Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys

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 lim- ited 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 carry- ing 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 rapamy- cin 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-muta- tion 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

Calcium mishandling in absence of primary mitochondrial dysfunction drives cellular pathology in Wolfram Syndrome

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

Leber's Hereditary Optic Neuropathy: A Report on Novel mtDNA Pathogenic Variants

Leber's hereditary optic neuropathy (LHON) is due to missense point mutations affecting mitochondrial DNA (mtDNA); 90% of cases harbor the m.3460G>A, m.11778G>A, and m.14484T>C primary mutations. Here, we report and discuss five families with patients affected by symptomatic LHON, in which we found five novel mtDNA variants. Remarkably, these mtDNA variants are located in complex I genes, though without strong deleterious effect on respiration in cellular models: this finding is likely linked to the tissue specificity of LHON. This study observes that in the case of a strong clinical suspicion of LHON, it is recommended to analyze the whole mtDNA sequence, since new rare mtDNA pathogenic variants causing LHON are increasingly identified

Respiratory chain complex I dysfunction in tumorigenesis

Diseases due to mutations in mitochondrial DNA probably represent the most common form of metabolic disorders, including cancer, as highlighted in the last years. Approximately 300 mtDNA alterations have been identified as the genetic cause of mitochondrial diseases and one-third of these alterations are located in the coding genes for OXPHOS proteins. Despite progress in identification of their molecular mechanisms, little has been done with regard to the therapy. Recently, a particular gene therapy approach, namely allotopic expression, has been proposed and optimized, although the results obtained are rather controversial. In fact, this approach consists in synthesis of a wild-type version of mutated OXPHOS protein in the cytosolic compartment and in its import into mitochondria, but the available evidence is based only on the partial phenotype rescue and not on the demonstration of effective incorporation of the functional protein into respiratory complexes. In the present study, we took advantage of a previously analyzed cell model bearing the m.3571insC mutation in MTND1 gene for the ND1 subunit of respiratory chain complex I. This frame-shift mutation induces in fact translation of a truncated ND1 protein then degraded, causing complex I disassembly, and for this reason not in competition with that allotopically expressed. We show here that allotopic ND1 protein is correctly imported into mitochondria and incorporated in complex I, promoting its proper assembly and rescue of its function. This result allowed us to further confirm what we have previously demonstrated about the role of complex I in tumorigenesis process. Injection of the allotopic clone in nude mice showed indeed that the rescue of complex I assembly and function increases tumor growth, inducing stabilization of HIF1α, the master regulator of tumoral progression, and consequently its downstream gene expression activation

Incomplete penetrance in mitochondrial optic neuropathies

Incomplete penetrance characterizes the two most frequent inherited optic neuropathies, Leber's Hereditary Optic Neuropathy (LHON) and dominant optic atrophy (DOA), due to genetic errors in the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA), respectively. For LHON, compelling evidence has accumulated on the complex interplay of mtDNA haplogroups and environmental interacting factors, whereas the nDNA remains essentially non informative. However, a compensatory mechanism of activated mitochondrial biogenesis and increased mtDNA copy number, possibly driven by a permissive nDNA background, is documented in LHON; when successful it maintains unaffected the mutation carriers, but in some individuals it might be hampered by tobacco smoking or other environmental factors, resulting in disease onset. In females, mitochondrial biogenesis is promoted and maintained within the compensatory range by estrogens, partially explaining the gender bias in LHON. Concerning DOA, none of the above mechanisms has been fully explored, thus mtDNA haplogroups, environmental factors such as tobacco and alcohol, and further nDNA variants may all participate as protective factors or, on the contrary, favor disease expression and severity. Next generation sequencing, complemented by transcriptomics and proteomics, may provide some answers in the next future, even if the multifactorial model that seems to apply to incomplete penetrance in mitochondrial optic neuropathies remains problematic, and careful stratification of patients will play a key role for data interpretation. The deep understanding of which factors impinge on incomplete penetrance may shed light on the pathogenic mechanisms leading to optic nerve atrophy, on their possible compensation and, thus, on development of therapeutic strategies

Liver transplant reverses biochemical imbalance in mitochondrial neurogastrointestinal encephalomyopathy

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Hypoxia influence the effect of metformin on Ewing sarcoma.

