Molekulární podstata deficitu F1Fo-ATP syntázy a jeho dopad na energetický metabolismus buňky

Mitochondria's primary function is to produce energy through the process of oxidative phosphorylation. ATP synthase is a macromolecular rotary machine located in the inner mitochondrial membrane that catalyzes the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). The mitochondrial disorders due to ATP synthase deficiency represent a heterogeneous group of diseases characterized by variable severity of the phenotype with onset at birth or later in life till adulthood. Mutations in both, mitochondrial or nucelar DNA encoded genes, may result in ATP synthase impairment, either isolated or combined with deficits of other complexes of oxidative phosphorylation. The aims of the thesis were to characterize TMEM70 protein, an ATP synthase assembly factor, and to analyze the impact of novel disease variants leading to ATP synthase deficiency in patients' derived samples. TMEM70 is a 21 kDa hairpin structure protein localized in the inner mitochondrial membrane, with both termini oriented into the matrix, which forms higher oligomer structures. Our results demonstrated that the absence of TMEM70 protein leads to an isolated deficiency of complex V followed in some stage by adaptive/compensatory effect of respiratory chain complexes. Different severities...Hlavní funkcí mitochondrií je produkce energie prostřednictvím procesu oxidační fosforylace. ATP syntáza je makromolekulární rotační stroj lokalizovaný ve vnitřní mitochondriální membráně katalyzující syntézu adenosintrifosfátu (ATP) z adenosindifosfátu (ADP) a anorganického fosfátu (Pi). Mitochondrialní poruchy ATP syntázy představují heterogenní skupinu onemocnění charakterizovanou různou závažností klinického fenotypu s nástupem od narození nebo v pozdějších fázích života. Mutace v mitochondriální nebo jaderné DNA mohou vést k poruše ATP syntázy, a to buď izolovaně, nebo v kombinaci s defekty dalších komplexů systému oxidační fosforylace. Cílem práce bylo charakterizovat protein TMEM70, asemblační faktor ATP syntázy, a studovat vliv nových variant vedoucích k deficitu ATP syntázy v pacientských vzorcích. TMEM70 je 21 kDa velký protein vnitřní mitochondriální membrány s orientací obou konců do mitochondriální matrix, který tvoří v membráně vyšší oligomerní struktury. Naše výsledky ukázaly, že absence proteinu TMEM70 vede ke vzniku izolovaného deficitu komplexu V, s přítomností adaptačního/kompenzačního efektu komplexů dýchacího řetězce. Ve svalových mitochondriích byly pozorovány různé stupně deficitu ATP syntázy způsobené extrémně vzácnými heteroplazmatickými variantami v genu MT-ATP6. Zatímco...Klinika pediatrie a dědičných poruch metabolismu 1. LF UK a VFNDepartment of Pediatrics and Inherited Metabolic Disorders First Faculty of Medicine Charles University and General University Hospital in PragueFirst Faculty of Medicine1. lékařská fakult

Molecular basis of deficit of F1Fo-ATP synthase and its impact on energy metabolism of a cell

Mitochondria's primary function is to produce energy through the process of oxidative phosphorylation. ATP synthase is a macromolecular rotary machine located in the inner mitochondrial membrane that catalyzes the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). The mitochondrial disorders due to ATP synthase deficiency represent a heterogeneous group of diseases characterized by variable severity of the phenotype with onset at birth or later in life till adulthood. Mutations in both, mitochondrial or nucelar DNA encoded genes, may result in ATP synthase impairment, either isolated or combined with deficits of other complexes of oxidative phosphorylation. The aims of the thesis were to characterize TMEM70 protein, an ATP synthase assembly factor, and to analyze the impact of novel disease variants leading to ATP synthase deficiency in patients' derived samples. TMEM70 is a 21 kDa hairpin structure protein localized in the inner mitochondrial membrane, with both termini oriented into the matrix, which forms higher oligomer structures. Our results demonstrated that the absence of TMEM70 protein leads to an isolated deficiency of complex V followed in some stage by adaptive/compensatory effect of respiratory chain complexes. Different severities..

Molecular basis of deficit of F1Fo-ATP synthase and its impact on energy metabolism of a cell

Mitochondria's primary function is to produce energy through the process of oxidative phosphorylation. ATP synthase is a macromolecular rotary machine located in the inner mitochondrial membrane that catalyzes the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). The mitochondrial disorders due to ATP synthase deficiency represent a heterogeneous group of diseases characterized by variable severity of the phenotype with onset at birth or later in life till adulthood. Mutations in both, mitochondrial or nucelar DNA encoded genes, may result in ATP synthase impairment, either isolated or combined with deficits of other complexes of oxidative phosphorylation. The aims of the thesis were to characterize TMEM70 protein, an ATP synthase assembly factor, and to analyze the impact of novel disease variants leading to ATP synthase deficiency in patients' derived samples. TMEM70 is a 21 kDa hairpin structure protein localized in the inner mitochondrial membrane, with both termini oriented into the matrix, which forms higher oligomer structures. Our results demonstrated that the absence of TMEM70 protein leads to an isolated deficiency of complex V followed in some stage by adaptive/compensatory effect of respiratory chain complexes. Different severities..

