Molecular, metabolic, and therapeutic aspects of respiratory complex III deficiency: Bcs1l mutant mice as an experimental model

Time: 16 October 2020 at 12:00

Place: Lecture Hall 3, Biomedicum Helsinki 1, Haartmaninkatu 8, 00290 Helsinki and via live stream:, password: 575575

Opponent: Professor Michael Murphy, MRC Mitochondrial Biology Unit, University of Cambridge, UK

Custos: Academy Professor Elina Ikonen



Mitochondrial disorders are rare diseases but collectively the most frequent group of inborn errors of metabolism. These disorders are genetically and phenotypically heterogenous and can manifest in any organ of the body with onset at any age. Mitochondrial functions are also diverse with the ATP production via the oxidative phosphorylation (OXPHOS) being the most notable. At the center of the OXPHOS machinery is the respiratory complex III (CIII, cytochrome bc1 complex). CIII deficiency in GRACILE syndrome belonging to the Finnish disease heritage causes a neonatal-lethal hepatorenal disease. The primary cause of GRACILE syndrome is a c.A232G (p.S78G) mutation in the BCS1L gene, which encodes a translocase required for Rieske Fe-S protein (RISP, UQCRFS1) incorporation into CIII. Homozygous Bcs1lp.S78G mice bearing the GRACILE syndrome mutation recapitulate the human syndrome, but unlike the patients they have a short asymptomatic period and relatively longer lifespan giving a window for therapeutic interventions. In this thesis project, we studied two potential therapies aiming to improve dysfunctional mitochondria in Bcs1lp.S78G mice: ketogenic diet and NAD+ repletion. We also utilized an alternative oxidase (AOX) transgene to bypass the electron-transfer blockade at CIII.

Ketogenic diets are low-carbohydrate high-fat diets causing nutritional ketosis. They have been proposed to induce a beneficial starvation-like adaptive mitochondrial response involving increased mitochondrial biogenesis. Bcs1lp.S78G mice tolerated the carbohydrate restriction of ketogenic diet, were able to utilize dietary fat as the main energy source and developed ketosis. Ketogenic diet attenuated the hepatic CIII assembly defect, increased CIII activity and corrected mitochondrial structural aberrations. Our results suggested that these changes were not due to increased mitochondrial biogenesis. In line with the improved CIII function, Bcs1l mutant mice showed attenuated hepatopathy as shown by delayed liver fibrosis, inhibited stellate cell activation and hepatic progenitor cell response, decreased cell death and plasma liver enzyme activities. Liver transcriptomics and subsequent histochemical analyses suggested altered macrophage activation and a normalizing effect by ketogenic diet.

In the second study, we characterized NAD+ metabolism in Bcs1lp.S78G mice. We found transcriptionally repressed NAD+ de novo biosynthesis and decreased hepatic NAD+ concentration. Changes in NAD+ consuming processes did not explain the decreased NAD+ levels. Aiming to replete the NAD+ levels, we fed the Bcs1lp.S78G mice a NAD+ precursor nicotinamide riboside (NR). In contrast to previous studies on mitochondrial myopathy models and mouse models with secondary mitochondrial dysfunctions, the hepatic NAD+ depletion of Bcs1lp.S78G mice was refractory to NR supplementation and the disease progression was unaltered. Cellular NAD+ levels regulate mitochondrial functions via sirtuin deacetylases, which are the main targets of NAD+ repletion therapies. Investigation of the upstream effectors of sirtuins showed that a starvation-like metabolic state of Bcs1lp.S78G mice is linked to AMP kinase and cAMP signaling, which likely counterbalances the repressive effect of decreased NAD+ levels on the activity of SIRT1 and SIRT3.

In the third study, we introduced Ciona intestinalis AOX transgene into the Bcs1lp.S78G mice. AOXs are non-mammalian enzymes that can bypass a blockade of the CIII-CIV segment of the respiratory electron transfer. The AOX-expressing Bcs1lp.S78G mice were viable, and their CIII-deficiency stimulated AOX-mediated respiration in isolated mitochondria. AOX expression tripled the median lifespan of Bcs1lp.S78G mice from 200 to 600 days. The extension of the lifespan was predominantly due to the complete prevention of late-onset cardiomyopathy. The effects of AOX were tissue specific. In the heart of Bcs1lp.S78G mice, it preserved normal tissue structure and function, mitochondrial morphology, respiratory electron transfer, and wild-type-like transcriptome. In contrast, AOX only minimally affected the late-stage liver disease. Whereas, in the kidneys, AOX normalized an atrophic kidney phenotype and some histological lesions but it did not normalize kidney function or cause global normalization of transcriptome changes. Our results suggest tissue-specific thresholds of CIII deficiency for in vivo AOX-mediated respiration in CIII deficiency. Moreover, our study demonstrates the value of AOX as a research tool to dissect the pathogenesis of CIII deficiency.