<p><b>A</b>) <i>In vivo</i> treatment with metformin in association or not with vincristine (VCR) against TC-71 xenografts in athymic nude mice. Treatments began when tumors started being measurable at day +7 after subcutaneous (s.c.) cell injection. <i>Left</i> graph, <i>in vivo</i> growth curves of TC-71 tumor volume (cm3) after s.c. injection of 5×10⁶ TC-71 cells in groups of 5 mice (treated groups) or 8 mice (Vehicle group). Vehicle, mice treated with PBS intratumor or water alone; metformin in drinking water, mice receiving metformin 200 mg/Kg in drinking water; metformin intratumor, mice receiving intratumor injection of metformin 200 mg/Kg daily, 5 days weekly. <i>Right</i> graph, <i>in vivo</i> growth curves of TC-71 tumor volume after s.c. injection of 7.5×10⁶ TC-71 cells in groups of 5 mice. Vehicle, mice treated with PBS; Metformin, mice treated daily, 5 days weekly with 500 mg/kg Metformin p.o. (gavage); VCR, mice treated with 1 mg/kg/d i.p. for 2 consecutive days; Metformin+VCR, combined therapy with Metformin 500 mg/Kg p.o. and VCR 1 mg/Kg/d i.p. <b>B</b>) Decrease responsiveness to metformin in hypoxia respect to normoxia environment. In hypoxia experiments, TC-71 and ASP-14 Ewing sarcoma cells were pre-treated for 18h with hypoxia mimetic agent CoCl2 (200 uM) than exposed for 48h to different concentration of metformin (10–30 mM) or left untreated. In combination experiments, TC-71 Ewing sarcoma cells were pre-treated for 18h with CoCl2 (200 uM) than exposed for 48h to metformin (10 mM) alone or in combination with different concentrations of vincristine (0.3–10 ng/ml). Effects of metformin on cell growth were assessed by trypan bleu assay and shown as percentage of survival over untreated control. *P < 0.05, ** P<0.01 statistically significant differences by Student’s t test. <b>C</b>) Western blotting analysis of AMPKα phosphorylation levels under hypoxia and normoxia conditions after metformin (10–30 mM) treatment. TC-71 and ASP-14 EWS cells were pre-treated for 18h with CoCl2 (200 uM) than exposed for 4h to different concentration of metformin (10–30 mM) or left untreated. Accumulation of HIF-1α protein confirmed the induction of hypoxia; β-actin was used as loading control. <b>D</b>) Immunohistochemical evaluation of HIF-1α and phospho-AMPKα in hypoxic (left panel) and normoxic areas (righ panel) in xenografts. Representative figures are shown (magnification X100).</p

<i>In vitro</i> combination study of metformin with conventional and targeted drugs in sarcoma cells.

<p><i>In vitro</i> combination study of metformin with conventional and targeted drugs in sarcoma cells.</p

Analysis of metformin targets in Ewing sarcoma cell lines.

<p><b>A</b>) protein expression levels of LKB1 in a panel of Ewing and osteosarcoma cell lines. β-Actin was used as loading control. <b>B</b>) <i>Left</i> Graph showing cellular ATP levels (RLUs) as a percentage of control in control and metformin treated ES cells. <i>Right</i> ES cells were treated as described in material and methods, and cellular lactate levels were measured using a colorimetric assay kit (Abcam). Graph shows L-lactate concentration. Values are expressed as mean ± SEM and significance (*) p<0.05; (**) p<0.01 as indicated. <b>C</b>) Metformin activity on AMPK and mTOR signaling on Ewing sarcoma cell lines. Western Blotting analysis of metformin-treated cells TC-71, SK-N-MC and 6647. Cells were treated with metformin (10 mM) for the indicated time points (30min-24h). Immunoblot analysis was carried out using antibodies against phosphorylated AMPKα (Thr¹⁷²), AMPKα, phosphorylated pS6K (Ser^240/244), pS6, and β-actin as normalization. <b>D</b>) Analysis of Metformin (10 mM) effects on cell cycle over 48h treatment in three Ewing sarcoma cell lines (TC-71, SK-N-MC, 6647), together with two anti-IGF1R agents resistant cells (TC/AVE, TC/AEW). <i>Left panel</i>, mean percentage of cells in different cell cycle phases as determined by flow cytometry analysis; <i>Righ panel</i>, Ki-67 positive cells (Ki-67 labeling index). Data are shown as percentages of mean of three independent experiments and significance (*) p<0.05 as indicated.</p

Metformin effects are independent to IGF system activation.

<p><b>A</b>) Cell growth was assessed by MTT assay after 72h exposure to Metformin (mM) in cells resistant to anti-IGF-1R agents (TC/AVE, TC/AEW, TC/CP) where recently we demonstrated an increased expression of IR-A with a concomitant downregulation of IGF-1R and displayed as a percentage of survival over controls. <i>Points</i>, mean of two independent experiments; <i>bars</i>, SE; <b>B</b>) After overnight starvation, TC-71 and 6647 EWS cells were exposed to indicate concentration of metformin and/or IGF-1 and/or Insulin in IMDM containing 1% FBS for 48h. Cell growth was assessed by trypan blue assay and shown as percentage of growth over untreated controls. <b>C</b>) Starved cells were exposed to metformin (10 mM) for 4h in IMDM plus 1% FBS than stimulated with IGF-1 (50 ng/ml) or Insulin (10 nM) for 15min. After harvesting, cells were lysed and prepared for immunoblot analyses for p-ERK and total ERK. β-Actin was used as loading control.</p