas do Brasil e EUA; 3) a compatibilidade reprodutiva entre isolinhagens norteamericanas e brasileira de T. pretiosum, avaliada por meio de uma abordagem integrativa e 4) o possível estabelecimento de uma linhagem de T. pretiosum proveniente da Colômbia num novo ecossistema, no Nordeste brasileiro. Com base nos resultados do presente trabalho conclui-se que a variabilidade genética de T. pretiosum exerce grande influência em parâmetros biológicos do parasitoide, uma vez que: 1) os diferentes desempenhos reprodutivos das isolinhagens em condições de laboratório foram correspondentes àqueles em condições de campo, sendo as sequências mitocondriais uma técnica de marcação precisa e eficiente para avaliação do desempenho de T. pretiosum em condições de campo; 2) para algumas isolinhagens, as condições ambientais do local onde se pretende liberar T. pretiosum, se distintas do hábitat natural do parasitoide, podem afetar negativamente o voo deste inseto; 3) foi registrada incompatibilidade reprodutiva leve e diferenças morfológicas mais pronunciadas entre as isolinhagens americanas e brasileira, geneticamente variáveis; 4) foram observados fortes indícios de que uma linhagem de T. pretiosum, introduzida em Petrolina, PE há 22 anos, trazida de Palmira, Colômbia, se estabeleceu naquela região.<br>The knowledge on genetic variability in populations of natural enemies, especially parasitoids, has a vital importance for the optimization of biological control programs. Thus, this study aimed to determine the influences of genetic variability on different biological parameters of Trichogramma pretiosum Riley, 1879, in laboratory and field experiments. We evaluated: 1) the effect of selection of T. pretiosum isofemale strains in laboratory conditions, marked by mitochondrial DNA, on the subsequent field performance; 2) the effects of RH on flight capacity isofemale lines, marked by the mitochondrial DNA, of T. pretiosum specimens from Brazil and the USA; 3) the reproductive compatibility between USA and Brazilian isofemale lines of T. pretiosum, through an integrative approach, and 4) the possible establishment of a T. pretiosum strain from Colombia in a new ecosystem in the Brazilian Northeast. The results allow to conclude that genetic variability of T. pretiosum has great influence on biological parameters of the parasitoid, since: 1) the different reproductive performance of isofemale strains in laboratory conditions corresponded to those under field conditions, and mitochondrial DNA was accurate and efficient, marking technique for the evaluation of T. pretiosum performance in field conditions; 2) for some isofemale lines, the environmental conditions of the release sites, if distinct from the natural habitat of the parasitoid, may adversely affect flight capacity of the parasitoid; 3) slight reproductive incompatibility and more pronounced morphological differences were observed between American and Brazilian isofemale lines, genetically variable; 4) there is strong evidence that T. pretiosum line introduced in Petrolina, PE, 22 years ago, brought from Palmira, Colombia, has established in that region.

La maladie de Parkinson (MP) est une affection neurodégénérative fréquente d’étiologie inconnue, touchant environ 5% de la population mondiale après 80 ans. Environ 10% des cas correspondent à des formes familiales à transmission mendélienne. Pendant longtemps, un dysfonctionnement mitochondrial a été soupçonné jouer un rôle dans la physiopathologie de la MP. Cette possibilité a été récemment corroborée par des découvertes majeures réalisées dans le cadre des formes autosomiques récessives. Parkine et PINK1, les produits de deux gènes associés à ces formes familiales, participent au sein d’une même voie moléculaire au contrôle de la qualité mitochondriale, par la régulation du transport, de la dynamique, de la biogenèse et de la clairance de ces organites.L’objectif de ce travail a été d’élucider certains des mécanismes moléculaires sous-jacents à la régulation de l’homéostasie mitochondriale par Parkine et PINK1. Nous avons utilisé un ensemble d’approches de biologie moléculaire et cellulaire, de biochimie et de microscopie confocale, afin d’identifier et de caractériser des interacteurs moléculaires de Parkine et PINK1 à la membrane mitochondriale externe (MME).Dans la première partie de ce travail, nous avons découvert que la Parkine et PINK1 s’associent sur la MME de mitochondries dysfonctionnelles à proximité de la translocase de la MME (TOM), un complexe dédié à l’import de la grande majorité des protéines mitochondriales. Nous avons montré que ces interactions protéiques jouent un rôle clé dans l’activation du programme de dégradation mitochondriale régulé par la voie PINK1/Parkine. Nous avons également observé que la GTPase de type dynamine Drp1, impliquée dans la fission mitochondriale, est recrutée au niveau de mitochondries endommagées à proximité de Parkine et PINK1 ; ainsi, les processus de fission et de dégradation mitochondriales pourraient être spatialement coordonnés. Dans la deuxième partie de ce projet, nous avons caractérisé l’interaction fonctionnelle entre la Parkine et l’enzyme neuroprotectrice multifonctionnelle de la matrice mitochondriale, 17B-hydroxystéroïde déshydrogénase de type 10 (HSD17B10), dont les taux s’étaient révélés être diminués chez la souris déficiente en Parkine. Nous avons mis en évidence un effet protecteur d’HSD17B10 vis-à-vis de la mitochondrie qui était indépendant de son activité catalytique. Nous avons de plus montré que la Parkine interagit directement avec HSD17B10 à proximité de la machinerie TOM et qu’elle régule positivement l’abondance mitochondriale de cette protéine ; cela suggère qu’elle pourrait promouvoir son import.Dans l’ensemble, ces résultats approfondissent notre connaissance des mécanismes moléculaires mis en jeu par la Parkine et PINK1 dans le contrôle de la qualité mitochondriale, élargissant ainsi notre compréhension de leur rôle dans la physiopathologie des formes autosomiques récessive de MP<br>Parkinson’s disease (PD) is a common neurodegenerative disorder of unknown etiology, affecting nearly 5% of the world population over the age of 80. Nearly 10% of PD cases are familial forms with Mendelian inheritance pattern. Mitochondrial dysfunction has long been suspected to play a role in the physiopathology of sporadic PD. This possibility has been recently corroborated by major discoveries in the field of autosomal recessive PD. Parkin and PINK1, the products of two genes associated with these forms, participate in a common molecular pathway focused on maintenance of mitochondrial quality, with roles in mitochondrial transport, dynamics, biogenesis and clearance.The aim of this work was to elucidate some of the molecular mechanisms underlying the regulation of mitochondrial homeostasis by Parkin and PINK1. We used a combination of approaches in molecular and cell biology, biochemistry and confocal microscopy to identify and characterize molecular interactors of Parkin and PINK1 on the outer mitochondrial membrane (OMM).In the first part of my project, we discovered that Parkin and PINK1 associate on dysfunctional mitochondria in proximity of the translocase of the OMM (TOM), a complex devoted to the mitochondrial import of the vast majority of the mitochondrial proteins. We provided evidence that these associations play a key role in activation of the mitochondrial degradation program mediated by the PINK1/Parkin pathway. We also observed that the dynamin-related GTPase Drp1, involved in mitochondrial fission is recruited to defective mitochondria in proximity of Parkin and PINK1, suggesting that mitochondrial fission occurs at sites where mitochondrial clearance is initiated.In the second part of my project, we characterized the functional interaction between Parkin and the multifunctional neuroprotective mitochondrial matrix enzyme 17B-hydroxysteroid dehydrogenase type 10 (HSD17B10), previously found by the team to be altered in abundance in Parkin-deficient mice. We demonstrated that HSD17B10 exerts a mitochondrion-protective function independent of its enzymatic activity. In addition, we provided evidence that Parkin directly interacts with HSD17B10 at the TOM machinery and that it positively regulates its mitochondrial levels, possibly through the regulation of its mitochondrial import.Altogether, these results provide novel insights into the molecular mechanisms by which Parkin and PINK1 control mitochondrial quality, and deepen our understanding of the role of these proteins in the physiopathology of autosomal recessive PD

