Annalinda Pisano

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber’s hereditary optic neuropathy

The mechanisms of incomplete penetrance in Leber’s hereditary optic neuropathy are elusive. Giordano et al. show that mitochondrial DNA content and mitochondrial mass are both increased in tissues and cells from unaffected mutation carriers relative to affected relatives and control individuals. Upregulation of mitochondrial biogenesis may represent a therapeutic target.

Isoleucyl-tRNA synthetase levels modulate the penetrance of a homoplasmic m.4277T>C mitochondrial tRNA Ile mutation causing hypertrophic cardiomyopathy

The genetic and epigenetic factors underlying the variable penetrance of homoplasmic mitochondrial DNA mutations are poorly understood. We investigated a 16-year-old patient with hypertrophic cardiomyopathy harboring a homoplasmic m.4277T>C mutation in the mt-tRNA(Ile) (MTTI) gene. Skeletal muscle showed multiple respiratory chain enzyme abnormalities and a decreased steady-state level of the mutated mt-tRNA(Ile). Transmitochondrial cybrids grown on galactose medium demonstrated a functional effect of this mutation on cell viability, confirming pathogenicity. These findings were reproduced in transmitochondrial cybrids, harboring a previously described homoplasmic m.4300A>G MTTI mutation. The pathogenic role of the m.4277T>C mutation may be ascribed to misfolding of the mt-tRNA molecule, as demonstrated by the altered electrophoretic migration of the mutated mt-tRNA. Indeed, structure and sequence analyses suggest that thymidine at position 4277 of mt-tRNA(Ile) is involved in a conserved tertiary interaction with thymidine at position 4306. Interestingly, the mutation showed variable penetrance within family members, with skeletal muscle from the patients clinically unaffected mother demonstrating normal muscle respiratory chain activities and steady-state levels of mt-tRNA(Ile), while homoplasmic for the m.4277T>C mutation. Analysis of mitochondrial isoleucyl-tRNA synthetase revealed significantly higher expression levels in skeletal muscle and fibroblasts of the unaffected mother when compared with the proband, while the transient over-expression of the IARS2 gene in patient transmitochondrial cybrids improved cell viability. This is the first observation that constitutively high levels of aminoacyl-tRNA synthetases (aaRSs) in human tissues prevent the phenotypic expression of a homoplasmic mt-tRNA point mutation. These findings extend previous observations on aaRSs therapeutic effects in yeast and human.

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‐tRNAIle 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‐tRNALeu(UUR) gene. Furthermore, we provide evidence that this small, non‐catalytic domain is able to directly and specifically interact in vitro with human mt‐tRNALeu(UUR) with high affinity and stability and, with lower affinity, with mt‐tRNAIle. 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.

Cardiomyopathies due to homoplasmic mitochondrial tRNA mutations: morphologic and molecular features☆

Isolated hypertrophic cardiomyopathy may represent the sole clinical feature of a mitochondrial disorder in adult patients. The clinical outcome is characterized by a rapid progression to dilation and failure. A mitochondrial etiology in these cases is not obvious at clinical investigation and may represent an unexpected finding at autopsy or after cardiac transplant. We describe the morphologic, biochemical, and molecular features of hearts from 3 transplanted patients with isolated mitochondrial cardiomyopathy caused by homoplasmic mutations in the MTTI gene, coding for mitochondrial isoleucine tRNA (mt-tRNA(Ile)). On gross examination, the 3 hearts showed a symmetric pattern of hypertrophy. At histology, cardiomyocytes were hypertrophic and showed sarcoplasmic vacuoles filled with granules that stain with antimitochondrial antibodies. On frozen sections, the combined cytochrome c oxidase (COX)/succinate dehydrogenase stain showed a large prevalence of COX-deficient cardiomyocytes. Mitochondrially encoded COX subunit I was almost absent on immunohistochemistry, whereas the nuclear-encoded COX subunit IV was normally expressed. Ultrastructural analysis confirmed the marked mitochondrial proliferation. Biochemical studies of cardiac homogenates revealed a combined respiratory chain defect. Quantitative restriction fragment length polymorphism analysis of DNA from cardiac homogenate confirmed that the mt-tRNA mutations were also detected in the patients blood. High-resolution Northern blot analysis showed a marked decrease in the steady-state level of mt-tRNA(Ile), confirming pathogenicity. In conclusion, pathologists play a major role in unraveling the mitochondrial etiology of isolated hypertrophic cardiomyopathies, provided that a detailed diagnostic flowchart is followed. Once the mitochondrial etiology is clearly defined, molecular analyses on the heart are an invaluable tool to assign mutation pathogenicity.

