Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Severe congenital lactic acidosis and hypertrophic cardiomyopathy caused by an intronic variant in NDUFB7.

Mutations in structural subunits and assembly factors of complex I of the oxidative phosphorylation system constitute the most common cause of mitochondrial respiratory chain defects. Such mutations can present a wide range of clinical manifestations, varying from mild deficiencies to severe, lethal disorders. We describe a patient presenting intrauterine growth restriction and anemia, which displayed postpartum hypertrophic cardiomyopathy, lactic acidosis, encephalopathy, and a severe complex I defect with fatal outcome. Whole genome sequencing revealed an intronic biallelic mutation in the NDUFB7 gene (c.113-10C>G) and splicing pattern alterations in NDUFB7 messenger RNA were confirmed by RNA Sequencing. The detected variant resulted in a significant reduction of the NDUFB7 protein and reduced complex I activity. Complementation studies with expression of wild-type NDUFB7 in patient fibroblasts normalized complex I function. Here we report a case with a primary complex I defect due to a homozygous mutation in an intron region of the NDUFB7 gene.

Clinical Presentation, Genetic Etiology, and Coenzyme Q10 Levels in 55 Children with Combined Enzyme Deficiencies of the Mitochondrial Respiratory Chain.

OBJECTIVES: To evaluate the clinical symptoms and biochemical findings and establish the genetic etiology in a cohort of pediatric patients with combined deficiencies of the mitochondrial respiratory chain complexes. STUDY DESIGN: Clinical and biochemical data were collected from 55 children. All patients were subjected to sequence analysis of the entire mitochondrial genome, except when the causative mutations had been identified based on the clinical picture. Whole exome sequencing/whole genome sequencing (WES/WGS) was performed in 32 patients. RESULTS: Onset of disease was generally early in life (median age, 6 weeks). The most common symptoms were muscle weakness, hypotonia, and developmental delay/intellectual disability. Nonneurologic symptoms were frequent. Disease causing mutations were found in 20 different nuclear genes, and 7 patients had mutations in mitochondrial DNA. Causative variants were found in 18 of the 32 patients subjected to WES/WGS. Interestingly, many patients had low levels of coenzyme Q10 in muscle, irrespective of genetic cause. CONCLUSIONS: Children with combined enzyme defects display a diversity of clinical symptoms with varying age of presentation. We established the genetic diagnosis in 35 of the 55 patients (64%). The high diagnostic yield was achieved by the introduction of massive parallel sequencing, which also revealed novel genes and enabled elucidation of new disease mechanisms.

TEFM regulates both transcription elongation and RNA processing in mitochondria.

Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.

CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels.

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.

DARS2 protects against neuroinflammation and apoptotic neuronal loss, but is dispensable for myelin producing cells.

Although mitochondria are ubiquitous, each mitochondrial disease has surprisingly distinctly different pattern of tissue and organ involvement. Congruently, mutations in genes encoding for different mitochondrial tRNA synthetases result in the development of a very flamboyant group of diseases. Mutations in some of these genes, including aspartyl-tRNA synthetase (DARS2), lead to the onset of a white matter disease-leukoencephalopathy with brainstem and spinal cord involvement, and lactate elevation (LBSL) characterized by progressive spastic ataxia and characteristic leukoencephalopathy signature with multiple long-tract involvements. Puzzled by the white matter disease phenotypes caused by DARS2 deficiency when numerous other mutations in the genes encoding proteins involved in mitochondrial translation have a detrimental effect predominantly on neurons, we generated transgenic mice in which DARS2 was specifically depleted in forebrain-hippocampal neurons or myelin-producing cells. Our results now provide the first evidence that loss of DARS2 in adult neurons leads to strong mitochondrial dysfunction and progressive loss of cells. In contrast, myelin-producing cells seem to be resistant to cell death induced by DARS2 depletion despite robust respiratory chain deficiency arguing that LBSL might originate from the primary neuronal and axonal defect. Remarkably, our results also suggest a role for early neuroinflammation in the disease progression, highlighting the possibility for therapeutic interventions of this process.

A multi-systemic mitochondrial disorder due to a dominant p.Y955H disease variant in DNA polymerase gamma.