La transcription des ARN messagers (ARNm), chez les eucaryotes est un processus complexe nécessitant de nombreuses étapes. En parallèle de ce mécanisme fondamentale, de nombreuses protéines vont se fixer à l’ARNm naissant afin de le maturer et de l’empaqueter pour former une particule ribonucléoprotéique (mRNP) compétente pour l’export vers le cytoplasme, où aura lieu la traduction. Les étapes de biogénèse de cette mRNP sont soumises à la surveillance par un système de contrôle qualité (QC) qui détectera tout évènement conduisant à une mauvaise formation de la particule. Les transcrits aberrants sont retenus au noyau pour y être dégradés par l’exosome. Afin de provoquer l’apparition massive d’ARNm aberrants dans le noyau de notre modèle d’étude, la levure S. cerevisiae, nous utilisons le facteur bactérien Rho. L’hélicase translocase Rho, une fois exprimée, va venir perturber l’assemblage co-transcriptionnel des mRNPs et ainsi générer des transcrits aberrants qui seront substrats de la machinerie de QC et dégradation. La présente étude étend les précédentes observations faites par l’équipe quant à l’implication de certaines protéines dans le système de QC par des approches globales (RNA-seq, ChIP-seq). De plus, l’étude du complexe THO, impliqué dans l’empaquetage et le transport de la mRNP, révèle l’implication d’une de ses sous-unités dans le marquage des ARNm aberrants ainsi qu’un lien de première importance dans le recrutement de l’exonucléase Rrp6 sur ces cibles à dégrader. Enfin, nous montrons, de manière globale l’existence, au sein du noyau des levures, d’une seconde voie de dégradation des ARNs aberrants distincte de la voie canonique passant par Rrp6<br>Eukaryotic transcription of messenger RNAs (mRNAs) is a complex multistep process. In parallel with this fundamental mechanism, many proteins will bind to the nascent mRNA in order to process and package it to form an export competent ribonucleoprotein particle (mRNP). These mRNP biogenesis steps are under the surveillance of a quality control system (QC) that will detect all the faulty events that can lead to the formation of an aberrant particle. Aberrant transcripts will be retained in the nucleus and degraded. To study the QC mechanisms, we previously implemented a powerful assay based on the global perturbation of mRNP biogenesis by the bacterial Rho factor. When expressed in the yeast nucleus, Rho will interfere with co-transcriptional mRNP assembly and generates aberrant transcripts which will be substrates for the QC and degradation system. This study extend the previous observations made by the team about implication of some proteins in the QC pathway by genome-wide methods (RNA-seq, ChIP-seq). Moreover, study of the THO complex, which is a packaging and export factor, shows that the Tho2 subunit is involved in the tagging of aberrant transcripts and in recruitment of the exonuclease Rrp6 on its targets. Finally, we are giving insights about the presence, in yeast, of a second degradation pathway for aberrant mRNPs different from the canonical pathway involving Rrp6

碩士<br>國立陽明大學<br>生物醫學資訊研究所<br>101<br>The function of mitochondria is highly correlated with mitochondrial morphology. Mitochondrial morphology and functions change during cell cycle, and these changes usually imply specific physiological conditions. In the mitotic phase, mitochondria become hyperfused to reach maximal activity. To analyze mitochondrial 3D morphological changes, we established a high content analysis system to apply to normal rat kidney cell image stacks. By using our system, we calculated mitochondrial morphological features to classify mitochondria into 5 different subtypes. And we extracted various cell-level mitochondrial morphological features to find the difference between cell cycle steps. In this study, we found the total mitochondrial volume increased to two-fold from G0 phase to S phase, abruptly drops to original size at G2 phase, and then increased again at M phase. Changes in mitochondrial cross section area and fluorescence intensity of fluorescence-tagged mitochondrial proteins show that the volume increase is not due to swelling of dysfunctional mitochondria. This indicated that mitochondrial biogenesis occurred at the transition from G0 phase to S phase and from G2 phase to M phase. Decrease of mitochondrial volume at G2 phase implies that mitochondrial quality control occurs at G2 phase. By classifying mitochondria into 5 representative morphological subtypes our system reveals specific mitochondrial morphological composition in different cell cycle steps of natural rat kidney cells. Moreover, using our system, we discovered that there are two phases of mitochondrial biogenesis and one phase of quality control during cell cycle implied by cell-level feature analysis. In this study, we show that mitochondrial morphology can be a potential biomarker for cell cycle.

Mitochondria are essential eukaryotic organelles required for diverse cellular functions including, energy homeostasis, iron-sulfur cluster biogenesis, and signaling. Therefore, the maintenance of organelle quality control is critical for cell survival, and any impairment in mitochondrial function is detrimental to cells. Proper mitochondrial functioning requires accurate and efficient transport of approximately 99% of proteins from the cytosol to their precise location into the mitochondria. This protein import is performed by sophisticated protein machinery present at the outer and inner mitochondrial membranes. The outer membrane contains the TOM complex, which serves as a general entry gate for most of the mitochondrial proteins. The inner membrane harbors two distinct import machinery types, namely, the presequence translocase (TIM23 complex) and the carrier translocase (TIM22 complex). The TIM23 complex is dedicated machinery for importing proteins containing N-terminal cleavable targeting signals into the matrix and inner membrane. On the other hand, the TIM22 complex facilitates the import of polytopic inner membrane proteins having internal hydrophobic targeting sequences. Tim22 forms the central channel of the carrier translocase with a twin pore structure. However, the role of the central channel forming Tim22 protein in modulating the assembly process of carrier translocase machinery coupled to protein import remains still elusive. In the present study, we report a novel set of conditional mutants isolated by an unbiased genetic screen from different regions of Tim22. Our genetic and biochemical analyses revealed a distinct functional role for different segments of Tim22 in the assembly of carrier translocase machinery. Further, we demonstrated that impairment in the TIM22 complex assembly process influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA. Overall, our results provide compelling evidence highlighting the functional significance of conserved regions of Tim22 in maintaining the TIM22 complex and mitochondrial integrity. As the substrates of the TIM22 pathway are highly hydrophobic, these proteins' turnover requires efficiently monitored to maintain proteostasis within the organelle. Mitochondria contain several proteases that provide protein quality control in different subcompartments. Yeast mitochondrial escape 1 (Yme1) is an inner membrane AAA class of metalloprotease, which regulates protein quality control with the aid of chaperone-like and proteolytic activities. The current study highlights a novel functional cross-talk between the TIM22 pathway and Yme1. Furthermore, our genetic and biochemical analyses provide compelling evidence for the role of the TIM22 complex and Yme1 in regulating inner membrane protein quality control and mitochondrial health.

Tese de doutoramento em Biologia apresentadas à Faculdade de Ciências e Tecnologia da Universidade de Coimbra<br>Through years of evolution and in order to maintain viable protein homeostasis, cells have developed defense mechanisms against the accumulation of misfolded proteins and aggregates. This mechanism comprises a complex network of specialized proteins designated as heat shock proteins (HSPs). Tumor necrosis factor receptor (TNFR)-associated protein 1 (TRAP1) is a 90 KDa HSP family member that has received large attention over the past years. The interest in TRAP1 originated from its identification as a mitochondrial chaperone whose expression is augmented in several human neoplasias. Since its identification, TRAP1 was described to play important anti-oxidant and anti-apoptotic roles, conferring tumor cells growth advantage. Despite the increasing knowledge, the mechanisms of TRAP1 cytoprotective actions are not yet fully understood. In fact, reports in literature are sometimes contradictory or describe alterations without providing a detailed mechanism of action. The present dissertation aims to contribute with new insights on the role of TRAP1 in conferring mitochondrial protection and regulating cellular quality control systems. We hypothesize that TRAP1 contributes to tumor homeostasis allowing cell growth and survival under stressful environments by preserving mitochondrial functionality and viability as well as through the regulation of cellular quality control systems, including autophagy and apoptosis. To test the hypothesis, the A549 lung carcinoma cell line was used due to its high expression of TRAP1. TRAP1 depletion in this system was achieved through small interference RNA. In addition, a parallel study was performed using MRC-5 cells, a normal lung fibroblast cell line, with low TRAP1 content. The use of the MRC-5 cell line would allow exploring the effects of TRAP1 silencing in a non-tumor cell line, with a low basal expression of that protein. We initially verified that TRAP1 localization in A549 cells was predominantly mitochondrial, whereas TRAP1 was localized in non-mitochondrial areas in MRC-5 cells. Overall, the results presented in this thesis regarding mitochondrial function are in agreement with previous observations showing that TRAP1 contributes to the maintenance of mitochondrial membrane potential and to decrease ROS production in tumor cells, while the same was not observed in normal MRC5 cells. Although TRAP1 function in mPTP modulation has been previously described, we show for the first time the direct effect of TRAP1 silencing on basal mPTP state. Surprisingly, and contrarily to what was expected, mPTP existed in a more closed conformation in A549 TRAP1-depleted cells. Another breakthrough of the present work regards ROS modulation in the tumor cell line, which according to our results may involve p66SHC phosphorylation in Ser36 residue. Additionally, TRAP1 silencing in A549 cells resulted in mitochondrial fragmentation, possibly involving DRP-1 fission protein. Although increased lysosome content (in A549 cells) and decreased p62 levels (in both cell lines) suggest an increased autophagic flux, our data showed a decrease in the expression of several macroautophagy markers in TRAP1-depleted cells. However, these apparently contradictory results are explained by a lower ubiquitin content and increased LAMP2A levels suggesting the activation of an alternative autophagy pathway, involving chaperone-mediated autophagy (CMA). Nonetheless, this activation of CMA was only observed in A549 cells. Moreover, incubation of TRAP1-silenced cells with the autophagy inducer rapamycin resulted in increased cellular growth, mainly in A549 cells, suggesting that autophagy signaling in these cells are pro-tumorigenic. Regarding TRAP1 silencing effects on apoptotic signaling, results for both cell lines showed an increase in caspase 3/7-like activity with no alterations in the apparent activity of initiator caspases (8, 9 and 12). Additionally, TRAP1 silencing shifts the BAX/BCL-xL balance in favor of apoptosis in A549 cells suggesting that these cells have an active apoptotic signaling, whereas MRC-5 cells are not affected. In conclusion, besides TRAP1 differential expression in normal versus cancer cells, its subcellular localization may contribute to the distinct effects observed after TRAP1 silencing (or chemical inhibition). The present work also suggests that p66SHC is a good candidate to mediate TRAP1 ROS modulation in cancer. Moreover, our data suggests that TRAP1 controls mitochondrial morphology through DRP1 content and, additionally, plays an important role in the maintenance of cellular quality control systems. Our results are relevant to clarify not only the role of TRAP1 as an anti-cancer target but also as to understand off-target effects of TRAP1 silencing/inhibition in non-tumor cells.

