Phd Dissertations

Pathomechanisms of Leigh Syndrome - Defects of Post-Transcriptional and Post-Translational Regulation of Mitochondrial Metabolism 

Time: 11 October 2019   12:00

Place: Biomedicum Helsinki, Lecture Hall 3, Haartmaninkatu 8, 00290 Helsinki

Opponent: MD, PhD, Professor Ingrid Tein, University of Toronto

Custos: Academy Professor Anu Wartiovaara



Leigh syndrome is a progressive mitochondrial encephalopathy manifesting in early childhood, with characteristic symmetric lesions of the brainstem and basal ganglia, and a spectrum of clinical findings. Often multi-organ manifestation is known to occur. The genetic background of Leigh syndrome is exceptionally wide, with over 75 known disease genes affecting mitochondrial function, in both the mitochondrial and nuclear genomes. The molecular characteristics of this clinically and genetically heterogenetic disease, however, remain largely unknown. In this study genetic diagnoses were found for patients with Leigh syndrome and the underlying molecular pathomechamisms were studied.

The disease found in two families was caused by a novel Scandinavian founder mutation in SUCLA2, causing deficiency of succinyl-CoA ligase (SCL) of the TCA cycle, a central metabolic pathway. The substrate for the reaction catalyzed by SCL is succinyl-CoA, also serving as the substrate for succinylation, a recently characterized post-translational modification with yet unknown biological significance. These results show SCL deficiency to lead to increased protein succinylation via accumulation of succinyl-CoA in cell lines of patients. Metabolic disturbances caused by the succinylation of hundreds of target lysine residues, found on a wide range of metabolic proteins in nearly all cell compartments, propose succinylation as a mechanism for the simultaneous control of several metabolic pathways.

Also, defects of the mitochondrial polynucleotide phosphorylase (PNPase) caused by PNPT1 mutations were established among the causative mechanisms of Leigh syndrome with next-generation sequencing in one patient with a progressive Leigh encephalomyopathy. Defective mitochondrial RNA metabolism due to loss of RNA degradation activity of PNPase is shown as a novel mechanism for mitochondrial disease.

The paths to molecular diagnoses for the patients in this study portray the recent advancements of molecular and genetic diagnostics, which have developed dramatically with the era of next-generation sequencing methods. Genetic diagnoses were provided for patients with Leigh syndrome of unknown molecular etiology, crucial for the well-being of the families and treatment of the patients. Simultaneously, patient-derived cell lines and tissues with a disarrangement of mitochondrial metabolism were utilized to increase our understanding of the related metabolic pathways and mitochondrial metabolism.

Interventions to improve mitochondrial function in a mouse model of GRACILE syndrome, a complex III disorder

Time: 05 April 2019 12:00

Place: Haartman Institute, Lecture Hall 2, Haartmaninkatu 3, 00290 Helsinki

Opponent: Professor Cristina Ugalde, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Spain

Custos: Professori Markku Heikinheimo


A rare homozygous BCS1Lc.A232G (Ser78Gly, p.S78G) mutation in infants causes GRACILE syndrome, which is a severe mitochondrial respiratory chain complex III (CIII) disorder resulting in multiple organ dysfunction and early lethality. Pathogenesis mechanisms have been studied using our viable Bcs1lp.S78G knock-in mouse model. The mouse model replicates most clinical phenotypes, such as growth restriction, hepatopathy, and tubulopathy.

Like patients, the survival of homozygous mice is reduced (to 35-45 days, P35-P45 in the C57BL/6JBomTac background), mainly because of severe hypoglycemia. Aiming to improve the glycemic balance we performed an intervention with a high sugar (60% dextrose) diet. This diet did not improve energy metabolism and resulted in slightly decreased survival despite apparent normalization of some plasma metabolites. For subsequent studies, we bred the Bcs1lc.A232G mutation into a C57BL/6JCrl background, in which the survival was five-fold longer (approximately 200 days). Moreover, the extended survival brought novel phenotypes, such as encephalopathy and late-onset cardiomyopathy. In this genetic background, we investigated the effect of ketogenic diet on disease progression. The ketogenic diet had a beneficial impact on liver disease, but it had adverse effects upon long-term feeding, resulting in shortened survival. In the third study, we introduced an alternative oxidase (AOX) transgene into the Bcs1lp.S78G mice to improve respiratory chain function. The ubiquitous expression of AOX, which should bypass electron transfer and relieve CIII blockade, prevented lethal cardiomyopathy and renal-tubular atrophy, and delayed focal astrogliosis in the somatosensory cortex of the brain. The beneficial effects of AOX extended the median survival of the homozygotes to median P590. The main conclusions from these studies are that the Bcs1lp.S78G mice in a C57BL/6JCrl background present with both the known early-onset manifestations of GRACILE syndrome and some later onset manifestations found in other CIII deficiencies.

