Browsing by Subject "mitochondria"

Autophagy is a cellular recycling and quality control process that eliminates cellular material in a non-selective or selective fashion. Macroautophagy is non-selective, and degrades macromolecules or damaged organelles to sustain cellular homeostasis. The selective autophagy of dysfunctional or excess mitochondria is known as mitophagy. The clinical importance of functional degradation is exemplified by the lysosomal storage disorders (LSDs), where lysosomal hydrolytic enzymes are absent or dysfunctional. Previous investigations of a rare infantile LSD indicated a change in autophagy and decreased mitochondrial content. The aim of this MSc thesis was to quantitatively compare macroautophagy and mitophagy in a cellular model of this rare LSD, by generating fluorescent macroautophagy and mitophagy reporter-expressing cell lines from patient material. Fibroblasts derived from patients diagnosed with a rare infantile LSD were transduced with lentiviruses carrying either mCherry-GFP-LC3 or mito-QC reporters, for the microscopic analysis of autophagy and mitophagy, respectively. I also monitored autophagic flux by traditional biochemistry in untreated and starved cells, in the presence or absence of lysosomal inhibitors (bafilomycin A1). Basal and iron-depletion induced mitophagy was profiled using confocal microscopy, quantitative cell biology and biochemistry. My findings suggest differential autophagic turnover in LSD patient-derived fibroblasts, with a marked accumulation of non-acidified autophagic structures. Basal mitophagy was elevated in two out of three LSD patient cell lines compared to unaffected controls. LSD patient cells exhibited altered mitochondrial content and network architecture compared to controls. These phenotypes were accompanied by distinct changes in the endo-lysosomal system and increased cell size. The patient-derived cells exhibit a profound accumulation of lysosomes and autophagic structures. My findings are in accordance with previous research in the field, suggesting perturbed macroautophagy in this rare LSD. The observations of altered mitochondrial homeostasis in this LSD provide a basis for future investigation. The reporter-expressing cells, generated as part of this MSc thesis project, will enable future studies of mechanisms that underlie phenotypic changes, and will complement essential in vivo work in this area.

Aging is the progressive accumulation of cellular dysfunction, stress and inflammation. The mitochondrial network plays a central role in the maintenance of cellular homeostasis, with a growing body of evidence assigning dysfunctional regulation of this network as cause or effect of age-related diseases including metabolic disorders, neuropathies, various forms of cancer and neurodegenerative diseases. Neuronal sensitivity to changes in energy supply and metabolic homeostasis make neurons especially susceptible to alterations in the mitochondrial network. Mitophagy, a specified form of autophagy, is the selective degradation and quality control mechanism of mitochondria by engulfment and fusion with acidic endolysosomal compartments of the cell. Mitophagy has been extensively characterised in cultured cells and short-lived model organisms. However, our understanding of physiological mitophagy during mammalian aging is unknown. This study utilizes mito-QC mitophagy reporter mice that enable in vivo detection and monitoring of mitochondrial turnover due to the distinct physicochemical properties of the tandem GFP-mCherry reporter. Using cohort groups of young and aged reporter mice, age-dependent alterations of mitophagy were quantified in the cerebellum and the outer nuclear layer (ONL) of the retina. Specific autophagy and mitophagy markers were used to assess the longitudinal alterations in the mitophagic landscape. Images of fixed brain tissue sections were attained by high-speed spinning disc confocal microscopy for the quantitative and histological analysis. This study characterises the longitudinal alterations of mitophagy in distinct regions of the central nervous system (CNS) of mitophagy reporter mice, demonstrating tissue-specific alterations in mitochondrial turnover throughout physiological time. Åldrande kan definieras som den successiva ackumuleringen av cellulär dysfunktion, stress och inflammation. I upprätthållandet av cellens funktioner och homeostas har det mitokondriella nätverket en central roll. Omfattande forskning visar att åldersrelaterade sjukdomar såsom neuropati, ämnesomsättningssjukdomar, olika cancerformer samt neurodegenerativa sjukdomar föranleds av mitokondriell dysfunktion. Neuroner är beroende av oavbruten energitillförsel och upprätthållen metabolisk homeostas, vilket gör dem speciellt mottagliga för förändringar i det mitokondriella nätverket. Mitofagi är en selektiv form av autofagi som degenererar och kvalitetskontrollerar mitokondrier genom att leverera dem till lysosomer där de bryts ned av hydrolytiska enzymer. Den aktuella kunskapen inom regleringen av och mekanismerna bakom mitofagi baserar sig på gedigen forskning av kortlivade organismer och cellkulturer. Däremot är vår kunskap inom åldrandets inverkan på mitofagi i däggdjur begränsad. I denna studie används musmodellen mito-QC vars rapportörgen består av ett binärt GFP-mCherry-komplex som besitter olika fysikaliska och kemikaliska egenskaper, vilket möjliggör upptäckt och analys av mitofagi in vivo. En kvantitativ jämförelse av mitofagi i unga och åldrande möss genomfördes i vävnadssnitt av cerebellum och av det yttre nukleära lagret av retinan. Specifika autofagi- och mitofagimarkörer användes för att utvärdera de longitudinella förändringarna i mitokondriell degenerering. Bilder för kvantitativ och histologisk analys erhölls med höghastighets spinning-disk-konfokalmikroskop. Denna forskning karaktäriserar de longitudinella förändringarna av mitofagi i definierade regioner av det centrala nervsystemet i musmodellen mito-QC och presenterar vävnadsspecifika förändringar i degenereringen av mitokondrier under åldrandets framskridande.

