PhD defenses from Molecular Neurology Research Program
Time: 07 October, 2017 12:00
Place: Biomedicum Helsinki, Lecture Hall 2, Haartmaninkatu 8, 00290 Helsinki
Defects of mitochondrial DNA (mtDNA) replication underlie common metabolic disorders. Despite mtDNA is degraded and synthesised in all cells containing mitochondria, mtDNA replication stress typically causes generation of mtDNA deletions or depletion of mtDNA copy number in muscle and brain, which manifest as mitochondrial myopathy (MM) or neurodegeneration, respectively. MtDNA replication defects, however, do not affect highly proliferative tissues, such as blood or intestine, despite their reliance on robust mtDNA replication to sustain high rates of proliferation. The mechanisms behind the tissue-specific manifestations of mtDNA replication defects remain unknown.
In this thesis, we aimed to identify the metabolic response pathways for mtDNA replication stress caused by a dominant Twinkle mtDNA helicase (TWNK) mutation leading to adult-onset MM. The study revealed that MM induces a metabolic stress response in muscle which we found to be orchestrated by one master regulator, mechanistic target of rapamycin complex I (mTORC1). The mTORC1-mediated stress response appeared to promote disease progression, and an mTORC1 inhibitor, rapamycin, remarkably improved the mitochondrial muscle disease. It ameliorated the typical hallmarks of MM: the number of ragged red fibers (RRFs) and the amount of mtDNA deletions were reduced after rapamycin treatment.
In the second part of this thesis, we studied the transcription regulation of mtDNA replication machinery. We identified a complex regulatory locus for DNA polymerase gamma (POLG) by in silico predictions, which were verified in vivo. The regulatory non-coding locus drives POLG expression specifically in the sensory interneurons of the spinal cord and oculomotor nucleus, which we found to degenerate in POLG patients. The death of these neurons might be the underlying cause of sensory neuropathy and progressive external ophthalmoplegia (PEO), which are typical clinical findings in POLG disorders. The identified regulatory locus is the first non-coding locus for a mitochondrial disease gene and offers the first candidate region for pathogenic non-coding mutations.
In conclusion, our work has identified novel contributors in the tissue-specific manifestations of mitochondrial diseases and offers multiple novel treatment targets for mitochondrial disorders, which currently lack effective treatment options.
Time: 22 September, 2017 12:00
Place: Haartman Institute, Lecture Hall 1, Haartmaninkatu 3, 00290 Helsinki
Opponent: Professor Rita Horvath, Newcastle University, United Kingdom
Mitochondrial diseases are inheritable diseases, where the function of the ATP
(adenosine triphosphate) producing organelle of the cell, is compromised. This
leads to a wide variety of phenotypes, known to arise from defects in over 200
genes. Typically manifesting organs are brain, heart, muscle, liver, endocrine
organs and sense organs. Mitochondrial disorders are often progressive, they
can manifest in multiple organs in one person and due to inheritance, other
family members might also be affected. Careful clinical assessment with
investigation of family history helps to predict the cause, but other diagnostic
assessments play a central role in diagnosis.
Elevation of blood or cerebrospinal fluid lactate has traditionally been an
indicator of mitochondrial dysfunction, but this biomarker lacks sensitivity and
specificity, and more accurate biomarkers are required. Muscle biopsy sample
is the gold standard of mitochondrial disease diagnosis. Cytochrome c oxidase
(COX) negative, succinate dehydrogenase (SDH) positive fibers and raggedred
fibers (RRFs) are hallmarks of mitochondrial dysfunction. Reduced
respiratory chain enzyme activity in tissue verifies diagnosis. Solid diagnosis
requires genetic evidence, but the expanding number of disease-causing
genes makes it difficult to choose which genes to sequence.
We report here a novel diagnostic serum biomarker, fibroblast growth factor
21 (FGF21), which is more sensitive and specific to muscle-manifesting
mitochondrial disorders than any of the conventional biomarkers used before.
It correlates with COX-negative muscle fibers and is most likely produced and
secreted by them. We also studied another recently discovered serum
biomarker, growth differentiation factor 15 (GDF15). We report that both
FGF21 and GDF15 correctly distinguish mitochondrial myopathies from nonmitochondrial
myopathies and controls, making them the most accurate
biomarkers for mitochondrial myopathies to date. The trigger for induction of
these biomarkers seems to be upstream of respiratory chain defect, most likely
initiates from the mitochondrial translational machinery.