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2018<br>Protein coding genes are transcribed in the nucleus by RNA polymerase II (RNAPII) forming a precursor messenger RNA (pre-mRNA) that undergoes extensive processing including 5' capping, splicing, 3' end cleavage and polyadenylation to form a mature mRNA. Pre-mRNA processing takes place cotranscriptionally, potentiated by the carboxyl-terminal domain (CTD) of the largest subunit of RNAPII, in a way that transcription and processing machineries communicate with each other to coordinate mRNA biogenesis. After being released from the chromatin template, mRNAs diffuse through the nucleoplasm until they encounter a nuclear pore to be translocated to the cytoplasm where they are translated into proteins, the final outcome of gene expression. Mutations that alter the coding sequence or affect splicing often result in the introduction of premature termination codons (PTCs). If translated, the resulting mRNAs would give rise to truncated proteins with potential deleterious effect for the cell. However, this rarely occurs because eukaryotic cells are able to recognize and degrade mRNAs containing PTCs by a cytoplasmic pathway referred to as nonsense-mediated mRNA decay (NMD). NMD was the first reported example of a quality control mechanism of gene expression. The advantages of mRNA quality control started to be appreciated in the case of beta-thalassemia, as it was found that in most cases only homozygotes suffered from severe anemia. Heterozygotes tend to be phenotypically healthy because NMD prevents production of truncated forms of beta-globin. In addition, thalassemia-like beta-globin mutations resulting in mRNA processing defects induce a nuclear RNA surveillance mechanism that lead to the retention of RNAs near the transcription site. To study the quality control mechanisms that operate during mRNA biogenesis it is essential to fully understand the process of gene expression in health and disease. One pertinent question that was addressed in my PhD work was how general the co-transcriptional mRNA quality control mechanism is and what is its impact in human genetic diseases. To address this question, I used as model system lymphoblastoid cell lines from patients with genetic diseases caused by splicing mutations and mutations in the coding region that introduce a PTC. Quantification of nascent transcripts revealed that a subset of genes containing splicing mutations have reduced transcriptional activity. Inhibition of NMD did not alter the levels of chromatin-associated transcripts, suggesting that a transcription-coupled surveillance mechanism operates independently from NMD to reduce cellular levels of abnormal RNAs in the context of human genetic diseases. Disease-causing mutations that disrupt splicing are mostly localized in splice sites, however next-generation sequencing has revealed that mutations localized deep within introns (more than 100 base pairs away from exonintron junctions) can be the cause of human genetic diseases. Aiming to highlight the importance of studying variation in deep intronic sequences, I reviewed evidence from mRNA analysis and entire genomic sequencing indicating that deep-intronic pathogenic mutations are the cause of over 75 monogenic disorders as well as hereditary cancer syndromes. Interestingly, deep-intronic mutations most commonly create/activate non-canonical splice sites in the pre-mRNA molecule that subsequently lead to pseudo-exon inclusion in the mature mRNA. Since disruption of splicing causes approximately 30% of human genetic diseases, measurement of splicing efficiency is essential to understanding gene regulation in wild-type and splicing-mutated genes. A variety of approaches have been used to purify nascent transcripts and determine the efficiency of splicing. Specifically, purification of newly transcribed molecules using 4sU-tagging has been widely used. Classically, this approach relies on treatment with a thio-reactive reagent HPDP to biotinylate the tagged RNA, which is then affinity-purified with streptavidin. Taking advantage of an efficient biotinylation strategy that uses MTS reagent, I showed that nascent RNA purified with biotin-HPDP contains a significantly higher proportion of unspliced long introns compared to RNAs purified with MTS-biotin. This argues that the splicing kinetics of long introns may be selectively underestimated in studies using biotin-HPDP, which may lead to mis-calculation of processing efficiency in different biological contexts. Disruption of 3' end processing can also be the cause of many human disorders. However, compared to splicing, this step of mRNA biogenesis has been less studied. To further study 3' end processing and transcription xxvi termination, I used a live-cell and single-molecule approach, in which time of release of two different reporter transcripts from the transcription site (TS) was measured. By using two different RNA labelling methods, MS2 and PP7, I showed that β-globin and IgM transcripts are released within 15-25 seconds after transcription of the 3' end of the gene. Furthermore, I showed that downregulating the cleavage factor CPSF3 by RNAi increases time of permanence at TS of both transcripts. Using a different RNA labelling method inserted past the poly(A) site (λN22), I determined that the time of transcription termination ranges between 20-80 seconds, with an average of 30 seconds. These results have important implications for a mechanistic understanding of mRNA biogenesis, particularly at 3' end. Altogether, the original data that resulted in this dissertation detailed the processes involved in mRNA synthesis and decay in the contexts of health and disease.

This work discusses both mechanisms underlying mitochondrial quality control and cytokinesis in the budding yeast Saccharomyces cerevisiae. As these topics are quite different, their presentation has been divided into two parts, "Part I: Mitochondrial Remodeling Through the Proteasome is Critical for Mitochondrial Quality Control in Budding Yeast" and "Part II: Aim44p Regulates Phosphorylation of Hof1p to Promote Contractile Ring Closure During Cytokinesis in Budding Yeast." In Part I, we show that the proteasome is critical for cellular fitness in response to chronic, low levels of mitochondrial reactive oxygen species (ROS) in budding yeast. Deleting DOA1, which is required for ubiquitin-mediated degradation, UFD5, which promotes proteasome gene expression, or NAS2, which promotes proteasome regulatory particle assembly, increases the sensitivity of yeast to chronic, low levels of mitochondrial ROS. In contrast, deleting ATG32, a gene required for mitophagy, other autophagy genes, non-essential chaperones including prohibitins, or mitochondrial proteins including the Lon protease (Pim1p) or YME1, does not affect cellular fitness under these conditions. Doa1p binds with Cdc48p and Vms1p, which associates with mitochondria and promotes extraction of ubiquitinated proteins from the organelle for proteasomal degradation in a pathway called mitochondria-associated degradation (MAD). Elevated mitochondrial ROS increases protein ubiquitination, ubiquitination of the mitochondrial protein aconitase and expression of key MAD proteins. Interestingly, down-regulating ER-associated degradation (ERAD), which shares some common proteins with MAD, can promote cell growth under conditions of elevated mitochondrial ROS. Finally, deletion of DOA1 results in increased sensitivity of yeast and yeast mitochondria to oxidative stress. Mitochondria in doa1 null cells are more oxidized than mitochondria in wild-type or atg32 null cells under conditions of elevated mitochondrial ROS. Moreover, deletion of DOA1 results in a decrease in chronological lifespan. These findings support a critical role for the proteasome and MAD in mitochondrial quality control, which in turn affects cellular fitness, in response to chronic, low levels of mitochondrial ROS. In Part II, we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p co-localizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p co-immunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation, and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for re-localization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44 null cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p.

碩士<br>國立陽明大學<br>藥理學研究所<br>104<br>Acrolein, a ubiquitous environmental pollutant, can be found in cigarette smoke, car exhausts, and overheated cooking oils, which are known as risk factors of lung diseases including asthma, chronic obstructive pulmonary disease (COPD) and lung cancer. Our previous studies have been shown that acrolein induces mutagenic DNA adducts and inhibits DNA repair, which plays an important role in lung carcinogenesis. Mitochondrial homoeostasis is crucial through mitochondrial fission/ fusion cycle, biogenesis, and mitophagy, a selective mitochondrial autophagy. It is not clear that the effect of acrolein on mitochondrial quality control and whether mitochondrial dynamics and mitophagy pathway are involved in acrolein–induced mitochondrial dysfunction. In the present study, our results show that acrolein induces mitochondrial oxidative stress, alteration of mtDNA copy number, and mitochondrial fission which results in mitochondrial dysfunction. Furthermore, mitophagy pathway is induced by acrolein by PINK1 stabilization on mitochondrial membrane and LC3 cleavage. These results suggest that acrolein-induced oxidative stress in mitochondria triggers mitochondrial fission and mitophagy to remove damaged mitochondria as a pro-survival role. However, if a substantial proportion of mitochondrial are damaged, apoptosis ensues. This research may help understand the mechanism of acrolein-induced mitochondrial dysfunction and provide the insight for prevention of acrolein-induced lung diseases.

