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Objective Mounting evidence links neurodegenerative disorders such as Parkinson disease and Alzheimer disease with mitochondrial dysfunction, and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link recently emerged, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in postmitotic tissues. Here, we report 2 Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia. Methods Patients were extensively studied by optical coherence tomography (OCT) to assess retinal nerve fibers, and underwent muscle and brain magnetic resonance spectroscopy (MRS), and muscle biopsy and fibroblasts were analyzed. Candidate genes were sequenced, and mtDNA was analyzed for rearrangements. Results Affected individuals displayed a slowly progressive syndrome characterized by CPEO, mitochondrial myopathy, sensorineural deafness, peripheral neuropathy, parkinsonism, and/or cognitive impairment, in most cases without visual complains, but with subclinical loss of retinal nerve fibers at OCT. Muscle biopsies showed cytochrome c oxidase-negative fibers and mtDNA multiple deletions, and MRS displayed defective oxidative metabolism in muscle and brain. We found 2 heterozygous OPA1 missense mutations affecting highly conserved amino acid positions (p.G488R, p.A495V) in the guanosine triphosphatase domain, each segregating with affected individuals. Fibroblast studies showed a reduced amount of OPA1 protein with normal mRNA expression, fragmented mitochondria, impaired bioenergetics, increased autophagy and mitophagy. Interpretation The association of CPEO and parkinsonism/dementia with subclinical optic neuropathy widens the phenotypic spectrum of OPA1 mutations, highlighting the association of defective mitochondrial dynamics, mtDNA multiple deletions, and altered mitophagy with parkinsonism. Ann Neurol 2015;78:21-38 [ABSTRACT FROM AUTHOR]

Autosomal dominant cerebellar ataxia-deafness and narcolepsy (ADCA-DN) and Hereditary sensory neuropathy with dementia and hearing loss (HSN1E) are two rare, overlapping neurodegenerative syndromes that have been recently linked to allelic dominant pathogenic mutations in the DNMT1 gene, coding for DNA (cytosine-5)- methyltransferase 1 (DNMT1). DNMT1 is the enzyme responsible for maintaining the nuclear genome methylation patterns during the DNA replication and repair, thus regulating gene expression. The mutations responsible for ADCA-DN and HSN1E affect the replication foci targeting sequence domain, which regulates DNMT1 binding to chromatin. DNMT1 dysfunction is anticipated to lead to a global alteration of the DNA methylation pattern with predictable downstream consequences on gene expression. Interestingly, ADCA-DN and HSN1E phenotypes share some clinical features typical of mitochondrial diseases, such as optic atrophy, peripheral neuropathy, and deafness, and some biochemical evidence of mitochondrial dysfunction. The recent discovery of a mitochondrial isoform of DNMT1 and its proposed role in methylating mitochondrial DNA (mtDNA) suggests that DNMT1 mutations may directly affect mtDNA and mitochondrial physiology. On the basis of this latter finding the link between DNMT1 abnormal activity and mitochondrial dysfunction in ADCA-DN and HSN1E appears intuitive, however, mtDNA methylation remains highly debated. In the last years several groups demonstrated the presence of 5-methylcytosine in mtDNA by different approaches, but, on the other end, the opposite evidence that mtDNA is not methylated has also been published. Since over 1500 mitochondrial proteins are encoded by the nuclear genome, the altered methylation of these genes may well have a critical role in leading to the mitochondrial impairment observed in ADCA-DN and HSN1E. Thus, many open questions still remain unanswered, such as why mtDNA should be methylated, and how this process is regulated and executed? [ABSTRACT FROM AUTHOR]

