Abstract Alternative splicing provides a versatile mechanism by which cells can generate proteins with different or even antagonistic properties. Herein we describe a novel splice variant of the hypoxia-inducible death gene Bnip3. Sequence analysis of the new Bnip3 protein revealed an N-terminus that was identical to Bnip3 but contained an Endoplasmic reticulum (ER) retention motif within the C-terminus, therefore we designated the new Bnip3 isoform NIPLET for (Nip-Like ER Target). While Bnip3 was predominately localized to mitochondria and promoted mitochondrial perturbations and cell death, NIPLET was preferentially localized to the ER and opposed the cytotoxic actions of Bnip3. Interestingly, NIPLET suppressed mitochondrial injury from Bnip3 activation and mitochondrial permeability transition pore opening by a mechanism dependent upon the dynamin motor protein Mitofusin-2 (MFN2). Notably, mutations of NIPLET within the critical ER retention motif rendered NIPLET defective for interacting with MFN2 and suppressed necrosis induced by Bnip3 or hypoxia. Hence, our findings reveal a novel signaling pathway that functionally couples ER and mitochondria for cell survival to a mechanism that is mutually dependent and obligatorily linked to a novel BNIP3 protein in cardiac myocytes. / May 2016

No description available.

Thesis advisor: Thomas N. Seyfried / Mitochondria are the key regulators of the bioenergetic state of the cell. Damage to mitochondrial protein, DNA, or membrane lipids can result as the cause or affect of disease pathology. Regardless, this damage can impair mitochondrial function resulting in a decreased ability to produce ATP to support cellular viability. This thesis research examined the mitochondrial lipidome by shotgun lipidomics in different populations of C57BL/6J (B6) brain mitochondria (non-synaptic and synaptic) and correlated lipid changes to differences in electron transport chain (ETC) activities. Furthermore, a comparison was made for non-synaptic mitochondria between the B6 and the VM mouse strain. The VM strain has a 1.5% incidence of spontaneous brain tumors, which is 210 fold greater than the B6 strain. I determined that differences in the brain mitochondrial lipidome existed in the VM strain compared to the B6 strain, likely corresponding to an increased rate of spontaneous brain tumor formation. Analysis of the mitochondrial genome in the CT-2A, EPEN, VM-NM1, and VM-M3 brain tumors compared to their syngeneic controls mouse strains, C57BL/6J (B6) and VM mice, was examined to determine if mutations existed in experimental brain cancer models. No pathogenic mtDNA mutations were discovered that would likely cause a decrease in the mitochondrial functionality. A novel hypothesis was devised to examine the tumor mitochondrial lipidome to determine if quantitative or molecular species differences existed that could potentially alter the functionality of the ETC. Brain tumor mitochondria were examined from tumors grown in vivo as well as in vitro. Numerous lipid differences were found in the mitochondria of brain tumors, of which the most interesting involved the unique molecular speciation of cardiolipin. ETC activities were significantly decreased in the primary ETC complexes which contribute protons to the gradient as well as the linked complexes of brain tumor mitochondria compared to controls. Taken together, it is likely that differences in the mitochondrial lipidome of brain tumors results in severe impairment of the mitochondria’s ability to produce ATP through the ETC. This research has provided a new understanding of the role of mitochondrial lipids in brain as well as brain cancer and offers an alternative explanation for metabolic dysfunction in cancer. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Hematopoietic stem cells (HSCs) take on the extraordinary role of sustaining life-long production of blood cells. Despite their indisputable therapeutic potential, HSC biology is poorly understood, and the field remains limited by the inability to maintain, expand, or generate HSCs in vitro. The aim of this study was to elucidate a particular gap in our understanding of the organellar cell biology of HSCs, specifically the role and function of the mitochondria. Several signaling pathways and biological processes converge onto the mitochondria, yet these organelles were found to be largely dispensable in HSCs on the basis of their predominantly glycolytic metabolism and reports of low mitochondrial content. Our studies show that MitoTracker Green (MTG), a frequently used fluorescent dye to measure mitochondrial mass in hematopoietic populations, is effluxed by HSCs resulting in their systematic and deceptive enrichment in the subset of cells with the lowest MTG fluorescence. Using dye-independent methods we discovered that HSCs have elevated mitochondrial content despite their reliance on glycolysis for ATP production. Moreover, mechanisms of mitochondrial quality control and clearance by autophagy appear to be comparatively lower in HSCs than in any other hematopoietic population we analyzed, suggesting HSCs maintain their mitochondria over time. To investigate the function of mitochondria in HSCs we generated mice with disruption of mitofusins (MFN) 1 and 2. These proteins are key mediators of mitochondrial fusion, a process that in coordination with mitochondrial fission regulates mitochondrial size, number, and function. Mice with deletion of Mfn1 and Mfn2 (DKO) die perinatally, are pale in appearance and their HSCs show complete loss of regenerative capacity. Several processes linked to dysfunctional mitochondrial fusion and known to be tightly regulated in HSCs are altered in these mutants, including mitochondrial morphology, mitochondrial mass, proliferation, and altered metabolism. Interestingly, one allele of Mfn1 is sufficient to rescue the hematopoietic function and lethality of DKO mice, while one allele of Mfn2 only rescues myeloid reconstitution. Taken together, our findings highlight the importance and complexity of mitochondrial function and dynamics in HSCs and have contributed to the recently increased appreciation of a vital role for mitochondria in HSCs.