During our investigations, we observed approximately 5-fold difference in the lifespan of the Bcs1lp.S78G mice on two closely related congenic backgrounds. In the fourth study, we tracked the difference to a spontaneous homoplasmic mitochondrial DNA (mtDNA) variant (mt-Cybp.D254N) in an isolated congenic Lund University mouse colony. The variant changes a highly conserved negative amino acid residue in the only mtDNA-encoded subunit of CIII, cytochrome b (MT-CYB). A crossbreeding experiment utilizing the maternal inheritance of mtDNA verified the novel variant as the determinant of the survival difference. Functional studies showed that the variant exacerbated complex III deficiency in all assessed tissues. In otherwise wild-type mice, it also decreased cardiac CIII activity, caused a slight disturbance in mitochondrial bioenergetics, and decreased whole-body energy expenditure. Molecular dynamics simulations and their verification in isolated mutagenized Rhodobacter capsulatus cytochrome bc1 complex showed that the mt-Cybp.D254N variant restricts the mobility of RISP head domain movement.

In summary, these studies provided novel mechanistic and therapeutic insights into CIII deficiency at genetic, molecular, and metabolic level. The results highlight the importance of knowing the underlying tissue-specific pathology and metabolic adaptations when designing therapies for mitochondrial diseases. The genetic epistasis between Bcs1lp.S78G and mt-Cybp.D254N also highlights the role of mitochondrial DNA background as a modifier of mitochondrial disease phenotypes.

Lipogenic subdomains of the ER : the role of seipin

Time: 14 April 2020 at 18:00

Place: Porthania, Yliopistonkatu 3, 00100 Helsinki, live stream provided

Opponent: Professor Robert V. Farese, Jr., Harvard University, USA



Cells store excess energy mostly as neutral lipids (NLs), inside fat-specialized organelles called lipid droplets (LDs). LDs are metabolic hubs involved in many cellular processes, such as lipid metabolism and endoplasmic reticulum (ER) homeostasis. There is increasing evidence linking LDs and human pathologies, including e.g. cardiovascular and fatty liver disease. Thus, detailed understanding of LD biology should be beneficial for tackling these disease states.

LDs consist of a core of NLs surrounded by a phospholipid monolayer. Their biogenesis begins in the ER, with accumulation of NL lenses within the ER bilayer. These lenses subsequently grow and bud into the cytosol. All this likely occurs in specialized ER subdomains, but a detailed understanding of the earliest steps of LD biogenesis is still lacking. After formation, LDs retain an intimate relationship with the ER, through ER-LD contacts. Seipin is an oligomeric ER transmembrane protein implicated in LD assembly and adipocyte development. Mutations in seipin give rise to three disease states: a severe form of congenital lipodystrophy (BSCL2), a motor neuron disease called seipinopathy and a fatal neurodegenerative disease called Celia’s encephalopathy. The molecular function of seipin is unclear.

In this thesis, using advanced cell biological techniques, we sought to expand the understanding of the function of seipin and how mutations in seipin lead to human disease.

In the first study, we focused on the seipinopathy-linked seipin mutant, N88S-seipin, which is known to form misfolded aggregates. We found that overexpression of N88S-seipin leads to ER stress, decreased NL storage and voluntary swimming in zebrafish larvae. Upon increasing NL stores, these defects were alleviated, and the mutant protein was translocated from the ER to regions flanking LDs. Increasing cellular LDs also alleviated ER stress induced by tunicamycin. We propose that increasing LDs may aid cells in coping with misfolded proteins.