<p>Advances in modern medicine have helped to prolong human life. These advancements coupled with an ever-increasing population means that diseases associated with aging will become more prevalent in the coming years. As such, it is critical to understand the pathogenesis of disease where aging is the main risk factor. While not widely known, age is in fact a large risk factor in development of obesity and metabolic syndrome. More widely known and discussed are the neurodegenerative diseases that occur late in life. While age as a risk factor is a common point between these types of pathology, there are other similarities, such as the interaction between lipid metabolism and mitochondrial health. </p><p>To study the overlap between obesity and neurodegeneration, we investigated two pathways that regulate both. First, we find that loss of cytoplasmic deacetylase HDAC6 leads to aberrant accumulation of lipid in vitro and in vivo. HDAC6 knock-out (KO) mice gain more weight than WT counterparts after a high-fat diet regimen. Additionally, the intermediary metabolism of cells lacking HDAC6 is disrupted as they increase glucose uptake while downregulating fatty acid oxidation. HDAC6 not only plays a role in lipid metabolism, but regulates mitochondrial dynamics. Upon glucose-withdrawal, HDAC6 KO cells fail to elongate their mitochondria and display increased levels of mitochondrial toxic by-products. Therefore, HDAC6 has critical roles in lipid homeostasis and mitochondrial health. </p><p>The other pathway we investigated is critical in neurodegenerative disease, Parkinson's disease. Parkin, an E3 ubiquitin ligase, flags damaged mitochondria for destruction so they do not poison the other functional organelles. We found that Parkin promotes lipid remodeling at the surface of the mitochondria. Phosphatidic acid (PA) accumulates shortly after mitochondrial damage while diacylglycerol (DAG) appears several hours later. This lipid accumulation is dependent upon Parkin's translocation and E3 ligase activity. Additionally, we found that lipin-1, a PA phosphatase, and endophilin B1 (EndoB1) are critical for DAG accumulation and effective mitochondrial clearance. </p><p>Through this work, we show that two proteins critical in quality control mechanisms also play significant roles in energy homeostasis. We aim to highlight this overlap and posit that common diseases of aging, though presenting differently, might have disruptions in the same basic process.</p><br>Dissertation

碩士<br>國立陽明大學<br>生命科學系暨基因體科學研究所<br>102<br>Mitochondria are dynamic organelles that undergo fusion, fission and mitochondria quality control (mitophagy). Proper mitochondrial dynamics regulates mitochondrial damages to ensure cellular integrity. During cell division, not only genetic materials but also organelles are evenly distributed to daughter cells. Current model for mitochondrial inheritance in mammalian cells suggested that mitochondria undergo complete fission for even partition to daughter cells. Paradoxically, extensive mitochondrial fission may result in too low production of energy to support enough energy for mitosis; mitotic cells may still keep tubular at a particular time point of mitosis. Nowadays, no quantitative method for mitochondrial morphology, exact cell cycle-dependent mitochondrial morphological changes are not well-defined and the mechanism how bioenergetics status and mitochondrial quality control affects mitochondrial morphology is still unclear. In this study, we used 3D confocal image stacks of CHO-K1 cells to establish the automated system to extract morphological features and calculate distribution of morphological subtypes for numerical 3D mitochondrial morphological analysis. Combining literature information, we employed Gaussian Mixture Model (GMM) and Bayesian Information Criterions (BIC) to identify 6 major mitochondrial morphological subtypes. Using this system, we can know that how the dynamics work and biogenesis status and quality control during cell division. To shorten the time of cell images acquirement, using serum starvation to synchronize cell in G0 phase, and get each cell cycle step time point to reduce mitochondrial damage in excessive laser exposure. From the result of cell synchronization, we observed the mitochondrial fusion occur rapidly, and mitochondrial fragments change into mitochondrial tubular networks in G0-G1 phase. From our 3D time-lapsed videos, we find mitochondrial morphology at specific to different cell cycle stages. Especially, the mitochondrial morphology of mitotic cell is in smaller tubular network instead of fragmentation. Mitochondrial fission usually occurs at the division furrow. Besides, intensity of GFP-tagged mitochondrial matrix protein and total mitochondrial volume are reduced at early mitotic phase and gradually increased in anaphase and telophase, and imply biogenesis status and quality control are changed in mitosis.

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.

Summary: The maintenance of healthy and functional mitochondria is essential for cellular homeostasis; a first check point is provided by the organelle itself through the mitochondrial quality control and through the mitochondrial dynamics. Mitochondrial dynamics involve the Drp1 protein, a large dynamin-like GTPases encoded by DNM1L gene, that is responsible for fission of mitochondria. Mutations in DNM1L gene have been associated with several neurological disorders (Schmid et al. 2019). Furthermore, one of the main players of the mitochondrial quality control is the Lon protease encoded by LONP1 gene, involved in mitochondrial proteostasis and in the maintenance of mitochondrial DNA. Mutations in LONP1 were associated with a multisystem disorder called CODAS (Cerebral, Ocular, Dental, Auricular, Skeletal) syndrome (Strauss et al. 2015) and, more recently, with a classical mitochondrial disease phenotype (Peter et al. 2018; Nimmo et al. 2019). Through the use of Next Generation Sequencing (NGS) technology, we identified new mutations in genes involved in mitochondrial quality control and dynamics. In five patients, we identified de novo dominant DNM1L variants, two of which have been never reported. Patients’ fibroblast displayed defects in mitochondrial morphology; interestingly, we observed, in muscle biopsies, changes in mitochondrial distribution. To date, no peculiar histochemical alterations have been reported in DNM1L-mutated patients and this can represent a diagnostic tool. In one patient, we found three different mutations in LONP1, never described before. These variants cause both energy defects and alterations in mitochondrial network too.

Dues à leur importance croissante dans la dégénérescence musculaire, les mitochondries sont de plus en plus étudiées en relation à diverses myopathies. Leurs mécanismes de contrôle de qualité sont reconnus pour leur rôle important dans la santé mitochondrial. Dans cette étude, nous tentons de déterminer si le déficit de mitophagie chez les souris déficiente du gène Parkin causera une exacerbation des dysfonctions mitochondriales normalement induite par la doxorubicine. Nous avons analysé l’impact de l’ablation de Parkin en réponse à un traitement à la doxorubicine au niveau du fonctionnement cardiaque, des fonctions mitochondriales et de l’enzymologie mitochondriale. Nos résultats démontrent qu’à l’état basal, l’absence de Parkin n’induit pas de pathologie cardiaque mais est associé à des dysfonctions mitochondriales multiples. La doxorubicine induit des dysfonctions respiratoires, du stress oxydant mitochondrial et une susceptibilité à l’ouverture du pore de transition de perméabilité (PTP). Finalement, contrairement à notre hypothèse, l’absence de Parkin n’accentue pas les dysfonctions mitochondriales induites par la doxorubicine et semble même exercer un effet protecteur.<br>Mitochondria are becoming the focus of many studies because of their increasingly important role in cellular damage and related myopathies. Their endogenous quality control mechanisms are recognized for their crucial role in mitochondrial health. In our study, we attempted to determine if the deficit of mitophagy in Parkin deficient mice would cause an exacerbation of mitochondrial dysfunctions usually induced by doxorubicin. We have analyzed the impact of the ablation of Parkin in response to treatment with doxorubicin at the level of cardiac functions, mitochondrial functions as well as mitochondrial enzymology. Our results demonstrated that at baseline, the absence of Parkin didn’t induce cardiac pathologies but was associated with many mitochondrial dysfunctions. Doxorubicin induced respiratory dysfunctions, mitochondrial oxidative stress as well as greater susceptibility to permeability transition pore (PTP) opening. Finally, contrary to our hypothesis, the absence of Parkin, didn’t exacerbate mitochondrial dysfunctions induced by doxorubicin and seemed to have a protective effect.