Targeting estrogen receptor β as preventive therapeutic strategy for Leber's hereditary optic neuropathy

Lebers hereditary optic neuropathy (LHON) is a maternally inherited blinding disease characterized by degeneration of retinal ganglion cells (RGCs) and consequent optic nerve atrophy. Peculiar features of LHON are incomplete penetrance and gender bias, with a marked male prevalence. Based on the different hormonal metabolism between genders, we proposed that estrogens play a protective role in females and showed that these hormones ameliorate mitochondrial dysfunction in LHON through the estrogen receptors (ERs). We also showed that ERβ localize to the mitochondria of RGCs. Thus, targeting ERβ may become a therapeutic strategy for LHON specifically aimed at avoiding or delaying the onset of disease in mutation carriers. Here, we tested the effects of ERβ targeting on LHON mitochondrial defective metabolism by treating LHON cybrid cells carrying the m.11778G>A mutation with a combination of natural estrogen-like compounds that bind ERβ with high selectivity. We demonstrated that these molecules improve cell viability by reducing apoptosis, inducing mitochondrial biogenesis and strongly reducing the levels of reactive oxygen species in LHON cells. These effects were abolished in cells with ERβ knockdown by silencing receptor expression or by using specific receptor antagonists. Our observations support the hypothesis that estrogen-like molecules may be useful in LHON prophylactic therapy. This is particularly important for lifelong disease prevention in unaffected LHON mutation carriers. Current strategies attempting to combat degeneration of RGCs during the acute phase of LHON have not been very effective. Implementing a different and preemptive approach with a low risk profile may be very helpful.

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.

Homozygous NOTCH3 null mutation and impaired NOTCH3 signaling in recessive early‐onset arteriopathy and cavitating leukoencephalopathy

Notch signaling is essential for vascular physiology. Neomorphic heterozygous mutations in NOTCH3, one of the four human NOTCH receptors, cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Hypomorphic heterozygous alleles have been occasionally described in association with a spectrum of cerebrovascular phenotypes overlapping CADASIL, but their pathogenic potential is unclear. We describe a patient with childhood‐onset arteriopathy, cavitating leukoencephalopathy with cerebral white matter abnormalities presented as diffuse cavitations, multiple lacunar infarctions and disseminated microbleeds. We identified a novel homozygous c.C2898A (p.C966*) null mutation in NOTCH3 abolishing NOTCH3 expression and causing NOTCH3 signaling impairment. NOTCH3 targets acting in the regulation of arterial tone (KCNA5) or expressed in the vasculature (CDH6) were downregulated. Patients vessels were characterized by smooth muscle degeneration as in CADASIL, but without deposition of granular osmiophilic material (GOM), the CADASIL hallmark. The heterozygous parents displayed similar but less dramatic trends in decrease in the expression of NOTCH3 and its targets, as well as in vessel degeneration. This study suggests a functional link between NOTCH3 deficiency and pathogenesis of vascular leukoencephalopathies.

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 hyperenhancement 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 etiology at autopsy. We examined 281 consecutive cases of sudden death of subjects aged 1 to 35 years. NLVS was defined as a thin, gray 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 fibroadipose tissue. Right ventricular involvement was always present. Patchy lymphocytic infiltrates were frequent. Genetic and molecular analyses clarified the etiology of NLVS in a subset of cases. Electrocardiographic (ECG) recordings were available in more than 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.

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 (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNALeu(UUR) or with mutations in the mt-tRNAIle, 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-tRNALys, 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-tRNALeu(UUR) and mt-tRNALys, and stabilize mutant mt-tRNALeu(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.

Potential role for the VDR agonist elocalcitol in metabolic control: Evidences in human skeletal muscle cells.

Vitamin D plays a pivotal role to maintain skeletal muscle integrity and health. Vitamin D deficiency characterizes inflammatory myopathy (IM) and diabetes, often overlapping diseases involving skeletal muscle damage. Vitamin D receptor (VDR) agonists likely exert beneficial effects in both IM and metabolic disturbances. We aim to evaluate in vitro the effect of elocalcitol, a non-hypercalcemic VDR agonist, on the biomolecular metabolic machinery of human skeletal muscle cells (Hfsmc), vs. insulin (I). We analyzed GLUT4, Flotillin-1, Caveolin-3 and Caveolin-1 cell expression/localization; mTOR, AKT, ERK and 4E-BP1 phosphorylation; IL-6 myokine release; VDR expression. We investigated in vivo vitamin D status in IM subjects, evaluating VDR muscular expression and serum vitamin D with metabolism-related parameters, as glycemia, triglycerides, cholesterol, resistin and adiponectin. In Hfsmc, elocalcitol exerted an I-like effect, promoting GLUT4 re-localization in Flotillin-1, Caveolin-3 and Caveolin-1 positive sites and mTOR, AKT, ERK, 4E-BP1 activation; it enhanced IL-6 myokine release. IM subjects, all normoglycemic, showed VDR/vitamin D deficiency that, together with high lipidemic and resistin profile, possibly increases the risk to develop metabolic diseases. VDR agonists as elocalcitol may be therapeutic tools for skeletal muscle integrity/function maintenance, an indispensable condition for health homeostasis.