Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome.

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.

Pharmacotherapeutic management of epilepsy in MERRF syndrome.

INTRODUCTION: Epilepsy is a prominent feature of myoclonic epilepsy with ragged-red fibers (MERRF)-syndrome. The most frequent seizure type is myoclonic seizures, of which the treatment is challenging and empiric. AREAS COVERED: Herein, the author summarises and discusses previous and recent findings of antiepileptic drug (AED) treatment in MERRF-syndrome. EXPERT OPINION: MERRF-syndrome is a predominantly maternally inherited, multisystem mitochondrial disorder caused by pathogenic variants predominantly of the mitochondrial DNA (mtDNA). Canonical clinical features of MERRF include myoclonus, epilepsy, ataxia, and myopathy. Additionally, other manifestations in the CNS, peripheral nerves, eyes, ears, heart, gastrointestinal tract, and endocrine organs may occur (MERRF-plus). Today, MERRF is considered rather as myoclonic ataxia than as myoclonic epilepsy. Genotypically, MERRF is due to mutations in 13 mtDNA-located genes and 1 nDNA-located gene. According to the modified Smith-score, the strongest gene-disease relationship exists for MT-TK, MT-TL1, and POLG1. Epilepsy is the second most frequent phenotypic feature of MERRF. Seizure-types associated with MERRF include focal myoclonic, focal clonic, and focal atonic seizures, generalized myoclonic, tonic-clonic, atonic, and myoclonic-atonic seizures, or typical absences. Treatment of myoclonic epilepsy relies on expert judgments recommending levetiracetam, together with clonazepam, or topiramate, zonisamide, or piracetam in monotherapy as the first line AEDs.

Antimyoclonic Effect of Levetiracetam and Clonazepam Combined Treatment on Myoclonic Epilepsy with Ragged-Red Fiber Syndrome with m.8344A>G Mutation.

BACKGROUND: Treatment of myoclonic seizures in myoclonic epilepsy with ragged-red fibers (MERRFs) has been empirical and ineffective. Guideline on this disease is not available. Additional trials must be conducted to find more suitable treatments for it. In this study, the antimyoclonic effects of monotherapies, including levetiracetam (LEV), clonazepam (CZP), valproic acid (VPA), and topiramate (TPM) compared to combination therapy group with LEV and CZP on MERRF, were evaluated to find a more advantageous approach on the treatment of myoclonic seizures. METHODS: Treatments of myoclonic seizures with VPA, LEV, CZP, and TPM were reported as monotherapies in 17 MERRF patients from Qilu Hospital between 2003 and 2016, who were diagnosed through clinical data and genetic testing. After 1-4 months of follow-up (mean: 82.9 ± 28.1 days), 12 patients that exhibited poor responses to monotherapy were given a combined treatment consisting of LEV and CZP subsequently. The follow-up period was 4-144 months (mean: 66.3 ± 45.3 months), the effective rates of monotherapy group (17 patients) and combination therapy group (12 patients) were analyzed by Chi-square test. RESULTS: The m.8344 A>G mutation was detected in all patients. There were four patients with partial response (4/17, two in the CZP group and two in the LEV group), ten patients with stable disease (10/17, six in the CZP group, three in the LEV group, and one in the TPM group), and three patients with progressive disease (3/18, two in the VPA group and one in the TPM group). Twelve of the patients with LEV combined with CZP showed a positive effect and good tolerance (12/12), eight of them demonstrated improved cognition and coordination. There was a significant difference between the monotherapy group and combination therapy group in the efficacy of antimyoclonic seizures (χ2 = 13.7, P < 0.001). CONCLUSIONS: LEV in combination with CZP is an efficient and safe treatment for myoclonic seizures in patients with this disease exhibiting the m.8344A>G mutation.

mitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels.

Pathogenic mitochondrial DNA (mtDNA) mutations often co-exist with wild-type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70-90%). Previously, our laboratory showed that mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY-YIG homing nuclease from T4 phage (I-TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev-TALEs) could specifically bind and cleave mtDNA of patient-derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged-red fibers (MERRF). We tested two mitoTev-TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev-TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev-TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues.