Objective: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an extremely rare disease caused by a genetically-driven thymidine phosphorylase (TP) defect. TP converts the nucleosides thymidine and deoxyuridine into respective nucleotides. In MNGIE, these nucleosides accumulate promoting tissue damage and mtDNA abnormalities responsible for neurological impairment and severe gastrointestinal dysmotility. The resulting clinical phenotype has a tremendous impact on patients' poor quality of life and fatal outcome.1;2 One possible treatment strategy to MNGIE patients is the stable reduction of circulating toxic nucleosides via liver transplantation. This approach has been successfully achieved in MNGIE patients in Italy and in many Western Countries.3 Although the liver has never been described as a target organ of the disease, explanted livers of MNGIE patients show clear macroscopic changes. Aim: The objective of our study was to provide an accurate morphologic and metabolic analysis of MNGIE liver tissue vs. controls (CTR). Methods: Liver tissue biopsies (1x1 cm each sample) were collected from n=7 MNGIE (2F; 20-38 yrs) and n=7 CTR (2F; 22-67 yrs) subjects. Formalin fixed-paraffin embedded tissue sections were processed for: 1) Hematoxylin and Eosin staining to evaluate architectural changes; 2) Sirius Red/Fast Green collagen assay to localize and quantify fibrosis; 3) Orcein, vascular endothelial cells growth factor and CD31 immunostaining to assess vascular changes; 4) hypoxia-inducible-factor-1a (HIF-1a) immunostaining to test hypoxia. Part of the liver tissue biopsies (0.5x0.5 cm each sample) were collected and snap frozen from n=3 MNGIE and n=3 CTR for metabolomic NMR assay. Results: MNGIE liver was characterized by intense steatosis. Fibrosis increases (P=0.0025) with patient age leading to the formation of porto-portal and porto-central collagen septa. The vascular and biliary branches were altered and these findings correlated with the increased hypoxia in hepatocytes, as indicated by HIF-1a positive staining. Preliminary analysis performed with NMR indicated a rearrangement characterized by triglyceride production and a significant decline in aerobic metabolism. Conclusions: Our data, showing marked morphological and metabolic changes, indicate that the liver is a target organ of MNGIE. Liver alterations should be carefully taken into account as a compromised liver may interfere with the clinical response to targeted treatments. [ABSTRACT FROM AUTHOR]

Mitochondrial neuro-gastro-intestinal encephalomyopathy (MNGIE) is a rare and unavoidably fatal disease due to mutations in thymidine phosphorylase (TP). Clinically it is characterized by gastrointestinal dysfunction, malnutrition/cachexia and neurological manifestations. MNGIE diagnosis remains a challenge mainly because of the complexity and rarity of the disease. Thus, our purposes were to promote a better knowledge of the disease in Emilia-Romagna region (ERR) by creating an accurate and dedicated network; to establish the minimal prevalence of MNGIE in Italy starting from ERR. Blood TP activity level was used as screening test to direct candidates to complete diagnostic work-up. During the study period of 1 year, only 10/71 units of ERR recruited 14 candidates. Their screening did not show TP activity changes. An Italian patient not resident in ERR was actually proved to have MNGIE. At the end of study in Italy there were nine cases of MNGIE; thus, the Italian prevalence of the disease is ~0.15/1,000,000 as a gross estimation. Our study confirms that MNGIE diagnosis is a difficult process which reflects the rarity of the disease and, as a result, a low level of awareness among specialists and physicians. Having available novel therapeutic options (e.g., allogenic hematopoietic stem cell transplantation and, more recently, liver transplantation) and an easy screening test, an early diagnosis should be sought before tissue damage occurs irreversibly. [ABSTRACT FROM AUTHOR]

Introduction: Chronic intestinal pseudo-obstruction (CIPO) is a rare syndrome characterized by severe gastrointestinal (GI) dysmotility that mimics a GI obstruction in the absence of any mechanical cause CIPO is due to muscular-neuronal-glial-interstitial cells of Cajal abnormalities ¹ and can be idiopathic or secondary to a genetic cause Recently we documented that changes in the microvasculature supplying the gut contribute to GI abnormalities in a genetically driven form of CIPO2 Aim: The object of this study was to investigate if jejunal microvasculature alterations may occur and contribute to the disease also in idiopathic forms of CIPO. Methods: Full thickness jejunal biopsies were collected from n=23 CIPO and n=10 controls. Formalin fixed-paraffin embedded tissue sections were 1) stained with orcein to identify, meas- ure and count blood vessels, that were subdivided in >300μm (large), 300-101μm (medium), 100-51μm (small) and <50μm (very small), 2) stained with Sirius Red/Fast Green- collagen- assay to measure longitudinal muscle layer thickness and to spectrophotometrically determine fibrosis. Frozen tissue was used to assess by western blot Thymidine Phosphorylase (TP) protein expression, as this enzyme is involved in physio-pathological angiogenesis and was found absent in the previously documented form of CIPO secondary to a genetic disorder ² ; and the expression of HIF-1α as marker of hypoxia. Results: CIPO patients showed a lower vascular area with more vessels/mm² submucosa vs. controls. Vessels <50 μm increased at the expense of medium and large vessels. As com- pared to controls, CIPO showed, a higher fi brosis index, hypoxia, a decreased thickness of the longitudinal muscle layer and a decreased number of myenteric neurons. Finally, TP enzyme expression was found decreased in the jejunum of CIPO patients. Conclusion: Our results indicate that the jejunum of CIPO patients is characterized by quantitative and qualitative vascular changes, muscular and neuronal atrophy, fibrosis and hypoxia. Thus, this study allows us to hypothesize that abnormal GI vascularization might be a possible mechanism underlying gut dysfunction in CIPO. [ABSTRACT FROM AUTHOR]

The article presents a study on recovery of mtDNA depletion in the ileum of a Mitochondrial Nero-Gastrointestinal Encephalomyopathy (MNGIE) patient receiving liver transplant. Topics include information on mitochondrial neurogastrointestinal encephalomyopathy (MNGIE); gene encoding for thymidine phosphorylase (TP); and imbalance of mitochondrial nucleotide pool impaired mtDNA replication and depletion.

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

Variants found in the respiratory complex I (CI) subunit genes encoded by mitochondrial DNA can cause severe genetic diseases. However, it is difficult to establish a priori whether a single or a combination of CI variants may impact oxidative phosphorylation. Here we propose a computational approach based on coarse-grained molecular dynamics simulations aimed at investigating new CI variants. One of the primary CI variants associated with the Leber hereditary optic neuropathy (m.14484T>C/ MT-ND6) was used as a test case and was investigated alone or in combination with two additional rare CI variants whose role remains uncertain. We found that the primary variant positioned in the E-channel region, which is fundamental for CI function, stiffens the enzyme dynamics. Moreover, a new mechanism for the transition between π- and α-conformation in the helix carrying the primary variant is proposed. This may have implications for the E-channel opening/closing mechanism. Finally, our findings show that one of the rare variants, located next to the primary one, further worsens the stiffening, while the other rare variant does not affect CI function. This approach may be extended to other variants candidate to exert a pathogenic impact on CI dynamics, or to investigate the interaction of multiple variants. • CI variants found in mtDNA encoded subunit genes can cause severe genetic diseases. • Here we propose a CG MD approach aimed at the study of new CI variants. • We successfully applied our approach on three LHON variants. • The primary m.14484 T > C/ MT-ND6 variant stiffens CI dynamics in the E-channel region. • One of the other variants further worsens the stiffening of CI dynamics. [ABSTRACT FROM AUTHOR]

Background: Genetic and metabolic heterogeneity are well-known features of cancer and tumors can be viewed as an evolving mix of subclonal populations, subjected to selection driven by microenvironmental pressures or drug treatment. In previous studies, anti-VEGF therapy was found to elicit rewiring of tumor metabolism, causing marked alterations in glucose, lactate ad ATP levels in tumors. The aim of this study was to evaluate whether differences in the sensitivity to glucose starvation existed at the clonal level in ovarian cancer cells and to investigate the effects induced by anti-VEGF therapy on this phenotype by multi-omics analysis. Methods: Clonal populations, obtained from both ovarian cancer cell lines (IGROV-1 and SKOV3) and tumor xenografts upon glucose deprivation, were defined as glucose deprivation resistant (GDR) or glucose deprivation sensitive (GDS) clones based on their in vitro behaviour. GDR and GDS clones were characterized using a multi-omics approach, including genetic, transcriptomic and metabolic analysis, and tested for their tumorigenic potential and reaction to anti-angiogenic therapy. Results: Two clonal populations, GDR and GDS, with strikingly different viability following in vitro glucose starvation, were identified in ovarian cancer cell lines. GDR clones survived and overcame glucose starvation-induced stress by enhancing mitochondrial oxidative phosphorylation (OXPHOS) and both pyruvate and lipids uptake, whereas GDS clones were less able to adapt and died. Treatment of ovarian cancer xenografts with the anti-VEGF drug bevacizumab positively selected for GDR clones that disclosed increased tumorigenic properties in NOD/SCID mice. Remarkably, GDR clones were more sensitive than GDS clones to the mitochondrial respiratory chain complex I inhibitor metformin, thus suggesting a potential therapeutic strategy to target the OXPHOS-metabolic dependency of this subpopulation. Conclusion: A glucose-deprivation resistant population of ovarian cancer cells showing druggable OXPHOS-dependent metabolic traits is enriched in experimental tumors treated by anti-VEGF therapy. [ABSTRACT FROM AUTHOR]