In another study, we used next generation sequencing to search for a
pathogenic mutation in a patient with fatal infantile Alpers
hepatoencephalopathy. We identified two compound heterozygous mutations
in a novel disease gene, FARS2. This gene encodes for a protein,
mitochondrial phenylalanyl-tRNA synthetase (mtPheRS), responsible for the
charging of mitochondrial phenylalanyl-tRNA with its cognate amino acid.
Structural prediction of the mutated proteins together with functional studies in
E. coli showing decreased activity of mutant mtPheRS, verified the diagnosis.
Our results strongly support the use of FGF21 and GDF15 as first line
diagnostic assessments. Rapid progression to next-generation sequencing is
advised if both of these biomarkers are elevated, with positive predictive value
being 95%. This would reduce the need for invasive diagnostic tests.
Time: 24 March, 2017 12:00
Place: Biomedicum 1, lecture hall 3, Haartmaninkatu 8, 00290 Helsinki
Proteins, consisting of amino acids, work as building blocks in the cells. In addition, they carry out vast amounts of cellular functions. Accurate protein synthesis is thus crucial for the normal function of cells. The first step of protein synthesis is the charging of transfer-RNAs (tRNAs) with their cognate amino acids. Evolutionarily conserved and extremely old proteins, aminoacyl-tRNA synthetases (aaRSs), carry out this step and each amino acid-tRNA pair has its own synthetase for the task. However, in some cases the amino acids are so similar in size and structure that they cannot be separated well enough by the aaRSs. To avoid mischarging and the subsequent protein misfolding, some of the synthetases have an editing domain, which recognizes and hydrolyses incorrect amino acid-tRNA pairs. In addition, cells have other important quality control mechanisms to ensure protein homeostasis, the capacity of cells to maintain internal stability of the proteome. Patient mutations in genes connected to protein synthesis and quality control are found to cause different diseases, the mechanisms of which are not yet, however, well known. Research on these topics is thus important. The aim of this thesis was to study the molecular mechanisms of different tRNA-charging defects and protein quality control mechanisms in both protein-translating compartments of a eukaryotic cell, cytosol and mitochondria.
The first part of the thesis describes the molecular mechanism and the clinical phenotype of a special cytosolic tRNA charging defect caused by mutations in SEPSECS gene. The corresponding protein is involved in charging of the 21st amino acid, selenocysteine, to its tRNA. We identified mutations in this gene and showed them to lead to a decreased amount of selenocysteine-containing proteins, selenoproteins, in the brain of a patient with a severe early onset encephalopathy. Our study also indicated increased protein oxidation in the patient brain. This study extends the clinical phenotypes connected to SEPSECS mutations, and indicates that selenoprotein synthesis defect can resemble mitochondrial disease with lactate elevation.
In the second part of this thesis, the potential of an amino acid analogue of arginine, canavanine, to induce protein misfolding in mitochondria was studied. The results demonstrated that mitochondrial protein translation machinery does not distinguish canavanine from arginine. The amino acid analog was incorporated into mitochondrially encoded proteins causing protein instability and formation of aberrant polypeptides. Surprisingly, however, canavanine did not induce mitochondrial unfolded protein response (UPRmt), a previously described signalling pathway induced by accumulation of misfolded proteins inside mitochondria. The study showed that none of the protein quality control mechanisms were able to solve protein misfolding caused by canavanine, which led to a severe respiratory chain defect. Canavanine has been used previously in a large number of studies to induce cytosolic protein misfolding, but the impact of canavanine for mitochondrial function has been largely ignored. Canavanine can be used in future as a tool to study further the consequences of protein misfolding in mitochondria, and for studying how mitochondria solve stalled ribosomes, which was also detected to be a consequence of canavanine in our study.
The goal of the third part of the thesis was to study UPRmt in an animal model. The purpose was to generate a mouse model that has a mutation in the editing domain of mitochondrial alanyl-tRNA synthetase, leading to amino acid mischarging and formation of unfolded proteins inside mitochondria in vivo. The result of the study indicated for the first time the importance of amino acid editing by a tRNA synthetase as an essential quality control mechanism in mammalian mitochondria. The work presented in this thesis provides new information concerning the mechanisms of different tRNA charging defects and their consequences for the cell and organism. Special emphasis was on mitochondrial function.