Drug-induced liver injury (DILI) is a relatively rare hepatic condition that can be classified as predictable and unpredictable. However, DILI is a primary reason for drug withdrawals, post-marketing warnings, and restrictions of use. DILI is a problem for the drug users but also for the pharmaceutical industry and regulatory bodies. From the perspective of patients' and clinicians', DILI is the major cause of acute liver injury. At present, a major problem predicting DILI in drug discovery is a poor understanding of its mechanisms as well as the complexity of DILI pathogenicity. The main mechanism behind DILI are alterations in bile acid homeostasis, oxidative stress, and mitochondrial dysfunction. More than 50 % of drugs causing DILI are causing mitochondrial impairment. If the normal function of mitochondria is disturbed, the energy production of the cell decreases, and cell function decline leading eventually to the cell death. In this study prediction of mitochondrial toxicity was studied using cryopreserved primary hepatocytes of humans and rats. The aim of the study was to clarify if there are interspecies differences in the prediction of toxicity but also investigate possible differences in the mechanisms behind hepatotoxicity by using three well-known compounds toxic to mitochondria. To determine these differences, total cellular ATP was measured after 2- and 24- hour exposure time to gain information on overall viability and possible adaptive responses. Mitochondrial energy pathways were studied as a real-time monitoring acute exposure of test compounds. Morphology, location, and possible adaptive response of mitochondria were studied using a fluorescent probe and antibody staining combined with high content imaging (HCI). Overall, primary rat hepatocytes were more sensitive to the test compounds than human hepatocytes. Also, there were differences between human hepatocyte batches that may reflect the metabolic differences between hepatocyte donors. Immunolabeling did not bring any additional values compared to the fluorescent probe staining in the study of morphology of mitochondria. Additionally, it was noticed that treatment with paraformaldehyde significantly changed the hepatocyte mitochondria morphology. Overall, more effort is needed to develop image analysis of mitochondria morphology. Finally, studying mitochondrial morphology has proven to be difficult, and this study did not unfortunately reveal any information about the adaptive responses of mitochondria for drug-induced liver injury.

Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.

Tausta: Mitokondriaalisen ATP6-geenin koodaama proteiini on yksi ATP-syntaasin alayksiköistä. Tämän geenin mutaatiot aiheuttavat useita neurologisia sairauksia vaihtelevin fenotyypein. Näitä ovat esimerkiksi Leighn syndrooma ja NARP sekä erilaiset ataksiat ja neuropatiat. ATP-syntaasi on avainasemassa solujen ATP:n tuotannossa ja lisäksi sen dimerisaatio on oleellista mitokondrioiden kristojen muodostumisen kannalta. Tavoitteet: Tutkimme uuden heteroplasmisen MT-ATP6-geenin mutaation (m.9154C>T) toiminnallisia vaikutuksia solutasolla. Metodit: Mutaatiota kantavan potilaan fenotyyppiin kuuluivat perifeerinen neuropatia, ataksia, sekä IgA-nefropatia. Keräsimme potilaalta solunäytteitä ja mallinsimme mutaatiota erilaistamalla potilaalle spesifejä indusoituja pluripotentteja kantasoluja motoneuroneiksi. Lisäksi arvioimme mutaation vaikutuksia molekyylirakenteeseen ja -stabiliteettiin simulaatiomenetelmin. Tulokset: MT-ATP6-geenin mutaation seurauksena ATP-syntaasi ei muodostunut normaalisti ja aiheutti muutoksia mitokondriaalisten kristojen morfologiassa. Tutkimuksessamme havaittiin useita heteroplasmian raja-arvoja, joiden ylittyessä ilmeni muutoksia solujen uudelleen ohjelmoinnissa, neurogeneesissä Notch-geenin yliaktivaatioon liittyen sekä kypsien motoneuronien laktaattimetaboliassa. Johtopäätökset: Nämä tulokset viittaavat siihen, että ATP-syntaasin säätelemällä mitokondrioiden morfologialla on kriittinen rooli ihmisen solujen erilaistumisprosessissa, sekä motoneuronien metaboliassa. Tämä saattaa olla oleellista mitokondrioiden roolin ja mekanismien ymmärtämisessä perifeeristen neuropatioiden yhteydessä.