My research focuses on understanding the importance of human mitochondrial Hsp70 (Grp75) chaperone machinery for the maintenance of protein quality control inside the mitochondrial matrix. The investigations carried out during this study have been addressed towards gaining better insights into the working of Grp75 chaperone folding machinery in association with its diverse set of co-chaperones residing in human mitochondria. Additionally, the research also focuses on explaining the various modes of Grp75 participation leading to multiple disease conditions. The thesis has been divided into the following sections as follows: Chapter I: An introduction to the mitochondrial import machinery and role of mitochondrial Hsp70 chaperone folding machinery for the maintenance of protein quality control: Mitochondrion is an essential organelle present in the eukaryotic cell and requires more than 1500 proteins for its proper functioning. Although, mitochondria harbour their own genome, it encodes for only 13 proteins in humans. The rest of the entire proteome is encoded by the nuclear genome and requires proper targeting of proteins to different compartments of mitochondria. Remarkably, mitochondrial matrix alone requires more than 60% of the proteome for its suitable functioning. Briefly, the mitochondrial matrix destined polypeptide passes through the outer membrane translocon; the ‘TOM’ complex and then enters the TIM23 translocon present in the inner membrane of mitochondria. The complete translocation of the polypeptide into the mitochondrial matrix side requires the assistance of mtHsp70 based motor system present on the matrix side which pulls the polypeptide into the matrix in an ATP-dependent manner and with the assistance of various co-chaperones. Subsequently, the unfolded polypeptide is to be folded back to its native state, which is ensured again by the mtHsp70 based chaperone folding machinery. Importantly, while 20% of mtHsp70 is involved in protein import, 80% of mtHsp70 is dedicated for protein folding. In addition to mtHsp70, the chaperone folding machinery consists of various soluble co-chaperones such as the J-proteins which stimulate the ATP hydrolysis rate of Hsp70. Furthermore, another co-chaperone termed as a nucleotide exchange factor ensures binding of fresh ATP molecule onto Hsp70 ensuring multiple rounds of folding cycles. To understand the relevance of mitochondrial Hsp70 chaperone folding machine in the maintenance of protein quality control, Chapter I of the thesis has been divided into multiple sections as follows: Briefly, the initial portion of Chapter I provide a glimpse of the translocon components present in mitochondria for targeting of proteins to outer membrane, inner membrane and inter-membrane space. Owing to the vast proteome size of the mitochondrial matrix, the following section describes the detailed mechanism and translocation process of the mitochondrial matrix targeted proteins. Additionally, subsequent sections of Chapter I provide a comprehensive description of each of the mtHsp70 chaperone folding components, which maintain the protein quality control in the matrix. The players that constitute the chaperone folding machines are mitochondrial Hsp70, J-proteins, nucleotide exchange factors and the newly discovered human escort protein. Essentially, the section provides information about the cellular distribution, structure and function of each of these players constituting the mtHsp70 chaperone folding machine. Loss of regulation between these players leads to defects in protein folding. Imbalance in protein homeostasis is one of the primary causes for mitochondrial dysfunction leading to various diseases. Importantly, recent literature has highlighted the involvement of mtHsp70 chaperone folding players in Parkinson’s disease (PD), Myelodysplastic syndrome (MDS) and cancer. In accordance, the last section of the Chapter I has been dedicated to describe the basic cell biology and proposed mechanisms for the above diseased states. Interestingly, in comparison to yeast and bacteria, the composition of mtHsp70 chaperone folding machinery in humans is unique and distinctly different. Owing to a lack of information about the functioning of human mitochondrial Hsp70 chaperone folding machinery and with an emphasis on understanding its role in various disease manifestations, the objectives that were laid for my PhD thesis are as follows: 1) Functional in vitro reconstitution of the human Grp75 chaperone folding machinery by purifying all the Grp75 chaperone folding machinery players namely; Grp75 (human mtHsp70), hTid-1L and hTid-1S (J-proteins), GrpEL1 (nucleotide exchange factor) and Human escort protein (Hep). 2) Dissection of the intrinsic biochemical defects associated with the variants of Grp75 reported in Parkinson’s disease (PD). 3) To understand the correlation between elevated levels of Grp75 and its contribution to malignancy. In conclusion, the current study has highlighted some of the key features of human Grp75 chaperone folding machinery and its regulation in the maintenance of human mitochondrial matrix protein quality control, failure of which leads to pathological conditions. Chapter II: Reconstitution of the human Grp75 chaperone folding machinery to understand the functional interplay between the multiple protein components: The mitochondrial Heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 towards mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5-peptide as compared to the other Hsp70’s. We observed that the two human mitochondrial matrix J-protein splice-variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hep possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C-terminus of mtHsp70 in a full-length context, and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C-terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thereby promoting a similar conformational change that leads to ATPase stimulation. Moreover, we have also dissected out the inter-domain defective nature associated with the point mutant of Grp75 implicated in Myelodysplastic syndrome thus providing an explanation for the loss of function of Grp75 eventually leading to loss of protein quality control in the diseased state. Chapter III: Enhanced J-protein interaction and compromised protein stability of Grp75 variants leads to mitochondrial dysfunction in Parkinson’s disease: Parkinson’s disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multi-factorial in nature, a recent proteomic screen involving PD-patients revealed two mitochondrial Hsp70 variants (P509S and R126W) that are implicated in PD-pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD-variants are centrally involved in PD-progression is totally elusive. In this report, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD-variants. Biochemically, R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, P509S variant exhibits significantly enhanced interaction with J-protein co-chaperones involved in folding and import machinery, thus altering the overall regulation of chaperone mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD-mutations at the cellular level, we have developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD-mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with ‘mitochondrial dysfunction’ such as mitochondrial DNA (mtDNA) loss and increased susceptibility to oxidative stress recapitulating the cellular features of dopaminergic neurons similar to those reported in other PD-models. Together, our observations for both the variants strongly indicate a definite involvement of mtHsp70 as a susceptibility factor in Parkinson’s disease. Chapter IV: To understand the correlation between elevated levels of Grp75 and its contribution to malignancy: Multiple studies carried out by various groups have reported the presence of elevated levels of Grp75 in cancer cells. Furthermore, proteomic screens show a positive correlation with the higher levels of Grp75 and the aggressive or metastatic nature of cancer. Importantly, cancer cells also exhibit altered mitochondrial metabolism and are found to be under constant oxidative stress pressure. Moreover, Grp75 actively participates in maintenance of mitochondrial function and as well is reported to interact with many putative oncoproteins. However, there is little information available on the possible role of Grp75 in modulating the cellular niche which might favor towards increased malignant transformation of cells. To identify pathways for explaining the correlation between Grp75 and cancer, our initial attempts have focused on monitoring the multiple cellular changes influenced by elevated levels of Grp75 in a cell line based system. To our surprise, transient transfection of cells with Grp75 led to a tremendous increase in the reactive oxygen species levels. Furthermore, a strong positive correlation between the extent of increased levels of Grp75 and the amount of ROS generated in these cells was established. As expected, increased ROS levels observed in Grp75 overexpressing cells also resulted in reduced cell viability. Notably, mitochondrial superoxide generation was found to be the major source for the observed increment in ROS levels in Grp75 expressing cells. In addition, the localization profile of the exogenously expressed Grp75 protein highlighted the fact that the protein was found to be predominantly targeted to mitochondria. Strikingly, the elevated Grp75 levels led to an increase in mitochondrial mass and also displayed a higher proportion of circular and fragmented mitochondria in these cells. Together, the above preliminary observations hint towards a strong correlation between the levels of Grp75 and its influence on the redox biology of cells providing an additional and a possible explanation of the mode of participation of Grp75 in generation and progression of malignancy.