The Bacterial Protein CNF1 as a Potential Therapeutic Strategy against Mitochondrial Diseases: A Pilot Study.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1), which acts on the Rho GTPases that are key regulators of the actin cytoskeleton, is emerging as a potential therapeutic tool against certain neurological diseases characterized by cellular energy homeostasis impairment. In this brief communication, we show explorative results on the toxin's effect on fibroblasts derived from a patient affected by myoclonic epilepsy with ragged-red fibers (MERRF) that carries a mutation in the m.8344A>G gene of mitochondrial DNA. We found that, in the patient's cells, besides rescuing the wild-type-like mitochondrial morphology, CNF1 administration is able to trigger a significant increase in cellular content of ATP and of the mitochondrial outer membrane marker Tom20. These results were accompanied by a profound F-actin reorganization in MERRF fibroblasts, which is a typical CNF1-induced effect on cell cytoskeleton. These results point at a possible role of the actin organization in preventing or limiting the cell damage due to mitochondrial impairment and at CNF1 treatment as a possible novel strategy against mitochondrial diseases still without cure.

Expanded phenotypic spectrum of the m.8344A>G "MERRF" mutation: data from the German mitoNET registry.

The m.8344A>G mutation in the MTTK gene, which encodes the mitochondrial transfer RNA for lysine, is traditionally associated with myoclonic epilepsy and ragged-red fibres (MERRF), a multisystemic mitochondrial disease that is characterised by myoclonus, seizures, cerebellar ataxia, and mitochondrial myopathy with ragged-red fibres. We studied the clinical and paraclinical phenotype of 34 patients with the m.8344A>G mutation, mainly derived from the nationwide mitoREGISTER, the multicentric registry of the German network for mitochondrial disorders (mitoNET). Mean age at symptom onset was 24.5 years ±10.9 (6-48 years) with adult onset in 75 % of the patients. In our cohort, the canonical features seizures, myoclonus, cerebellar ataxia and ragged-red fibres that are traditionally associated with MERRF, occurred in only 61, 59, 70, and 63 % of the patients, respectively. In contrast, other features such as hearing impairment were even more frequently present (72 %). Other common features in our cohort were migraine (52 %), psychiatric disorders (54 %), respiratory dysfunction (45 %), gastrointestinal symptoms (38 %), dysarthria (36 %), and dysphagia (35 %). Brain MRI revealed cerebral and/or cerebellar atrophy in 43 % of our patients. There was no correlation between the heteroplasmy level in blood and age at onset or clinical phenotype. Our findings further broaden the clinical spectrum of the m.8344A>G mutation, document the large clinical variability between carriers of the same mutation, even within families and indicate an overlap of the phenotype with other mitochondrial DNA-associated syndromes.

When should MERRF (myoclonus epilepsy associated with ragged-red fibers) be the diagnosis?

Myoclonic epilepsy associated with ragged red fibers (MERRF) is a rare mitochondrial disorder. Diagnostic criteria for MERRF include typical manifestations of the disease: myoclonus, generalized epilepsy, cerebellar ataxia and ragged red fibers (RRF) on muscle biopsy. Clinical features of MERRF are not necessarily uniform in the early stages of the disease, and correlations between clinical manifestations and physiopathology have not been fully elucidated. It is estimated that point mutations in the tRNALys gene of the DNAmt, mainly A8344G, are responsible for almost 90% of MERRF cases. Morphological changes seen upon muscle biopsy in MERRF include a substantive proportion of RRF, muscle fibers showing a deficient activity of cytochrome c oxidase (COX) and the presence of vessels with a strong reaction for succinate dehydrogenase and COX deficiency. In this review, we discuss mainly clinical and laboratory manifestations, brain images, electrophysiological patterns, histology and molecular findings as well as some differential diagnoses and treatments.

When should MERRF (myoclonus epilepsy associated with ragged-red fibers) be the diagnosis? / Quando o diagnóstico deveria ser MERRF (epilepsia mioclônica associada com fibras vermelhas rasgadas)?

Myoclonic epilepsy associated with ragged red fibers (MERRF) is a rare mitochondrial disorder. Diagnostic criteria for MERRF include typical manifestations of the disease: myoclonus, generalized epilepsy, cerebellar ataxia and ragged red fibers (RRF) on muscle biopsy. Clinical features of MERRF are not necessarily uniform in the early stages of the disease, and correlations between clinical manifestations and physiopathology have not been fully elucidated. It is estimated that point mutations in the tRNALys gene of the DNAmt, mainly A8344G, are responsible for almost 90% of MERRF cases. Morphological changes seen upon muscle biopsy in MERRF include a substantive proportion of RRF, muscle fibers showing a deficient activity of cytochrome c oxidase (COX) and the presence of vessels with a strong reaction for succinate dehydrogenase and COX deficiency. In this review, we discuss mainly clinical and laboratory manifestations, brain images, electrophysiological patterns, histology and molecular findings as well as some differential diagnoses and treatments.