A Novel MTTK Gene Variant m.8315A>C as a Cause of MERRF Syndrome

In this study, we report on a novel heteroplasmic pathogenic variant in mitochondrial DNA (mtDNA). The studied patient had myoclonus, epilepsy, muscle weakness, and hearing impairment and harbored a heteroplasmic m.8315A>C variant in the MTTK gene with a mutation load ranging from 71% to >96% in tested tissues. In muscle mitochondria, markedly decreased activities of respiratory chain complex I + III and complex IV were observed together with mildly reduced amounts of complex I and complex V (with the detection of V*- and free F1-subcomplexes) and a diminished level of complex IV holoenzyme. This pattern was previously seen in other MTTK pathogenic variants. The novel variant was not present in internal and publicly available control databases. Our report further expands the spectrum of MTTK variants associated with mitochondrial encephalopathies in adults

Case report: A rare variant m.4135T>C in the MT-ND1 gene leads to Leber hereditary optic neuropathy and altered respiratory chain supercomplexes

Leber hereditary optic neuropathy is a primary mitochondrial disease characterized by acute visual loss due to the degeneration of retinal ganglion cells. In this study, we describe a patient carrying a rare missense heteroplasmic variant in MT-ND1, NC_012920.1:m.4135T>C (p.Tyr277His) manifesting with a typical bilateral painless decrease of the visual function, triggered by physical exercise or higher ambient temperature. Functional studies in muscle and fibroblasts show that amino acid substitution Tyr277 with His leads to only a negligibly decreased level of respiratory chain complex I (CI), but the formation of supercomplexes and the activity of the enzyme are disturbed noticeably. Our data indicate that although CI is successfully assembled in the patient’s mitochondria, its function is hampered by the m.4135T>C variant, probably by stabilizing CI in its inactive form. We conclude that the m.4135T>C variant together with a combination of external factors is necessary to manifest the phenotype

Deterioration of mitochondrial bioenergetics and ultrastructure impairment in skeletal muscle of a transgenic minipig model in the early stages of Huntington's disease

Skeletal muscle wasting and atrophy is one of the more severe clinical impairments resulting from the progression of Huntington's disease (HD). Mitochondrial dysfunction may play a significant role in the etiology of HD, but the specific condition of mitochondria in muscle has not been widely studied during the development of HD. To determine the role of mitochondria in skeletal muscle during the early stages of HD, we analyzed quadriceps femoris muscle from 24-, 36-, 48- and 66-month-old transgenic minipigs that expressed the N-terminal portion of mutated human huntingtin protein (TgHD) and age-matched wild-type (WT) siblings. We found altered ultrastructure of TgHD muscle tissue and mitochondria. There was also significant reduction of activity of citrate synthase and respiratory chain complexes (RCCs) I, II and IV, decreased quantity of oligomycin-sensitivity conferring protein (OSCP) and the E2 subunit of pyruvate dehydrogenase (PDHE2), and differential expression of optic atrophy 1 protein (OPA1) and dynamin-related protein 1 (DRP1) in the skeletal muscle of TgHD minipigs. Statistical analysis identified several parameters that were dependent only on HD status and could therefore be used as potential biomarkers of disease progression. In particular, the reduction of biomarker RCCII subunit SDH30 quantity suggests that similar pathogenic mechanisms underlie disease progression in TgHD minipigs and HD patients. The perturbed biochemical phenotype was detectable in TgHD minipigs prior to the development of ultrastructural changes and locomotor impairment, which become evident at the age of 48 months. Mitochondrial disturbances may contribute to energetic depression in skeletal muscle in HD, which is in concordance with the mobility problems observed in this model

A transgenic minipig model of Huntington’s disease shows early signs of behavioral and molecular pathologies

Huntington's disease (HD) is a monogenic, progressive, neurodegenerative disorder with currently no available treatment. The Libechov transgenic minipig model for HD (TgHD) displays neuroanatomical similarities to humans and exhibits slow disease progression, and is therefore more powerful than available mouse models for the development of therapy. The phenotypic characterization of this model is still ongoing, and it is essential to validate biomarkers to monitor disease progression and intervention. In this study, the behavioral phenotype (cognitive, motor and behavior) of the TgHD model was assessed, along with biomarkers for mitochondrial capacity, oxidative stress, DNA integrity and DNA repair at different ages (24, 36 and 48 months), and compared with age-matched controls. The TgHD minipigs showed progressive accumulation of the mutant huntingtin (mHTT) fragment in brain tissue and exhibited locomotor functional decline at 48 months. Interestingly, this neuropathology progressed without any significant age-dependent changes in any of the other biomarkers assessed. Rather, we observed genotype-specific effects on mitochondrial DNA (mtDNA) damage, mtDNA copy number, 8-oxoguanine DNA glycosylase activity and global level of the epigenetic marker 5-methylcytosine that we believe is indicative of a metabolic alteration that manifests in progressive neuropathology. Peripheral blood mononuclear cells (PBMCs) were relatively spared in the TgHD minipig, probably due to the lack of detectable mHTT. Our data demonstrate that neuropathology in the TgHD model has an age of onset of 48 months, and that oxidative damage and electron transport chain impairment represent later states of the disease that are not optimal for assessing interventions