Mutations in the mitochondrial DNA polymerase, POLG, are associated with a variety of clinical presentations, ranging from early onset fatal brain disease in Alpers syndrome to chronic progressive external ophthalmoplegia. The majority of mutations are linked with disturbances of mitochondrial DNA (mtDNA) integrity and maintenance. On a molecular level, depending on their location within the enzyme, mutations either lead to mtDNA depletion or the accumulation of multiple mtDNA deletions, and in some cases these molecular changes can be correlated to the clinical presentation. We identified a patient with a dominant p.Y955H mutation in POLG, presenting with a severe, early-onset multi-systemic mitochondrial disease with bilateral sensorineural hearing loss, cataract, myopathy, and liver failure. Using a combination of disease models of Drosophila melanogaster and in vitro biochemistry analysis, we compare the molecular consequences of the p.Y955H mutation to the well-documented p.Y955C mutation. We demonstrate that both mutations affect mtDNA replication and display a dominant negative effect, with the p.Y955H allele resulting in a more severe polymerase dysfunction.

Mitochondrial Polyadenylation Is a One-Step Process Required for mRNA Integrity and tRNA Maturation.

Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. We studied knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in Drosophila melanogaster and demonstrate that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, our results show that mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs.

Intra-mitochondrial Methylation Deficiency Due to Mutations in SLC25A26.

S-adenosylmethionine (SAM) is the predominant methyl group donor and has a large spectrum of target substrates. As such, it is essential for nearly all biological methylation reactions. SAM is synthesized by methionine adenosyltransferase from methionine and ATP in the cytoplasm and subsequently distributed throughout the different cellular compartments, including mitochondria, where methylation is mostly required for nucleic-acid modifications and respiratory-chain function. We report a syndrome in three families affected by reduced intra-mitochondrial methylation caused by recessive mutations in the gene encoding the only known mitochondrial SAM transporter, SLC25A26. Clinical findings ranged from neonatal mortality resulting from respiratory insufficiency and hydrops to childhood acute episodes of cardiopulmonary failure and slowly progressive muscle weakness. We show that SLC25A26 mutations cause various mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation, and the biosynthesis of CoQ10 and lipoic acid.

Cyclophilin D, a target for counteracting skeletal muscle dysfunction in mitochondrial myopathy.

Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca(2+) uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca(2+) handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy.

SUV3 helicase is required for correct processing of mitochondrial transcripts.

Mitochondrial gene expression is largely regulated by post-transcriptional mechanisms that control the amount and translation of each mitochondrial mRNA. Despite its importance for mitochondrial function, the mechanisms and proteins involved in mRNA turnover are still not fully characterized. Studies in yeast and human cell lines have indicated that the mitochondrial helicase SUV3, together with the polynucleotide phosphorylase, PNPase, composes the mitochondrial degradosome. To further investigate the in vivo function of SUV3 we disrupted the homolog of SUV3 in Drosophila melanogaster (Dm). Loss of dmsuv3 led to the accumulation of mitochondrial mRNAs, without increasing rRNA levels, de novo transcription or decay intermediates. Furthermore, we observed a severe decrease in mitochondrial tRNAs accompanied by an accumulation of unprocessed precursor transcripts. These processing defects lead to reduced mitochondrial translation and a severe respiratory chain complex deficiency, resulting in a pupal lethal phenotype. In summary, our results propose that SUV3 is predominantly required for the processing of mitochondrial polycistronic transcripts in metazoan and that this function is independent of PNPase.

Loss of UCP2 attenuates mitochondrial dysfunction without altering ROS production and uncoupling activity.

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.

LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs.

Regulation of mtDNA expression is critical for maintaining cellular energy homeostasis and may, in principle, occur at many different levels. The leucine-rich pentatricopeptide repeat containing (LRPPRC) protein regulates mitochondrial mRNA stability and an amino-acid substitution of this protein causes the French-Canadian type of Leigh syndrome (LSFC), a neurodegenerative disorder characterized by complex IV deficiency. We have generated conditional Lrpprc knockout mice and show here that the gene is essential for embryonic development. Tissue-specific disruption of Lrpprc in heart causes mitochondrial cardiomyopathy with drastic reduction in steady-state levels of most mitochondrial mRNAs. LRPPRC forms an RNA-dependent protein complex that is necessary for maintaining a pool of non-translated mRNAs in mammalian mitochondria. Loss of LRPPRC does not only decrease mRNA stability, but also leads to loss of mRNA polyadenylation and the appearance of aberrant mitochondrial translation. The translation pattern without the presence of LRPPRC is misregulated with excessive translation of some transcripts and no translation of others. Our findings point to the existence of an elaborate machinery that regulates mammalian mtDNA expression at the post-transcriptional level.

Rescue of primary ubiquinone deficiency due to a novel COQ7 defect using 2,4-dihydroxybensoic acid.

BACKGROUND: Coenzyme Q is an essential mitochondrial electron carrier, redox cofactor and a potent antioxidant in the majority of cellular membranes. Coenzyme Q deficiency has been associated with a range of metabolic diseases, as well as with some drug treatments and ageing. METHODS: We used whole exome sequencing (WES) to investigate patients with inherited metabolic diseases and applied a novel ultra-pressure liquid chromatography-mass spectrometry approach to measure coenzyme Q in patient samples. RESULTS: We identified a homozygous missense mutation in the COQ7 gene in a patient with complex mitochondrial deficiency, resulting in severely reduced coenzyme Q levels We demonstrate that the coenzyme Q analogue 2,4-dihydroxybensoic acid (2,4DHB) was able to specifically bypass the COQ7 deficiency, increase cellular coenzyme Q levels and rescue the biochemical defect in patient fibroblasts. CONCLUSION: We report the first patient with primary coenzyme Q deficiency due to a homozygous COQ7 mutation and a potentially beneficial treatment using 2,4DHB.

MTERF3 regulates mitochondrial ribosome biogenesis in invertebrates and mammals.

Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression.

Rapid pulsed whole genome sequencing for comprehensive acute diagnostics of inborn errors of metabolism.

BACKGROUND: Massively parallel DNA sequencing (MPS) has the potential to revolutionize diagnostics, in particular for monogenic disorders. Inborn errors of metabolism (IEM) constitute a large group of monogenic disorders with highly variable clinical presentation, often with acute, nonspecific initial symptoms. In many cases irreversible damage can be reduced by initiation of specific treatment, provided that a correct molecular diagnosis can be rapidly obtained. MPS thus has the potential to significantly improve both diagnostics and outcome for affected patients in this highly specialized area of medicine. RESULTS: We have developed a conceptually novel approach for acute MPS, by analysing pulsed whole genome sequence data in real time, using automated analysis combined with data reduction and parallelization. We applied this novel methodology to an in-house developed customized work flow enabling clinical-grade analysis of all IEM with a known genetic basis, represented by a database containing 474 disease genes which is continuously updated. As proof-of-concept, two patients were retrospectively analysed in whom diagnostics had previously been performed by conventional methods. The correct disease-causing mutations were identified and presented to the clinical team after 15 and 18 hours from start of sequencing, respectively. With this information available, correct treatment would have been possible significantly sooner, likely improving outcome. CONCLUSIONS: We have adapted MPS to fit into the dynamic, multidisciplinary work-flow of acute metabolic medicine. As the extent of irreversible damage in patients with IEM often correlates with timing and accuracy of management in early, critical disease stages, our novel methodology is predicted to improve patient outcome. All procedures have been designed such that they can be implemented in any technical setting and to any genetic disease area. The strategy conforms to international guidelines for clinical MPS, as only validated disease genes are investigated and as clinical specialists take responsibility for translation of results. As follow-up in patients without any known IEM, filters can be lifted and the full genome investigated, after genetic counselling and informed consent.

Altered dopamine metabolism and increased vulnerability to MPTP in mice with partial deficiency of mitochondrial complex I in dopamine neurons.