My research focuses on understanding the importance of human mitochondrial Hsp70 (Grp75) chaperone machinery for the maintenance of protein quality control inside the mitochondrial matrix. The investigations carried out during this study have been addressed towards gaining better insights into the working of Grp75 chaperone folding machinery in association with its diverse set of co-chaperones residing in human mitochondria. Additionally, the research also focuses on explaining the various modes of Grp75 participation leading to multiple disease conditions. The thesis has been divided into the following sections as follows: Chapter I: An introduction to the mitochondrial import machinery and role of mitochondrial Hsp70 chaperone folding machinery for the maintenance of protein quality control: Mitochondrion is an essential organelle present in the eukaryotic cell and requires more than 1500 proteins for its proper functioning. Although, mitochondria harbour their own genome, it encodes for only 13 proteins in humans. The rest of the entire proteome is encoded by the nuclear genome and requires proper targeting of proteins to different compartments of mitochondria. Remarkably, mitochondrial matrix alone requires more than 60% of the proteome for its suitable functioning. Briefly, the mitochondrial matrix destined polypeptide passes through the outer membrane translocon; the ‘TOM’ complex and then enters the TIM23 translocon present in the inner membrane of mitochondria. The complete translocation of the polypeptide into the mitochondrial matrix side requires the assistance of mtHsp70 based motor system present on the matrix side which pulls the polypeptide into the matrix in an ATP-dependent manner and with the assistance of various co-chaperones. Subsequently, the unfolded polypeptide is to be folded back to its native state, which is ensured again by the mtHsp70 based chaperone folding machinery. Importantly, while 20% of mtHsp70 is involved in protein import, 80% of mtHsp70 is dedicated for protein folding. In addition to mtHsp70, the chaperone folding machinery consists of various soluble co-chaperones such as the J-proteins which stimulate the ATP hydrolysis rate of Hsp70. Furthermore, another co-chaperone termed as a nucleotide exchange factor ensures binding of fresh ATP molecule onto Hsp70 ensuring multiple rounds of folding cycles. To understand the relevance of mitochondrial Hsp70 chaperone folding machine in the maintenance of protein quality control, Chapter I of the thesis has been divided into multiple sections as follows: Briefly, the initial portion of Chapter I provide a glimpse of the translocon components present in mitochondria for targeting of proteins to outer membrane, inner membrane and inter-membrane space. Owing to the vast proteome size of the mitochondrial matrix, the following section describes the detailed mechanism and translocation process of the mitochondrial matrix targeted proteins. Additionally, subsequent sections of Chapter I provide a comprehensive description of each of the mtHsp70 chaperone folding components, which maintain the protein quality control in the matrix. The players that constitute the chaperone folding machines are mitochondrial Hsp70, J-proteins, nucleotide exchange factors and the newly discovered human escort protein. Essentially, the section provides information about the cellular distribution, structure and function of each of these players constituting the mtHsp70 chaperone folding machine. Loss of regulation between these players leads to defects in protein folding. Imbalance in protein homeostasis is one of the primary causes for mitochondrial dysfunction leading to various diseases. Importantly, recent literature has highlighted the involvement of mtHsp70 chaperone folding players in Parkinson’s disease (PD), Myelodysplastic syndrome (MDS) and cancer. In accordance, the last section of the Chapter I has been dedicated to describe the basic cell biology and proposed mechanisms for the above diseased states. Interestingly, in comparison to yeast and bacteria, the composition of mtHsp70 chaperone folding machinery in humans is unique and distinctly different. Owing to a lack of information about the functioning of human mitochondrial Hsp70 chaperone folding machinery and with an emphasis on understanding its role in various disease manifestations, the objectives that were laid for my PhD thesis are as follows: 1) Functional in vitro reconstitution of the human Grp75 chaperone folding machinery by purifying all the Grp75 chaperone folding machinery players namely; Grp75 (human mtHsp70), hTid-1L and hTid-1S (J-proteins), GrpEL1 (nucleotide exchange factor) and Human escort protein (Hep). 2) Dissection of the intrinsic biochemical defects associated with the variants of Grp75 reported in Parkinson’s disease (PD). 3) To understand the correlation between elevated levels of Grp75 and its contribution to malignancy. In conclusion, the current study has highlighted some of the key features of human Grp75 chaperone folding machinery and its regulation in the maintenance of human mitochondrial matrix protein quality control, failure of which leads to pathological conditions. Chapter II: Reconstitution of the human Grp75 chaperone folding machinery to understand the functional interplay between the multiple protein components: The mitochondrial Heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 towards mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5-peptide as compared to the other Hsp70’s. We observed that the two human mitochondrial matrix J-protein splice-variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hep possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C-terminus of mtHsp70 in a full-length context, and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C-terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thereby promoting a similar conformational change that leads to ATPase stimulation. Moreover, we have also dissected out the inter-domain defective nature associated with the point mutant of Grp75 implicated in Myelodysplastic syndrome thus providing an explanation for the loss of function of Grp75 eventually leading to loss of protein quality control in the diseased state. Chapter III: Enhanced J-protein interaction and compromised protein stability of Grp75 variants leads to mitochondrial dysfunction in Parkinson’s disease: Parkinson’s disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multi-factorial in nature, a recent proteomic screen involving PD-patients revealed two mitochondrial Hsp70 variants (P509S and R126W) that are implicated in PD-pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD-variants are centrally involved in PD-progression is totally elusive. In this report, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD-variants. Biochemically, R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, P509S variant exhibits significantly enhanced interaction with J-protein co-chaperones involved in folding and import machinery, thus altering the overall regulation of chaperone mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD-mutations at the cellular level, we have developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD-mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with ‘mitochondrial dysfunction’ such as mitochondrial DNA (mtDNA) loss and increased susceptibility to oxidative stress recapitulating the cellular features of dopaminergic neurons similar to those reported in other PD-models. Together, our observations for both the variants strongly indicate a definite involvement of mtHsp70 as a susceptibility factor in Parkinson’s disease. Chapter IV: To understand the correlation between elevated levels of Grp75 and its contribution to malignancy: Multiple studies carried out by various groups have reported the presence of elevated levels of Grp75 in cancer cells. Furthermore, proteomic screens show a positive correlation with the higher levels of Grp75 and the aggressive or metastatic nature of cancer. Importantly, cancer cells also exhibit altered mitochondrial metabolism and are found to be under constant oxidative stress pressure. Moreover, Grp75 actively participates in maintenance of mitochondrial function and as well is reported to interact with many putative oncoproteins. However, there is little information available on the possible role of Grp75 in modulating the cellular niche which might favor towards increased malignant transformation of cells. To identify pathways for explaining the correlation between Grp75 and cancer, our initial attempts have focused on monitoring the multiple cellular changes influenced by elevated levels of Grp75 in a cell line based system. To our surprise, transient transfection of cells with Grp75 led to a tremendous increase in the reactive oxygen species levels. Furthermore, a strong positive correlation between the extent of increased levels of Grp75 and the amount of ROS generated in these cells was established. As expected, increased ROS levels observed in Grp75 overexpressing cells also resulted in reduced cell viability. Notably, mitochondrial superoxide generation was found to be the major source for the observed increment in ROS levels in Grp75 expressing cells. In addition, the localization profile of the exogenously expressed Grp75 protein highlighted the fact that the protein was found to be predominantly targeted to mitochondria. Strikingly, the elevated Grp75 levels led to an increase in mitochondrial mass and also displayed a higher proportion of circular and fragmented mitochondria in these cells. Together, the above preliminary observations hint towards a strong correlation between the levels of Grp75 and its influence on the redox biology of cells providing an additional and a possible explanation of the mode of participation of Grp75 in generation and progression of malignancy.

