Developmental Consequences of Defective ATG7-Mediated Autophagy in Humans.

BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare. METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast. RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7. CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

A Yeast-Based Screening Unravels Potential Therapeutic Molecules for Mitochondrial Diseases Associated with Dominant ANT1 Mutations.

Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.

FDXR Mutations Cause Sensorial Neuropathies and Expand the Spectrum of Mitochondrial Fe-S-Synthesis Diseases.

Hearing loss and visual impairment in childhood have mostly genetic origins, some of them being related to sensorial neuronal defects. Here, we report on eight subjects from four independent families affected by auditory neuropathy and optic atrophy. Whole-exome sequencing revealed biallelic mutations in FDXR in affected subjects of each family. FDXR encodes the mitochondrial ferredoxin reductase, the sole human ferredoxin reductase implicated in the biosynthesis of iron-sulfur clusters (ISCs) and in heme formation. ISC proteins are involved in enzymatic catalysis, gene expression, and DNA replication and repair. We observed deregulated iron homeostasis in FDXR mutant fibroblasts and indirect evidence of mitochondrial iron overload. Functional complementation in a yeast strain in which ARH1, the human FDXR ortholog, was deleted established the pathogenicity of these mutations. These data highlight the wide clinical heterogeneity of mitochondrial disorders related to ISC synthesis.

Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy.

Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.

Mutations in MDH2, Encoding a Krebs Cycle Enzyme, Cause Early-Onset Severe Encephalopathy.

MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized hypotonia, psychomotor delay, refractory epilepsy, and elevated lactate in the blood and cerebrospinal fluid. Functional studies in fibroblasts from affected subjects showed both an apparently complete loss of MDH2 levels and MDH2 enzymatic activity close to null. Metabolomics analyses demonstrated a significant concomitant accumulation of the MDH substrate, malate, and fumarate, its immediate precursor in the Krebs cycle, in affected subjects' fibroblasts. Lentiviral complementation with wild-type MDH2 cDNA restored MDH2 levels and mitochondrial MDH activity. Additionally, introduction of the three missense mutations from the affected subjects into Saccharomyces cerevisiae provided functional evidence to support their pathogenicity. Disruption of the Krebs cycle is a hallmark of cancer, and MDH2 has been recently identified as a novel pheochromocytoma and paraganglioma susceptibility gene. We show that loss-of-function mutations in MDH2 are also associated with severe neurological clinical presentations in children.

Biallelic PPA2 Mutations Cause Sudden Unexpected Cardiac Arrest in Infancy.

Sudden unexpected death in infancy occurs in apparently healthy infants and remains largely unexplained despite thorough investigation. The vast majority of cases are sporadic. Here we report seven individuals from three families affected by sudden and unexpected cardiac arrest between 4 and 20 months of age. Whole-exome sequencing revealed compound heterozygous missense mutations in PPA2 in affected infants of each family. PPA2 encodes the mitochondrial pyrophosphatase, which hydrolyzes inorganic pyrophosphate into two phosphates. This is an essential activity for many biosynthetic reactions and for energy metabolism of the cell. We show that deletion of the orthologous gene in yeast (ppa2Δ) compromises cell viability due to the loss of mitochondria. Expression of wild-type human PPA2, but not PPA2 containing the mutations identified in affected individuals, preserves mitochondrial function in ppa2Δ yeast. Using a regulatable (doxycycline-repressible) gene expression system, we found that the pathogenic PPA2 mutations rapidly inactivate the mitochondrial energy transducing system and prevent the maintenance of a sufficient electrical potential across the inner membrane, which explains the subsequent disappearance of mitochondria from the mutant yeast cells. Altogether these data demonstrate that PPA2 is an essential gene in yeast and that biallelic mutations in PPA2 cause a mitochondrial disease leading to sudden cardiac arrest in infants.

Combined use of Saccharomyces cerevisiae, Caenorhabditis elegans and patient fibroblasts leads to the identification of clofilium tosylate as a potential therapeutic chemical against POLG-related diseases.

Mitochondria are organelles that have their own DNA (mitochondrial DNA, mtDNA) whose maintenance is necessary for the majority of ATP production in eukaryotic cells. Defects in mtDNA maintenance or integrity are responsible for numerous diseases. The DNA polymerase γ (POLG) ensures proper mtDNA replication and repair. Mutations in POLG are a major cause of mitochondrial disorders including hepatic insufficiency, Alpers syndrome, progressive external ophthalmoplegia, sensory neuropathy and ataxia. Mutations in POLG are also associated with parkinsonism. To date, no effective therapy is available. Based on the conservation of mitochondrial function from yeast to human, we used Saccharomyces cerevisiae and Caenorhabditis elegans as first pass filters to identify a chemical that suppresses mtDNA instability in cultured fibroblasts of a POLG-deficient patient. We showed that this unsuspected compound, clofilium tosylate (CLO), belonging to a class of anti-arrhythmic agents, prevents mtDNA loss of all yeast mitochondrial polymerase mutants tested, improves behavior and mtDNA content of polg-1-deficient worms and increases mtDNA content of quiescent POLG-deficient fibroblasts. Furthermore, the mode of action of the drug seems conserved as CLO increases POLG steady-state level in yeast and human cells. Two other anti-arrhythmic agents (FDA-approved) sharing common pharmacological properties and chemical structure also show potential benefit for POLG deficiency in C. elegans. Our findings provide evidence of the first mtDNA-stabilizing compound that may be an effective pharmacological alternative for the treatment of POLG-related diseases.