A variety of observations support the hypothesis that deficiency of complex I [reduced nicotinamide-adenine dinucleotide (NADH):ubiquinone oxidoreductase] of the mitochondrial respiratory chain plays a role in the pathophysiology of Parkinson's disease (PD). However, recent data from a study using mice with knockout of the complex I subunit NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (Ndufs4) has challenged this concept as these mice show degeneration of non-dopamine neurons. In addition, primary dopamine (DA) neurons derived from such mice, reported to lack complex I activity, remain sensitive to toxins believed to act through inhibition of complex I. We tissue-specifically disrupted the Ndufs4 gene in mouse heart and found an apparent severe deficiency of complex I activity in disrupted mitochondria, whereas oxidation of substrates that result in entry of electrons at the level of complex I was only mildly reduced in intact isolated heart mitochondria. Further analyses of detergent-solubilized mitochondria showed the mutant complex I to be unstable but capable of forming supercomplexes with complex I enzyme activity. The loss of Ndufs4 thus causes only a mild complex I deficiency in vivo. We proceeded to disrupt Ndufs4 in midbrain DA neurons and found no overt neurodegeneration, no loss of striatal innervation and no symptoms of Parkinsonism in tissue-specific knockout animals. However, DA homeostasis was abnormal with impaired DA release and increased levels of DA metabolites. Furthermore, Ndufs4 DA neuron knockouts were more vulnerable to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Taken together, these findings lend in vivo support to the hypothesis that complex I deficiency can contribute to the pathophysiology of PD.

Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo.

Mitochondrial dysfunction is heavily implicated in Parkinson disease (PD) as exemplified by the finding of an increased frequency of respiratory chain-deficient dopamine (DA) neurons in affected patients. An inherited form of PD is caused by impaired function of Parkin, an E3 ubiquitin ligase reported to translocate to defective mitochondria in vitro to facilitate their clearance. We have developed a reporter mouse to assess mitochondrial morphology in DA neurons in vivo and show here that respiratory chain deficiency leads to fragmentation of the mitochondrial network and to the formation of large cytoplasmic bodies derived from mitochondria. Surprisingly, the dysfunctional mitochondria do not recruit Parkin in vivo, and neither the clearance of defective mitochondria nor the neurodegeneration phenotype is affected by the absence of Parkin. We also show that anterograde axonal transport of mitochondria is impaired in respiratory chain-deficient DA neurons, leading to a decreased supply of mitochondria to the axonal terminals.

The bicoid stability factor controls polyadenylation and expression of specific mitochondrial mRNAs in Drosophila melanogaster.

The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation.

PARKIN is not required to sustain OXPHOS function in adult mammalian tissues.

Loss-of-function variants in the PRKN gene encoding the ubiquitin E3 ligase PARKIN cause autosomal recessive early-onset Parkinson's disease (PD). Extensive in vitro and in vivo studies have reported that PARKIN is involved in multiple pathways of mitochondrial quality control, including mitochondrial degradation and biogenesis. However, these findings are surrounded by substantial controversy due to conflicting experimental data. In addition, the existing PARKIN-deficient mouse models have failed to faithfully recapitulate PD phenotypes. Therefore, we have investigated the mitochondrial role of PARKIN during ageing and in response to stress by employing a series of conditional Parkin knockout mice. We report that PARKIN loss does not affect oxidative phosphorylation (OXPHOS) capacity and mitochondrial DNA (mtDNA) levels in the brain, heart, and skeletal muscle of aged mice. We also demonstrate that PARKIN deficiency does not exacerbate the brain defects and the pro-inflammatory phenotype observed in mice carrying high levels of mtDNA mutations. To rule out compensatory mechanisms activated during embryonic development of Parkin-deficient mice, we generated a mouse model where loss of PARKIN was induced in adult dopaminergic (DA) neurons. Surprisingly, also these mice did not show motor impairment or neurodegeneration, and no major transcriptional changes were found in isolated midbrain DA neurons. Finally, we report a patient with compound heterozygous PRKN pathogenic variants that lacks PARKIN and has developed PD. The PARKIN deficiency did not impair OXPHOS activities or induce mitochondrial pathology in skeletal muscle from the patient. Altogether, our results argue that PARKIN is dispensable for OXPHOS function in adult mammalian tissues.