The phenotypic expression of mitochondrial tRNA-mutations can be modulated by either mitochondrial leucyl-tRNA synthetase or the C-terminal domain thereof

Mutations in mitochondrial (mt) DNA determine important human diseases. The majority of the known pathogenic mutations are located in transfer RNA (tRNA) genes and are responsible for a wide range of currently untreatable disorders. Experimental evidence both in yeast and in human cells has shown that the detrimental effects of mt-tRNA point mutations can be attenuated by increasing the expression of the cognate mt-aminoacyl-tRNA synthetases (aaRSs). In addition, constitutive high levels of isoleucyl-tRNA syntethase have been shown to reduce the penetrance of a homoplasmic mutation in mt-tRNAIle in a small kindred. More recently, we showed that the isolated carboxy-terminal domain of human mt-leucyl tRNA synthetase (LeuRS-Cterm) localizes to mitochondria and ameliorates the energetic defect in transmitochondrial cybrids carrying mutations either in the cognate mt-tRNALeu(UUR) or in the non cognate mt-tRNAIle gene. Since the mt-LeuRS-Cterm does not possess catalytic activity, its rescuing ability is most likely mediated by a chaperon-like effect, consisting in the stabilization of the tRNA structure altered by the mutation. All together, these observations open potential therapeutic options for mt-tRNA mutations-associated diseas

Nonischemic left ventricular scar and cardiac sudden death in the young

Nonischemic Left Ventricular Scar (NLVS) is a pattern of myocardial injury characterized by midventricular and/or subepicardial gadolinium hyper enhancement at cardiac magnetic resonance, in absence of significant coronary artery disease. We aimed to evaluate the prevalence of NLVS in juvenile sudden cardiac death and to ascertain its aetiology at autopsy. We examined 281 consecutive cases of sudden death of subjects aged 1 to 35 years of age. NLVS was defined as a thin, grey rim of subepicardial and/or midmyocardial scar in the left ventricular free wall and/or the septum, in absence of significant stenosis of coronary arteries. NLVS was the most frequent finding (25%) in sudden deaths occurring during sports. Myocardial scar was localized most frequently within the left ventricular posterior wall, and affected the subepicardial myocardium, often extending to the midventricular layer. On histology it consisted of fibrous or fibro-adipose tissue. Right ventricular involvement was always present. Patchy lymphocytic infiltrates were frequent. Genetic and molecular analyses clarified the aetiology of NLVS in a subset of cases. ECG recordings were available in over half of subjects. The most frequent abnormality was the presence of low QRS voltages (< 0,5 mV) in limb leads. In serial ECG tracings, the decrease in QRS voltages appeared, in some way progressive. NLVS is the most frequent morphologic substrate of juvenile cardiac sudden death in sports. It can be suspected based on ECG findings. Autopsy study and clinical screening of family members are required to differentiate between Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia and chronic acquired myocarditis

Impaired mitochondrial biogenesis is a common feature to myocardial hypertrophy and end-stage ischemic heart failure

Mitochondrial (mt) DNA depletion and oxidative mtDNA damage have been implicated in the process of pathological cardiac remodeling. Whether these features are present in the early phase of maladaptive cardiac remodeling, that is, during compensated cardiac hypertrophy, is still unknown. We compared the morphologic and molecular features of mt biogenesis and markers of oxidative stress in human heart from adult subjects with compensated hypertrophic cardiomyopathy and heart failure. We have shown that mtDNA depletion is a constant feature of both conditions. A quantitative loss of mtDNA content was associated with significant down-regulation of selected modulators of mt biogenesis and decreased expression of proteins involved in mtDNA maintenance. Interestingly, mtDNA depletion characterized also the end-stage phase of cardiomyopathies due to a primary mtDNA defect. Oxidative stress damage was detected only in failing myocardium

Novel compound mutations in the mitochondrial translation elongation factor (TSFM) gene cause severe cardiomyopathy with myocardial fibro-adipose replacement

Primary mitochondrial dysfunction is an under-appreciated cause of cardiomyopathy, especially when cardiac symptoms are the unique or prevalent manifestation of disease. Here, we report an unusual presentation of mitochondrial cardiomyopathy, with dilated phenotype and pathologic evidence of biventricular fibro-adipose replacement, in a 33-year old woman who underwent cardiac transplant. Whole exome sequencing revealed two novel compound heterozygous variants in the TSFM gene, coding for the mitochondrial translation elongation factor EF-Ts. This protein participates in the elongation step of mitochondrial translation by binding and stabilizing the translation elongation factor Tu (EF-Tu). Bioinformatics analysis predicted a destabilization of the EF-Ts variants complex with EF-Tu, in agreement with the dramatic steady-state level reduction of both proteins in the clinically affected myocardium, which demonstrated a combined respiratory chain enzyme deficiency. In patient fibroblasts, the decrease of EF-Ts was paralleled by up-regulation of EF-Tu and induction of genes involved in mitochondrial biogenesis, along with increased expression of respiratory chain subunits and normal oxygen consumption rate. Our report extends the current picture of morphologic phenotypes associated with mitochondrial cardiomyopathies and confirms the heart as a main target of TSFM dysfunction. The compensatory response detected in patient fibroblasts might explain the tissue-specific expression of TSFM-associated disease

The isolated carboxy-terminal domain of human mitochondrial leucyl-tRNA synthetase rescues the pathological phenotype of mitochondrial tRNA mutations in human cells.

Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA genes. Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no effective treatment is available at present. We show that three human mt aminoacyl-tRNA syntethases, namely leucyl-, valyl-, and isoleucyl-tRNA synthetase are able to improve both viability and bioenergetic proficiency of human transmitochondrial cybrid cells carrying pathogenic mutations in the mt-tRNA(Ile) gene. Importantly, we further demonstrate that the carboxy-terminal domain of human mt leucyl-tRNA synthetase is both necessary and sufficient to improve the pathologic phenotype associated either with these "mild" mutations or with the "severe" m.3243A>G mutation in the mt-tRNA(L)(eu(UUR)) gene. Furthermore, we provide evidence that this small, non-catalytic domain is able to directly and specifically interact in vitro with human mt-tRNA(Leu(UUR)) with high affinity and stability and, with lower affinity, with mt-tRNA(Ile). Taken together, our results sustain the hypothesis that the carboxy-terminal domain of human mt leucyl-tRNA synthetase can be used to correct mt dysfunctions caused by mt-tRNA mutations

Elucidating the molecular mechanisms underlying the biological activity of rescuing peptides in MELAS mitochondrial disease

Mitochondrial Encephalopathy with Lactic Acidosis and Stroke like Episodes (MELAS) is a mitochondrial disease caused by point mutations in the tRNALeu(UUR) with a prevalence in position 3243 (A&gt;G). These mutations cause severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA such as reduced aminoacylation and lack of post-transcriptional modification. It has been shown that overexpression of leucyl-tRNA-synthetase (mt-LeuRS) is able to rescue defects of pathogenic mutation of tRNALeu(UUR) in MELAS cybrids [1]. The rescuing activity of human mt-LeuRS resides in a small (&lt;70 amino acids) part of the carboxy-terminal domain (Cterm) of the enzyme and in short (15-16 aa) C-term-derived peptides. However, the molecular mechanisms underlying the rescuing process are still unknown. On these bases, we started a project aimed at elucidating the ability of mt-LeuRS C-term and its derived peptides to correct the pathological phenotype in MELAS. To address this issue we intend: i) to investigate in vivo the interactions of rescuing molecules with mutated mt-tRNA; ii) to study the effect of overexpression of rescuing molecules on the rate of synthesis and stability of de novo synthesized mt-polypeptides; iii) to investigate the effect of rescuing molecules on the steady-state level, the aminoacylation level and the post-transcriptional modifications of the mutated tRNA. To date we have characterized the profile of the de novo synthesized mt-polypeptides from MELAS cybrids, finding an overall decreased rate of synthesis and the appearance of aberrant mitochondrial translation product. We have also observed a decreased steady-state level of NDUFB8 (Complex I) and COXI (Complex IV). Finally, we have demonstrated in MELAS cybrids constitutively expressing C-term, the existence of a preferential interaction between Cterm polypeptide and tRNALeu(UUR) by means of RNA immunoprecipitation experiments

Isolated Distal Myopathy of the Upper Limbs Associated With Mitochondrial DNA Depletion and Polymerase gamma Mutations

Objective: To describe an unusual clinical phenotype in an adult harboring 2 compound heterozygous polymerase gamma (POLG) mutations. Design: Case report. Setting: University-based outpatient neurology clinic and pathology and genetics laboratory. Patient: A 27-year-old man presenting with isolated distal myopathy of the upper extremities in the absence of sensory disturbances. Results: Histochemical analysis of a muscle biopsy specimen showed numerous cytochrome c oxidase deficient fibers. Molecular analysis revealed marked depletion of muscle mitochondrial DNA in the absence of multiple mitochondrial DNA deletions. Sequence analysis of the POLG gene revealed heterozygous sequence variants in compound c.1156C>T (p.R386C) and c.2794C>T (p.H932Y) segregating with clinical disease in the family. The p.R386C change appears to be a novel mutation. Conclusion: Our case broadens the phenotypic spectrum of disorders associated with POLG mutations and highlights the complex relationship between genotype and phenotype in POLG-related disease

Short peptides from leucyl-trna synthetase rescue disease-causing mitochondrial trna point mutations

Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain of human mt-leucyl tRNA synthetase (Cterm) rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNA(Leu(UUR)) or with mutations in the mt-tRNA(Ile), both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs).Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNA(Lys), aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, β30_31 and β32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNA(Leu(UUR)) and mt-tRNA(Lys), and stabilize mutant mt-tRNA(Leu(UUR)). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs