Alan Diot

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.

Mitophagy plays a central role in mitochondrial ageing

The mechanisms underlying ageing have been discussed for decades, and advances in molecular and cell biology of the last three decades have accelerated research in this area. Over this period, it has become clear that mitochondrial function, which plays a major role in many cellular pathways from ATP production to nuclear gene expression and epigenetics alterations, declines with age. The emerging concepts suggest novel mechanisms, involving mtDNA quality, mitochondrial dynamics or mitochondrial quality control. In this review, we discuss the impact of mitochondria in the ageing process, the role of mitochondria in reactive oxygen species production, in nuclear gene expression, the accumulation of mtDNA damage and the importance of mitochondrial dynamics and recycling. Declining mitophagy (mitochondrial quality control) may be an important component of human ageing.

Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations

Objective: To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls. Methods: Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes. Results: Fibroblasts from 3 biallelic OPA1(−/−) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)–depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA–treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts. Conclusions: We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion.

The m.13051G>A mitochondrial DNA mutation results in variable neurology and activated mitophagy

Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in ∼1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment.

Is Placental Mitochondrial Function a Regulator that Matches Fetal and Placental Growth to Maternal Nutrient Intake in the Mouse

Background Effective fetal growth requires adequate maternal nutrition coupled to active transport of nutrients across the placenta, which, in turn requires ATP. Epidemiological and experimental evidence has shown that impaired maternal nutrition in utero results in an adverse postnatal phenotype for the offspring. Placental mitochondrial function might link maternal food intake to fetal growth since impaired placental ATP production, in response to poor maternal nutrition, could be a pathway linking maternal food intake to reduced fetal growth. Method We assessed the effects of maternal diet on placental water content, ATP levels and mitochondrial DNA (mtDNA) content in mice at embryonic (E) day 18 (E18). Females maintained on either low- (LPD) or normal- (NPD) protein diets were mated with NPD males. Results To investigate the possibility of an underlying mitochondrial stress response, we studied cultured human trophoblast cells (BeWos). High throughput imaging showed that amino acid starvation induces changes in mitochondrial morphology that suggest stress-induced mitochondrial hyperfusion. This is a defensive response, believed to increase mitochondrial efficiency, that could underlie the increase in ATP observed in placenta. Conclusions These findings reinforce the pathophysiological links between maternal diet and conceptus mitochondria, potentially contributing to metabolic programming. The quiet embryo hypothesis proposes that pre-implantation embryo survival is best served by a relatively low level of metabolism. This may extend to post-implantation trophoblast responses to nutrition.

Modulating mitochondrial quality in disease transmission: towards enabling mitochondrial DNA disease carriers to have healthy children

One in 400 people has a maternally inherited mutation in mtDNA potentially causing incurable disease. In so-called heteroplasmic disease, mutant and normal mtDNA co-exist in the cells of carrier women. Disease severity depends on the proportion of inherited abnormal mtDNA molecules. Families who have had a child die of severe, maternally inherited mtDNA disease need reliable information on the risk of recurrence in future pregnancies. However, prenatal diagnosis and even estimates of risk are fraught with uncertainty because of the complex and stochastic dynamics of heteroplasmy. These complications include an mtDNA bottleneck, whereby hard-to-predict fluctuations in the proportions of mutant and normal mtDNA may arise between generations. In ‘mitochondrial replacement therapy’ (MRT), damaged mitochondria are replaced with healthy ones in early human development, using nuclear transfer. We are developing non-invasive alternatives, notably activating autophagy, a cellular quality control mechanism, in which damaged cellular components are engulfed by autophagosomes. This approach could be used in combination with MRT or with the regular management, pre-implantation genetic diagnosis (PGD). Mathematical theory, supported by recent experiments, suggests that this strategy may be fruitful in controlling heteroplasmy. Using mice that are transgenic for fluorescent LC3 (the hallmark of autophagy) we quantified autophagosomes in cleavage stage embryos. We confirmed that the autophagosome count peaks in four-cell embryos and this correlates with a drop in the mtDNA content of the whole embryo. This suggests removal by mitophagy (mitochondria-specific autophagy). We suggest that modulating heteroplasmy by activating mitophagy may be a useful complement to mitochondrial replacement therapy.