Epilepsia mioclônica associada com fibras vermelhas rasgadas (MERRF) é uma rara doença mitocondrial. O critério diagnóstico para MERRF inclui as manifestações típicas da doença: mioclonia, epilepsia generalizada, ataxia cerebelar e fibras vermelhas rasgadas (RRF) na biópsia de músculo. Na fase inicial da doença, as manifestações clínicas podem não ser uniformes, e correlação entre as manifestações clínicas e fisiopatologia não estão completamente elucidadas. Estima-se que as mutações de ponto no gene tRNALys do DNAmt, principalmente a A8344G, sejam responsáveis por quase 90% dos casos de MERRF. As alterações morfológicas na biópsia muscular incluem uma grande proporção de RRF, fibras musculares com deficiência de atividade da citocromo c oxidase (COX) e presença de vasos com forte reação para succinato desidrogenase e deficiência da COX. Nesta revisão, são discutidas as principais manifestações clínicas e laboratoriais, imagens cerebrais, padrões eletrofisiológicos, histológicos e alterações moleculares, bem como, alguns dos diagnósticos diferenciais e tratamentos.

MERRF syndrome without ragged-red fibers: the need for molecular diagnosis.

We report a patient with myoclonic epilepsy who underwent muscle biopsy for suspected mitochondrial disease (myoclonic epilepsy with ragged-red fibers, MERRF). In spite of normal histochemical studies and of the absence of a severe COX deficiency, the molecular analysis showed the common MERRF mutation (A8344G) in the tRNA(Lys) gene on mitochondrial DNA. The case serves to illustrate the importance of pursuing the proposed mitochondrial genetic abnormality, even in patients with normal biopsy findings.

Antimyoclonic effect of levetiracetam in MERRF syndrome.

The treatment of progressive myoclonic epilepsy (PME) is largely empirical, even though valproic acid (VPA) is usually considered the drug of first choice. However, VPA should be used with caution in PME due to mitochondrial dysfunction, i.e. in MERRF (myoclonic epilepsy with ragged red fibers) syndrome, because of its interaction with mitochondrial respiration and metabolism. Levetiracetam (LEV) treatment was started in combination with VPA in a patient with typical clinical, histological, and biochemical features of MERRF due to a mutation on the tRNA of Phenilalanine gene. The average myoclonus score improved dramatically, as well as the quality of life and no side effects were observed, even after having withdrawn VPA. LEV may benefit myoclonus in PME of mitochondrial origin without altering mitochondrial function, and it could be considered the drug of first choice for the treatment of myoclonus in MERRF.

Levetiracetam in progressive myoclonic epilepsy: an exploratory study in 9 patients.

The authors conducted an open study of levetiracetam as add-on therapy in nine patients with well-defined progressive myoclonic epilepsies and refractory myoclonus. Myoclonus was evaluated semiquantitatively (territory, intensity, daily living activities). Five patients had improvement of their myoclonus score. Levetiracetam may benefit myoclonus in progressive myoclonic epilepsy.

Treatment of mitochondrial encephalomyopathy with a combination of cytochrome C and vitamins B1 and B2.

The therapeutic efficacy of a regimen consisting of intravenous injection of Cardiocrome, containing cytochrome c, flavin mononucleotide and thiamine diphosphate for mitochondrial encephalomyopathy (MEM) was examined. This combined therapy was applied to nine patients with MEM, including four with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. For the standard regimen, Cardiocrome was first injected daily, usually for 4 weeks, and later by means of intermittent injections for maintenance treatment. Clinical improvement was obtained in eight of the patients. Improvement was observed in the muscle symptoms of easy fatigability, motor disability and severity of stroke-like episodes, as well as in various other symptoms such as phosphate, tinnitus, headache, corneal edema, chilblains, thalamic pain, respiratory failure, and nystagmus. This clinical improvement was maintained for more than 1 year by additional intermittent injections. In conclusion, this therapy was fairly effective for the management of patients with MEM.

The treatment of mitochondrial myopathies and encephalomyopathies.

This paper briefly summarizes the results of a long-term, open pharmacotherapy trial in 16 patients with well-characterized mitochondrial disease. Outcome measures included repeated clinical evaluation, 31P-NMR spectroscopy and near-infrared spectroscopy. Treated patients appeared to survive longer with less functional disability and medical complications than typically seen in clinical practice.