The dietary interventions had limited benefits, probably because of a severe course of the disease. In contrast, bypassing the blocked electron flow using AOX had a robust beneficial effect, mainly in tissues or cells with high ATP demand such as the heart and renal proximal tubular cells.

The Metabolic and Molecular Consequences of Mitochondrial Dysfunction in Mitochondrial Disease and Acquired Obesity

Time: 9 February 2019   12:00

Place: Biomedicum Helsinki, Lecture Hall 2, Haartmaninkatu 8, 00290 Helsinki

Opponent: Professor Maria Judit Molnar, Semmelweis University

Custos: Academy Professor Anu Wartiovaara



Mitochondrial diseases are the most common group of inherited metabolic disorders. The clinical symptoms of mitochondrial disease patients are highly variable, which makes both the diagnosis and the management exceptionally challenging. The molecular mechanisms of tissue-specificity and clinical variability in mitochondrial disorders are unknown. Currently, an effective pharmacological treatment and reliable single biomarkers that would sufficiently detect mitochondrial disorders are lacking.

Due to the often severe neurological symptoms of mitochondrial disease patients, primary care and research often focus on the characterization and management of the neuromuscular manifestations, while the numerous and comparatively secondary metabolic complications remain neglected. Obesity and diabetes, for example, are common among certain mitochondrial disease groups, and therefore the contribution of mitochondrial dysfunction in metabolically active tissues is needed in mitochondrial medicine. In the first part of this thesis, the aim was to study the role of mitochondria in adipose tissue in mitochondrial disease and in acquired obesity. The metabolic and molecular consequences of mitochondrial dysfunction were analysed in 26 mitochondrial disease patients with different types of causative mutations and compared to 30 age-matched controls. The study revealed that patients with recessive mutations in mitochondrial DNA polymerase (mitochondrial recessive ataxia syndrome, MIRAS) were associated with central obesity, large adipocytes, insulin resistance and metabolic syndrome, whereas patients with a primary mitochondrial DNA mutation (mitochondrial myopathy, encephalopathy, lactate acidosis and stroke-like episodes, MELAS/ maternally inherited diabetes and deafness, MIDD) had diabetes, lower volume of adipose tissue and less adipocytes. The molecular analysis of adipose tissue showed a reduction of mitochondrial biogenesis and oxidative capacity in MIRAS patients, and to a lesser extend also in MELAS/MIDD patients. The effect of acquired obesity on mitochondrial function in adipose tissue was further studied in 26 rare monozygotic twins discordant for body weight. In adipose tissue of the obese co-twins, mitochondrial oxidative metabolism was reduced and associated with whole-body insulin resistance and inflammation, present already before the clinical diagnosis of diabetes and other related complications of acquired obesity.

In the second part of this thesis, the aim was to study metabolic changes of mitochondrial and other muscle-manifesting disease patients, and to identify potential metabolic biomarkers for mitochondrial disease diagnostics. Targeted metabolomics analysis of blood and/or muscle samples from 25 primary mitochondrial disease patients, 16 unaffected carriers, six inclusion body myositis patients, 15 non-mitochondrial neuromuscular disease patients, and 30 age-matched controls revealed different metabolic profiles that pointed to disease-specific mechanisms of pathogenesis. Changes in metabolites of transsulfuration pathway were specific for primary mitochondrial disease and inclusion body myositis patients, whereas creatine depletion marked neuromuscular diseases, inclusion body myositis and infantile-onset spinocerebellar ataxia patients. Low blood and muscle arginine was specific for MELAS/MIDD patients. The metabolomics data showed that blood metabolic fingerprints are potential multi-biomarkers for diagnostics. By combining a minimum of four metabolites (sorbitol, alanine, cystathionine and myoinositol), we created a metabolic multi-biomarker that distinguished primary mitochondrial disorders with sensitivity of 76% and specificity of 95%. Moreover, our results suggested that detected metabolites from affected pathways could be considered and further studied as disease therapy targets.

In conclusion, this thesis highlights the role of mitochondria in obesity. It reveals that different mitochondrial genetic defects provoke different consequences to systemic metabolism, leading to a disease-specific metabolic phenotype - obesity or leanness. The thesis further highlights the insufficiency of mitochondrial oxidative capacity in the adipose tissue of the obese subjects and its association with metabolic complications in acquired obesity. It also shows that targeted metabolomics analysis is a valuable tool in personalized medicine for suggesting metabolic targets for treatment and diet.