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

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

Ischaemia-reperfusion (IR) injury is caused by the re-introduction of oxygen to organs, following periods of reduced blood flow (ischaemia). Whilst re-establishing blood flow (reperfusion) to the heart following myocardial infarction is vital for organ survival, this paradoxically leads to tissue damage. Mitochondria are at the heart of IR injury, with succinate dehydrogenase (SDH) a major player in orchestrating the damage. Succinate accumulates during ischaemia and is rapidly oxidised by SDH upon reperfusion, producing reactive oxygen species (ROS), leading to cellular death. I have investigated the development of drugs, aimed at targeting succinate metabolism to ameliorate IR injury. I firstly screened a range of compounds for their ability to inhibit SDH, having been chosen for their similar structures to succinate or the classical SDH inhibitor, malonate. Interestingly, only malonate and oxaloacetate showed potent SDH inhibition, thus were selected for further development. Malonate ester prodrugs with different properties were characterised. Hydrolysis rates of the esters differed greatly, with tuned, labile, malonate esters releasing malonate much more rapidly. Malonate esters were taken up into cells and hydrolysed to release malonate to different extents. Additionally, mitochondria-targetedmalonatemono and diesters were developed, each differing in mitochondrial and cellular uptake andmalonate release. Targeted and nontargeted malonate esters distributed into tissues in vivo, with preliminary in vivo work carried out on IR injury models, to assess for protective effects of the compounds. In addition, the physiological role of the tricarboxylic acid cycle metabolite, itaconate, was investigated. In lipopolysaccharide stimulated macrophages, itaconate has been reported to exert its effects by inhibition of SDH however, I found itaconate was a relatively poor SDH inhibitor, indicating other mechanisms of action. Current prodrugs of itaconate have many non-specific effects, not attributable to itaconate. I therefore characterised a novel itaconate prodrug and found it to be a much better surrogate, which could be subsequently used to elucidate roles for itaconate. Overall, I have shown the importance of ester selection for the prodrug delivery of dicarboxylate molecules and developed methods to improve their biological delivery.

by Lee Fung Yee Janet. / Thesis (M.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 257-316). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Both an intuitive observation and maybe the most mysterious process of biology, aging describes the progressive deterioration of cellular functions with time. Asymmetric cell divisions stand at the center of ability to reset age in offspring and for stem cells to self-renew. This requires the asymmetric segregation of age-determinants, many of which have been identified in the budding yeast Saccharomyces cerevisiae. We here use budding yeast to explore fundamental aspects underlying the asymmetric inheritance of mitochondria and the concurrent rejuvenation of daughter cells. We show that in addition to the preferential inheritance of high-functioning mitochondria to daughter cells, a distinct population of high-quality organelles must also be retained within the mother cell. We find that both physical retention and qualitative maintenance of a distinct mitochondrial population at the mother cell tip depends on Mitochondrial F-box protein (Mfb1p) and that MFB1-deletion leads to premature aging. Our findings outline a critical balance between the need for daughter cell rejuvenation and the requirement to conserve replicative potential within the mother cell. The particular mechanism by which Mfb1p functions further lead us to uncover a critical role of globally maintained cellular polarity in form of an axial budding pattern in lifespan regulation, the functional significance of which thus far remained essentially unexplored. We also find that the asymmetric localization of Mfb1p depends on potentially novel structures of the actin cytoskeleton and the loss of Mfb1p-polarization with age may accurately predict remaining cellular lifespan.

No description available.