Dear Sirs, Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder causing selective loss of motor neurons in brain motor cortex and spinal cord in which muscle denervation and atrophy are associated with pyramidal involvement, typically spreading to contiguous muscular regions and leading eventually to a fatal paralysis. Here, we report two ALS cases carrying the m.11778A > G/ I MT-ND4 i (R340H) mitochondrial DNA (mtDNA) mutation pathogenic for Leber's hereditary optic neuropathy (LHON), a maternally inherited disease due to mtDNA mutations affecting complex I function and usually leading to isolated optic atrophy [[2]]. Pathogenic mtDNA mutations have been found in ALS phenocopies with a primary mitochondrial disorder [[12]] and, more recently, mutations in nuclear genes, such as I CHCHD10 i , have been associated with ALS and mitochondrial myopathy with accumulation of mtDNA multiple deletions [[13]]. [Extracted from the article]

Background: Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements. Methods: We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment. Results: The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB). Conclusions: Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF. [ABSTRACT FROM AUTHOR]

Background: Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements. Methods: We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment. Results: The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB). Conclusions: Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF. [ABSTRACT FROM AUTHOR]

Recent scientific evidence suggests a link between migraine and brain energy metabolism. In fact, migraine is frequently observed in mitochondrial disorders. We studied 46 patients affected by mitochondrial disorders, through a headache-focused semi-structured interview, to evaluate the prevalence of migraine among patients affected by mitochondrial disorders, the possible correlations between migraine and neuromuscular genotype or phenotype, comorbidities, lactate acid levels and brain magnetic resonance spectroscopy. We explored migraine-related disability, analgesic and prophylactic treatments. Diagnoses were achieved according to International Classification of Headache Disorders, 3rd edition. Lifetime prevalence of migraine was 61% (28/46), with high values in both sexes (68% in females, 52% in males) and higher than the values found in both the general population and previous literature. A maternal inheritance pattern was reported in 57% of cases. MIDAS and HIT6 scores revealed a mild migraine-related disability. The high prevalence of migraine across different neuromuscular phenotypes and genotypes suggests that migraine itself may be a common clinical manifestation of brain energy dysfunction. Our results provide new relevant indications in favour of migraine as the result of brain energy unbalance. [ABSTRACT FROM AUTHOR]

Wolfram syndrome (WS) is a recessive multisystem disorder defined by the association of diabetes mellitus and optic atrophy, reminiscent of mitochondrial diseases. The role played by mitochondria remains elusive, with contradictory results on the occurrence of mitochondrial dysfunction. We evaluated 13 recessive WS patients by deep clinical phenotyping, including optical coherence tomography (OCT), serum lactic acid at rest and after standardized exercise, brain Magnetic Resonance Imaging, and brain and muscle Magnetic Resonance Spectroscopy (MRS). Finally, we investigated mitochondrial bioenergetics, network morphology, and calcium handling in patient-derived fibroblasts. Our results do not support a primary mitochondrial dysfunction in WS patients, as suggested by MRS studies, OCT pattern of retinal nerve fiber layer loss, and, in fibroblasts, by mitochondrial bioenergetics and network morphology results. However, we clearly found calcium mishandling between endoplasmic reticulum (ER) and mitochondria, which, under specific metabolic conditions of increased energy requirements and in selected tissue or cell types, may turn into a secondary mitochondrial dysfunction. Critically, we showed that Wolframin (WFS1) protein is enriched at mitochondrial-associated ER membranes and that in patient-derived fibroblasts WFS1 protein is completely absent. These findings support a loss-of-function pathogenic mechanism for missense mutations in WFS1, ultimately leading to defective calcium influx within mitochondria. [ABSTRACT FROM AUTHOR]

Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology. [ABSTRACT FROM AUTHOR]

Idebenone is the only approved treatment for Leber's hereditary optic neuropathy (LHON). It promotes recovery of visual function in up to 50% of patients, but, based on the current knowledge, we can neither predict nor understand the non‐responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON. We explored the possibility that genetic variant in the NQO1 enzyme can impact on idebenone response. We found that two polymorphic variants drastically reduce NQO1 protein levels, when homozygous or in compound heterozygote combination affecting idebenone reduction. In its oxidized form idebenone inhibits complex I, decreasing respiratory function in cells. We also retrospectively analysed a large cohort of LHON treated patients, classified by their response to therapy, demonstrating that patients homozygous or compound heterozygote for the NQO1 variants are characterized by the worst therapy response, particularly if carrying the m.3460G>A/MT‐ND1 LHON mutation. In summary, these results support the concept that the response to idebenone therapy may be influenced by the NQO1 genotype and the different mitochondrial DNA mutations. [ABSTRACT FROM AUTHOR]

OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA “plus”. Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two “ad hoc” generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1 −/− MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA “plus” phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions. [ABSTRACT FROM AUTHOR]