Tese no âmbito do Doutoramento em Biologia Experimental e Biomedicina, ramo de especialização em Neurociências e Doença, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra.<br>Parkinson’s Disease (PD) is the most common movement disorder and the second most common neurodegenerative disorder, having a prevalence of ~2% in people older than 65 years old. Currently, PD has no cure and no early diagnostic method exists. Considering the increase in average age of population, PD prevalence is predicted to increase in the next years. Pathologically, PD is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) leading to a dopamine deficit in striatum. Although the exact mechanism by which PD develops and progresses is not still clear, there are evidences that mitochondrial dysfunction, impairment in quality control mechanism pathways and oxidative stress are implicated in PD pathogenesis. However, despite the increasing knowledge, there are still gaps that need to be filled for a better understanding, especially in the context of the PD sporadic form. Important questions remain unresolved, both regarding to an efficient diagnostic and therapeutics, as well as how others diseases might be a risk factor for PD. In this context, our first aim was to assess whether mitochondrial biology of human skin fibroblasts can be manipulated under standard or modified culture conditions. To do so, a glucose free/galactose/glutamine/pyruvate-containing media (OXPHOSm), that force cells to be more reliant on oxidative phophorylation (OXPHOS) for energy production, was used. The OXPHOSm could be used in a more reliable way to disclose mitochondrial liabilities of drug candidates or intrinsic metabolic differences in fibroblasts (Chapter 5). Our results showed that OXPHOSm forcing mitochondrial remodeling in human skin fibroblasts increased oxygen consumption rate, ATP levels, and mitochondria-related transcripts and proteins. Moreover, the metabolic remodeling towards a more oxidative state increased the susceptibility of fibroblasts to the cytotoxic effects of mitochondrial poisons. The chapter 5 highlights not only the importance of using human skin primary cells to study drug-induced mitochondrial toxicity, as reinforced the use of this tool to detect specific mitochondrial defects in skin fibroblasts from patients, including PD patients. Secondly, the sub-cellular events that lead to PD progression were investigate using non-neuronal cells, such as skin fibroblasts, in order to develop personalized interventions. These cells can be collected in a minimally invasive manner from diagnosed patients and be important tools to test pharmacological and non-pharmacological interventions aimed at improving mitochondrial function (Chapter 6). Human skin fibroblasts from sporadic PD (sPD) patients were used, as a cell proxy to detect metabolic and mitochondrial alterations. In this model, the same modified cell culture conditions previously described in Chapter 5 was used. Results demonstrated that fibroblasts from sPD patients show hyperpolarized and elongated mitochondrial networks paralled by an increased levels of mitochondrial reactive oxygen species (ROS) levels, as well as decreased ATP levels and glycolysis-related ECAR. Moreover, results also showed that abnormalities of fibroblasts from sPD patients became more evident when stimulating OXPHOS. Under these culture conditions, fibroblasts from sPD cells presented altered mitochondrial function, such as decreased basal respiration, ATP-linked OCR and maximal respiration, and increased mitochondria-targeting phosphorylation of DRP1 when compared to control cells. Chapter 6 validates the relevance of using fibroblasts from sPD patients to study cellular and molecular changes that are characteristic of dopaminergic neurodegeneration of PD and shows that forcing mitochondrial OXPHOS uncovers metabolic defects that were otherwise hidden. Taking into account the metabolic and mitochondrial defects in skin fibroblasts from sPD patients, a mitochondrial-directed intervention may significantly improve the cellular phenotype found in sPD (Chapter 7). Thus, a mitochondria-targeted hydroxycinnamic acid derivative (AntiOxCIN4), presenting antioxidant and iron-chelating properties, which showed to prevent oxidative stress-induced damage in several biological models of disease was evaluated in skin fibroblasts from sPD patients. The results demonstrated that treatment of human skin fibroblasts from sPD patients with a non-lethal concentration of AntiOxCIN4 restored mitochondrial membrane potential and mitochondrial fission, decreased autophagic flux, and improved cellular responses to oxidative stress by improving the cellular redox state (GSH levels) and decreasing ROS levels. In addition, fibroblasts from sPD patients treated with AntiOxCIN4 showed increased maximal respiration and metabolic activity, converting the phenotype of sPD fibroblasts more similar to their sex- and age-matched controls. Chapter 7 led to a more deep understanding on AntiOxCIN4 mechanism of action in skin fibroblasts from sPD patients and points out that mitochondria-targeted antioxidants based on a polyphenol scaffold are potential drug candidates for delaying PD progression. The data also validates the use of fibroblasts from sPD patients with more active OXPHOS as platforms for mitochondria-based drug candidates for delaying PD progression. The data also validates the use of fibroblasts from sPD patients with more active oxidative phosphorylation as platforms for mitochondria-based drug development. Lastly, the work focused on how others diseases, namely lysosomal storage disorders (LSDs) could be a risk factor for PD. In this context, the fourth aim was to evaluate whether lysosomal malfunction alters authophagy and triggers proteasomal saturation and accumulation of protein aggregates leading to molecular features which are characteristic from PD (Chapter 8). To do so, an acid α-glucosidase (GAA) knockout model was used. Preliminary data showed that lysosomal dysfunction associated with GAA deficiency triggers autophagic pathway in the brain and induces some pathological hallmarks of PD, such as α-synuclein protein content increased, decreased metabolic activity, and induction of mitophagy, suggesting mitochondrial alterations. Overall, the results obtained in this dissertation added novel and relevant knowledge not only by showing the importance of using human skin primary cells for personalized medicine, particularly to detect specific mitochondrial defects in sPD patients, as well as, to validate a mitochondria-directed antioxidant intervention based on dietary phenolic acid antioxidant as a potential therapeutic tool to revert some PD-associated mitochondrial defects. Additionaly, the work presented in this dissertation also added knowledge in how dysfunctional lysosomes impact quality control mechanisms and induce molecular features which are characteristic of PD, giving new insights of how others LSDs might be a risk factor for PD.<br>A doença de Parkinson (DP) é a doença mais comum a afetar a coordenação de movimentos e a segunda doença como doença neurodegenerativa, apresentando uma prevalência de ~ 2% em pessoas com mais de 65 anos. Actualmente, a DP não tem cura e nem existe nenhum método que permita um diagnóstico precoce. Considerando o aumento na idade média da população, prevê-se que a prevalência da DP aumente nos próximos anos. Patologicamente, a DP é caracterizada pela perda de neurónios dopaminérgicos na substantia nigra pars compacta (SNpc) levando a uma diminuição dos níveis de dopamina no corpo estriado. Embora o mecanismo exato pelo qual isso ocorre ainda não seja completamente conhecido, há evidências de que a disfunção mitocondrial, alterações nas vias dos mecanismos de controlo de qualidade celulares e o stress oxidativo contribuem para a patogénese da DP. Apesar do crescente conhecimento sobre a DP, várias lacunas precisam ser colmatadas para um melhor entendimento da patofisiologia desta doença, em particular para a forma esporádica. Questões pertinentes permanecem sem resposta, tanto no que diz respeito ao diagnóstico ou a uma terapêutica eficaz, como em relação a compreender se a existência de outras doenças pode ser um fator de risco para o desenvolvimento da DP. Neste contexto, o nosso primeiro objetivo foi avaliar a biologia mitocondrial de fibroblastos da pele humana em condições de cultura padrão ou modificadas usando um meio de cultura sem glucose e que contém galactose/glutamina/piruvato o qual força os fibroblastos a dependerem mais da fosforilação oxidativa para a produção de energia. Esta estratégia permitirá de um modo mais confiável descobrir disfunções mitocondriais na toxicidade de compostos candidatos a fármacos ou mesmo revelar diferenças metabólicas intrínsecas em fibroblastos de diferentes dadores (Capítulo 5). Os resultados mostraram que forçar a remodelação mitocondrial nos fibroblastos da pele humana causou um aumento da taxa de consumo de oxigénio, dos níveis de adenosina trifosfato (ATP) e dos transcritos e proteínas relacionados com as mitocôndrias. Além disso, a remodelação metabólica para um estado mais oxidativo intensificou a citotoxicidade de tóxicos mitocondriais. O capítulo 5 não só destacou a relevância do uso de células primárias da pele humana para o estudo da toxicidade mitocondrial induzida por possíveis fármacos, mas também reforça o uso desta ferramenta e abordagem para encontrar defeitos mitocondriais específicos em fibroblastos da pele de pacientes, incluindo pacientes com a DP. O segundo objetivo foi investigar os eventos subcelulares que causam a progressão da DP e desenvolver intervenções personalizadas, usando células não neuronais, recolhidas de uma forma menos invasiva, e que podem ser fundamentais para testar intervenções destinadas a melhorar a função mitocondrial (Capítulo 6). Usamos fibroblastos da pele humana de pacientes com a DP esporádica como modelo celular para detetar alterações metabólicas e mitocondriais. Neste modelo, usamos a estratégia de cultivo de células descrita no capítulo 5. Demonstramos que os fibroblastos de pacientes com a DP esporádica apresentam uma rede mitocondrial hiperpolarizada e alongada exibindo um conteúdo aumentado de espécies reativas de oxigênio mitocondriais, bem como uma diminuição dos níveis de ATP e da taxa de acidificação extracelular relacionado com a glicólise. Os nossos resultados também mostraram que as anomalias dos fibroblastos de pacientes com a DP esporádica tornaram-se mais evidentes quando a fosforilação oxidativa foi estimulada. Nessas condições de cultura celular, os fibroblastos das células de DP esporádica exibiram uma diminuição da respiração basal, da taxa de consumo de oxigénio associada à produção de ATP e da respiração máxima, e apresentaram um aumento da fosforilação da proteina DRP1, quando comparados às células de indivíduos do grupo controlo. Esta parte do trabalho validou a relevância do uso de fibroblastos da pele de pacientes com a DP esporádica para estudar alterações celulares e moleculares que são características da neurodegeneração dopaminérgica que ocorre na DP e demonstrou que forçar a fosforilação oxidativa mitocondrial revela defeitos metabólicos que de outra forma permaneceriam ocultos. Tendo em consideração as alteracões metabólicas e mitocondriais presentes nos fibroblastos de pacientes com a DP esporádica, uma intervenção direccionada à mitocôndria pode significativamente melhorar o fenótipo encontrado nessas mesmas células. Assim, um derivado do ácido hidroxicinâmico direcionado à mitocôndria (AntiOxCIN4), o qual apresenta propriedades antioxidantes e quelantes de ferro, que previne o stress oxidativo em diversos modelos biológicos de doença, foi testado em fibroblastos da pele de pacientes com a DP esporádica (Capítulo 7). Os nossos resultados demonstraram que o tratamento dos fibroblastos da pele de pacientes com a DP esporádica com uma concentração não letal de AntiOxCIN4 restaurou o potencial de membrana mitocondrial e a fissão mitocondrial, diminuiu o fluxo autofágico e melhorou a resposta celular ao stress oxidativo, melhorando o estado redox celular (níveis de glutationa reduzida, GSH) e diminuindo as espécies reativas de oxigénio (ERO). Além disso, os fibroblastos de pacientes com a DP esporádica tratados com o AntiOxCIN4 exibiram um aumento da respiração máxima e da atividade metabólica, convertendo o fenótipo dos fibroblastos com DP esporádica mais semelhantes aos seus controlos de igual sexo e idade. Estes dados apontaram um possível mecanismo de ação do AntiOxCIN4 permitindo um entendimento mais profundo de como o uso de antioxidantes direcionados para a mitocôndria, com base numa arcabouço de polifenol, apresentam potencial terapêutico para serem usados como novos fármacos para retardar a progressão da DP. Os resultados obtidos também validam o uso da estratégia do cultivo de fibroblastos de pacientes com DP esporádica em condições que forçam a fosforilação oxidativa como uma plataforma para o desenvolvimento de medicamentos que tenham a mitocôndria como alvo, visto que os seus efeitos pretendidos ou colaterais serão evidenciados. Por último, estudamos como outras doenças, particularmente doenças do armazenamento lisossomal (DALs), podem ser um fator de risco para o desenvolvimento da DP. Nesse contexto, o nosso quarto objetivo foi avaliar se a disfunção lisossomal, recorrendo a um modelo animal sem α-glucosidase ácida (silenciamento total por knockout), alterará o fluxo autofágico desencadeiando a saturação proteassomal e a acumulação de agregados proteicos causando padrões moleculares característicos da DP (Capítulo 8). Os nossos resultados preliminares evidenciaram que a disfunção lisossomal associada à deficiência da α-glucosidase ácida estimula a via autofágica no cérebro e induz algumas das características patológicas da DP, tais como o aumento do conteúdo da proteína α-sinucleína, a diminuição da atividade metabólica e a estimulação da mitofagia, sugerindo a ocorrência de alterações mitocondriais. Os resultados obtidos nesta tese acrescentaram conhecimento inovador e pertinente sobre a DP; não só validam a importância do uso de células primárias da pele humana para uma medicina personalizada, em particular para detectar disfunções mitocondriais em doentes com a DP e avaliar a eficácia de possíveis tratamentos, como também permitiram validar o uso de um composto com base num antioxidante presente na dieta humana como uma molécula com potencial terapêutico para reverter alguns dos defeitos das linhas celulares de DP possibilitando a utilização desse conhecimento no desenvolvimento de novos fármacos. Além disso, o trabalho apresentado nesta tese também acrescentou conhecimento de como a acumulação de lisossomas disfuncionais influencia os mecanismos de controlo de qualidade celular e induzem atributos moleculares característicos da DP, apontando que outras doenças e/ou DALs podem ser um fator de risco para o desenvolvimento da DP.