Acute nutritional stress during pregnancy affects placental efficiency, fetal growth and adult glucose homeostasis

Exposure to maternal malnutrition impairs postnatal health. Acute nutritional stress is less clearly implicated in intrauterine programming. We studied the effects of stressing pregnant mothers on perinatal growth and adult glucose homeostasis. We compared one group (“stressed”, mothers fasted for 16 hours) with controls (“unstressed”). We found that fasting stress had adverse effects on the weight of the fetuses conceived (p<0.005) and the placental efficiency (p<0.001) in stressed compared to unstressed offspring. Placental weight was increased (p<0.001) presumably in compensation. Stress affected the glucose homeostasis of the offspring when they became adults (p<0.005) when analysed as individuals. We previously linked nutritional stress throughout pregnancy with a mitochondrial stress response. We modelled placenta with cultured human trophoblast cells (BeWos) and fetal tissues with mouse embryonic fibroblasts (MEFs). High throughput imaging showed that the mitochondria of both cell types underwent a similar sequence of changes in morphology, induced by nutritional stresses. The contrasting stress responses on fetal and placental weight were not captured by the cellular models. The stress of maternal fasting may be an important determinant of perinatal outcome in the mouse and might be relevant to nutritional stress in human pregnancy.

Validating the RedMIT/GFP-LC3 Mouse Model by Studying Mitophagy in Autosomal Dominant Optic Atrophy Due to the OPA1Q285STOP Mutation

Background: Autosomal dominant optic atrophy (ADOA) is usually caused by mutations in the essential gene, OPA1. This encodes a ubiquitous protein involved in mitochondrial dynamics, hence tissue specificity is not understood. Dysregulated mitophagy (mitochondria recycling) is implicated in ADOA, being increased in OPA1 patient fibroblasts. Furthermore, autophagy may be increased in retinal ganglion cells (RGCs) of the OPA1Q285STOP mouse model. Aims: We developed a mouse model for studying mitochondrial dynamics in order to investigate mitophagy in ADOA. Methods: We crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse, harboring red fluorescent mitochondria and green fluorescent autophagosomes. Colocalization between mitochondria and autophagosomes, the hallmark of mitophagy, was quantified in fluorescently labeled organelles in primary cell cultures, using two high throughput imaging methods Imagestream (Amnis) and IN Cell Analyzer 1000 (GE Healthcare Life Sciences). We studied colocalization between mitochondria and autophagosomes in fixed sections using confocal microscopy. Results: We validated our imaging methods for RedMIT/GFP-LC3 mouse cells, showing that colocalization of red fluorescent mitochondria and green fluorescent autophagosomes is a useful indicator of mitophagy. We showed that colocalization increases when lysosomal processing is impaired. Further, colocalization of mitochondrial fragments and autophagosomes is increased in cultures from the OPA1Q285STOP/RedMIT/GFP-LC3 mice compared to RedMIT/GFP-LC3 control mouse cells that were wild type for OPA1. This was apparent in both mouse embryonic fibroblasts (MEFs) using IN Cell 1000 and in splenocytes using ImageStream imaging flow cytometer (Amnis). We confirmed that this represents increased mitophagic flux using lysosomal inhibitors. We also used microscopy to investigate the level of mitophagy in the retina from the OPA1Q285STOP/RedMIT/GFP-LC3 mice and the RedMIT/GFP-LC3 control mice. However, the expression levels of fluorescent proteins and the image signal-to-background ratios precluded the detection of colocalization so we were unable to show any difference in colocalization between these mice. Conclusions: We show that colocalization of fluorescent mitochondria and autophagosomes in cell cultures, but not fixed tissues from the RedMIT/GFP-LC3, can be used to detect mitophagy. We used this model to confirm that mitophagy is increased in a mouse model of ADOA. It will be useful for cell based studies of diseases caused by impaired mitochondrial dynamics.