We next focused on deciphering the function of seipin in LD formation and ER-LD contacts. Endogenously tagged seipin localized stably at ER-LD contact sites, but a BSCL2-linked mutant did not. Knockout (KO) of seipin led to the formation of numerous tiny LDs, which failed to grow and recruit protein and lipid cargo from the ER. Furthermore, whilst all LDs in control cells were connected to the ER, seipin KO lead to a subset of LDs completely detaching from the ER. The remaining LDs of seipin KO cells also had morphologically aberrant and dysfunctional ER-LD contacts. As similar defects were also evident in BSCL2 patient fibroblasts, we propose that seipin is crucial for the formation of ER-LD contacts and cargo delivery.

Finally, we found that seipin can determine the site where LDs start to form, as relocalization of seipin to a subdomain of the ER, the nuclear envelope, was sufficient to relocalize LD biogenesis to that site. We found that seipin-mediated ER-LD contact sites display a uniform neck-like architecture, suggesting that the seipin oligomer may structurally restrain this site. Seipin was also required for ER-LD contact maintenance, as acute removal of seipin from ER-LD contacts lead to strikingly heterogonous LD growth. This LD growth heterogeneity arises via a biophysical ripening process, with NLs partitioning from smaller to larger LDs via LD-ER contacts. These data suggest seipin-mediated ER-LD contacts function as a valve facilitating NL flux from the ER to LDs, thus allowing controlled LD growth.

Overall, this thesis provides insight into the function of seipin and mechanisms of LD formation. Our data suggest that the ER-LD nexus could be considered as a joint system, with continuous bidirectional trafficking of cargo occurring via ER-LD contacts mediated by seipin. Future work will be required to investigate the molecular intricacies of the lipid flux occurring at ER-LD contacts. A deeper understanding of LD biology will aid in the development of new therapeutics for a number of pathologies, including seipin-related disorders and common metabolic disturbances.

Mechanisms and dynamics of mitochondrial disease stress responses : special emphasis on FGF21

Time: 6 March 2020 at 12:00

Place: Biomedicum Helsinki, Lecture Hall 1, Haartmaninkatu 8, 00290 Helsinki

Opponent: Professor Kirsi Virtanen, University of Eastern Finland

Custos: Academy Professor Elina Ikonen



Mitochondria are best known for ATP production in oxidative phosphorylation (OXPHOS), dependent on nuclear factors and the organellar genome of the mitochondria, mtDNA. Mitochondrial disorders are categorically OXPHOS diseases, presenting with extreme variability of tissue-specific manifestation that cannot be explained by genetic causes or impaired ATP production. Comprehensive understanding of the pathophysiology is key for better diagnostics and therapy targets.

AdPEO is a mitochondrial disease characterized by mtDNA deletions and mitochondrial deficiency in muscles and brain. Studies on a transgenic mouse model of AdPEO, Deletor, have revealed similar pathology to human patients, and induction of a local stress response in muscle that involves expression of a metabolic hormone, FGF21. In physiological challenges, FGF21 is secreted by the liver to enhance systemic energy-metabolism. Therefore, chronic exposure to FGF21 in mitochondrial disease exposes the body to non-homeostatic regulation of metabolism. In this thesis, we have studied the stress responses upon mitochondrial myopathy with emphasis on the actions of FGF21. We utilized parallel analysis of patient samples and established mouse models, and generated a Deletor-FGF21 knockout model.

In the first part, we discovered novel rearrangements of whole-cellular metabolism in post-mitotic muscle with mtDNA maintenance defect, including induction of one-carbon metabolism and glucose-driven glutathione synthesis. We demonstrated that the transcriptional and metabolic stress programs progressed sequentially along the primary disease pathology, and that FGF21 expression was indisputable for initiation of the glucose-driven metabolic programs in the muscle. Systemically, FGF21 expectedly impaired maintenance of adiposity and altered tissue-level glucose preferences. Additionally, we discovered FGF21-dependent mitochondrial and metabolic pathology in CA2-region of hippocampus in Deletor. The second part highlighted that the mitochondrial stress responses dependent on the primary disease mechanisms, not on the fundamental OXPHOS-deficiency. Our clinical analyses showed that the circulating mitochondrial disease biomarkers FGF21 and GDF15 were specific for primary or secondary mitochondrial translation defects, not for isolated OXPHOS mutations.

In summary, this work has revealed clinically relevant local and systemic metabolic rearrangements in response to mtDNA maintenance and expression defects, and demonstrated tissue-specific regulation of pathophysiology by FGF21.


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.