<p>Nitric oxide (NO) is an endogenous gaseous signaling molecule produced by three NO synthase isoforms (NOS1, 2, 3) and important in host defense. The induction of NOS2 during bacterial sepsis is critical for pathogen clearance but its sustained activation has long been associated with increased mortality secondary to multiple organ dysfunction syndrome (MODS). High levels of NO produced by NOS2 incite intrinsic cellular dysfunction, in part by damaging macromolecules through nitration and/or nitrosylation. These include mitochondrial DNA (mtDNA) and enzymes of key mitochondrial pathways required for maintenance of normal O2 utilization and energy homeostasis. However, animal studies and clinical trials inhibiting NOS2 have demonstrated pronounced organ dysfunction and increased mortality in response to live bacterial infections, confirming that NOS2 confers pro-survival benefits. Of particular interest here, the constitutive NOS1 and NOS3 have been linked to the up-regulation of nuclear genes involved in mitochondrial biogenesis but no comparable role has been described for NOS2. <italic> Therefore, I hypothesized that NOS2 is indispensible for host protection but must be tightly regulated to ensure NO levels are high enough to activate mitochondrial and other pro-survival genes, but below the threshold for cellular damage.</italic></p><p>This hypothesis was explored with two major Aims. The <italic>first Aim</italic> was to define the role of NOS2 in the activation of mitochondrial biogenesis in the heart of <italic>E. coli</italic>-treated mice. The <italic>second</italic> was to investigate the ability of NOS2 to be transcriptionally regulated by an enzyme previously shown to induce mitochondrial biogenesis, heme oxygenase-1 (HO-1). This hypothesis was tested using an <italic>in vivo</italic> model of sublethal heat-killed <italic>E. coli</italic> (<italic>HkEC</italic>) peritonitis in C57B/L6 (Wt), NOS2-/-, and TLR4-/- mice. Additionally, <italic>in vitro</italic> systems of mouse AML-12 or Hepa 1-6 cells pretreated with HO-1 activators or <italic>Hmox1</italic> shRNA prior to inflammatory challenge with lipopolysaccharide (LPS) +/- tumor necrosis factor-&alpha; (TNF-&alpha;). For the first Aim, Wt, NOS2-/-, and TLR4-/- mice were treated with (<italic>HkEC</italic> and cardiac tissue analyzed for mitochondrial function, expression of nuclear and mitochondrial proteins needed for mitochondrial biogenesis, and histological expression of NOS2 and TLR4 relative to changes in mitochondrial mass. For the second Aim, Wt mice were pretreated with hemin or carbon monoxide (CO) to activate HO-1 prior to <italic>HkEC</italic>-peritonitis. Liver tissue in these animals was evaluated at four hours for HO-1 induction, <italic>Nos2</italic> mRNA expression, cytokine profiles, and nuclear factor (NF)-&kappa;B activation. Liver cell lines were pretreated with hemin, CO-releasing molecule (CORM), or bilirubin one hour before LPS exposure and the <italic>Nos2</italic> transcriptional response evaluated at two and 24 hours. The MTT assay was used to confirm that <italic>in vitro</italic> treatments were not lethal. </p><p>These studies demonstrated that <italic>HkEC</italic> induced mtDNA damage in the heart that was repaired in Wt mice but not in NOS2-deficient mice. In KO mice, sustained mtDNA damage was associated with the reduced expression of nuclear (NRF-1, PGC-1&alpha;) and mitochondrial (Tfam, Pol-&gamma;) proteins needed for mitochondrial biogenesis. The findings thus supported that NOS2 is required for mitochondrial biogenesis in the heart during Gram-negative challenge. Evaluation of the relationship between HO-1 and NOS2 in murine liver was more complex; HO-1 activation in <italic>HkEC</italic>-treated Wt mice attenuated 4-hour <italic>Nos2</italic> gene transcription. In liver cell lines, hemin, CORM, and bilirubin were unable to suppress <italic>Nos2</italic> expression at the time of maximal induction (2 hours). <italic>Nos2</italic> was, however, suppressed by 24 hours, suggesting that the regulatory impact of HO-1 induction was not engaged early enough to reduce <italic>Nos2</italic> transcription at 2 hours. It is concluded that NOS2 induction in bacterial sepsis optimizes the expression of the mitochondrial biogenesis transcriptional program, which subsequently can also be regulated by HO-1/CO in murine liver. This provides a potential new mechanism by which immune suppression and mitochondrial repair can occur in tandem during the acute inflammatory response.</p><br>Dissertation

Mitochondria are ubiquitous organelles placed at the nexus of several metabolic and signaling pathways essential for cell survival. Therefore, maintaining a healthy and functional organelle becomes paramount for the cell. The complex structural organization and the bi-genomic nature of the mitochondria pose a significant challenge in maintaining their homeostasis. In addition to the proteins encoded by the mitochondrial DNA (mtDNA), the mito-proteome primarily consists of nuclear-encoded proteins synthesized in the cytosol and subsequently translocated to the mitochondria. Thus, the biogenesis and functioning of the mitochondria are dependent on the efficient transport, folding, and localization of the nuclear-encoded proteins. Any disruptions in this chain of events can be detrimental to the mitochondria and, thereby, to the cell. As a result, several quality-control mechanisms have evolved that operate at multiple levels to abate any mitochondrial damage due to internal and external cellular stress. Within the mitochondria, the import, folding, targeting, and degradation of proteins are regulated by the molecular chaperones. Among these, the mitochondrial Hsp70 (mtHsp70) is a crucial mediator of protein quality control. In conjunction with multiple co-chaperones, mtHsp70 performs two critical functions: the vectorial import of the nascent polypeptides into the mitochondria and their subsequent folding within the matrix. At the organellar level, a quality check is monitored by the segregation and degradation of superfluous or dysfunctional mitochondria via the process of mitophagy. This is achieved by the concerted action of AuTophaGy (ATG) related proteins and the dynamics of the mitochondrial network. Interestingly, studies reveal that increased mitophagy mitigates the effects of mtHsp70 mutations identified in patients with Parkinson’s disease, thus, suggesting an overlap between the quality control pathways. However, the details and implications of this interaction remain unexplored. Thus, in the current study, we have employed an array of genetic and biochemical techniques in the yeast model system to understand the overlap between the quality checkpoints and, further, to delineate the involvement of mtHsp70-mediated quality control in the progression of Congenital Sideroblastic Anemia (CSA). We have explored how mtHsp70-mediated quality control engages and responds to the abrogation of mitophagy. Utilizing an unbiased genetic screen, we have identified mtHsp70 mutants that exhibit compromised growth without the mitophagy receptor, Atg32. This is accompanied by an alteration in the mitochondrial physiology, general autophagy, lipid homeostasis, and redox balance overall, resulting in a reduction in cellular lifespan. Our findings highlight the role of mtHsp70 in maintaining mitochondrial integrity under stress conditions and underscore the need for an overlap between the quality control pathways. Further, we have investigated the role of mtHsp70 in the onset and progression of Congenital Sideroblastic Anemia (CSA), a hereditary blood disorder characterized by the accumulation of iron-laden mitochondria. Preliminary analyses of analogous mutations in the yeast mtHsp70 reveal perturbations in the mitochondrial network and functionality. Further, we observe mutations in mtHsp70 impair its import and chaperone activity resulting in a loss of function that manifests as the disease phenotypes observed in Congenital Sideroblastic Anemia. The current study provides insights into the interaction between the various mitochondrial quality checkpoints and highlights the relevance of protein quality control in the context of Congenital Sideroblastic Anemia progression.<br>Indian Institute of Science