The impairment of MAGMAS function in human is responsible for a severe skeletal dysplasia.

Impairment of the tightly regulated ossification process leads to a wide range of skeletal dysplasias and deciphering their molecular bases has contributed to the understanding of this complex process. Here, we report a homozygous mutation in the mitochondria-associated granulocyte macrophage colony stimulating factor-signaling gene (MAGMAS) in a novel and severe spondylodysplastic dysplasia. MAGMAS, also referred to as PAM16 (presequence translocase-associated motor 16), is a mitochondria-associated protein involved in preprotein translocation into the matrix. We show that MAGMAS is specifically expressed in trabecular bone and cartilage at early developmental stages and that the mutation leads to an instability of the protein. We further demonstrate that the mutation described here confers to yeast strains a temperature-sensitive phenotype, impairs the import of mitochondrial matrix pre-proteins and induces cell death. The finding of deleterious MAGMAS mutations in an early lethal skeletal dysplasia supports a key role for this mitochondrial protein in the ossification process.

Overexpression of yeast thioredoxin TRX2 reduces p53-mediated cell death in yeast.

We have previously shown that overexpression of the human tumor suppressor protein P53 causes cell death of the yeast Saccharomyces cerevisiae. P53 overproduction led to transcriptional downregulation of some yeast genes, such as the TRX1/2 thioredoxin system, which plays a key role in cell protection against various oxidative stresses induced by reactive oxygen species (ROS). In the present work, the impact of TRX2 overexpression on apoptosis mediated by p53 overexpression in yeast is investigated. In yeast cells expressing P53 under an inducible promoter together with TRX2 under a strong constitutive promoter, we showed that Tr2p overproduction reduced the apoptotic effect exerted by P53 and increased the viability of the P53-overproducing cells. Furthermore, measurements of ROS amounts by flow cytometry and fluorescence microscopy indicated that the TRX2 protein acted probably through its increased detoxifying activity on the P53-generated ROS. The steady-state level and activity of P53 were not affected by TRX2 overexpression, as shown by western blotting and functional analysis of separated alleles in yeast (FASAY), respectively. The growth inhibitory effect of P53 was partially reversed by the antioxidant N-acetylcysteine. Our data strengthen the idea that overexpression of a single gene (trx2) decreases the p53-mediated cell death by decreasing ROS accumulation.

Mutations in the tricarboxylic acid cycle enzyme, aconitase 2, cause either isolated or syndromic optic neuropathy with encephalopathy and cerebellar atrophy.

BACKGROUND: Inherited optic neuropathy has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the disease causing gene remains unknown in many families. METHODS: We used exome sequencing in order to identify the gene responsible for isolated or syndromic optic atrophy in five patients from three independent families. RESULTS: We found homozygous or compound heterozygous missense and frameshift mutations in the gene encoding mitochondrial aconitase (ACO2), a tricarboxylic acid cycle enzyme, catalysing interconversion of citrate into isocitrate. Unlike wild type ACO2, all mutant ACO2 proteins failed to complement the respiratory growth of a yeast aco1-deletion strain. Retrospective studies using patient-derived cultured skin fibroblasts revealed various degrees of deficiency in ACO2 activity, but also in ACO1 cytosolic activity. CONCLUSIONS: Our study shows that autosomal recessive ACO2 mutations can cause either isolated or syndromic optic neuropathy. This observation identifies ACO2 as the second gene responsible for non-syndromic autosomal recessive optic neuropathies and provides evidence for a genetic overlap between isolated and syndromic forms, giving further support to the view that optic atrophy is a hallmark of defective mitochondrial energy supply.

Study of LPIN1, LPIN2 and LPIN3 in rhabdomyolysis and exercise-induced myalgia.

BACKGROUND: Recessive LPIN1 mutations were identified as a cause of severe rhabdomyolysis in pediatric patients. The human lipin family includes two other closely related members, lipin-2 and 3, which share strong homology and similar activity. The study aimed to determine the involvement of the LPIN family genes in a cohort of pediatric and adult patients (n = 171) presenting with muscular symptoms, ranging from severe (CK >10 000 UI/L) or moderate (CK <10 000 UI/L) rhabdomyolysis (n = 141) to exercise-induced myalgia (n = 30), and to report the clinical findings in patients harboring mutations. METHODS: Coding regions of LPIN1, LPIN2 and LPIN3 genes were sequenced using genomic or complementary DNAs. RESULTS: Eighteen patients harbored two LPIN1 mutations, including a frequent intragenic deletion. All presented with severe episodes of rhabdomyolysis, starting before age 6 years except two (8 and 42 years). Few patients also suffered from permanent muscle symptoms, including the eldest ones (≥ 40 years). Around 3/4 of muscle biopsies showed accumulation of lipid droplets. At least 40% of heterozygous relatives presented muscular myalgia. Nine heterozygous SNPs in LPIN family genes were identified in milder phenotypes (mild rhabdomyolysis or myalgia). These variants were non-functional in yeast complementation assay based on respiratory activity, except the LPIN3-P24L variant. CONCLUSION: LPIN1-related myolysis constitutes a major cause of early-onset rhabdomyolysis and occasionally in adults. Heterozygous LPIN1 mutations may cause mild muscular symptoms. No major defects of LPIN2 or LPIN3 genes were associated with muscular manifestations.

Biallelic variants in TAMM41 are associated with low muscle cardiolipin levels, leading to neonatal mitochondrial disease.

Mitochondrial disorders are clinically and genetically heterogeneous, with variants in mitochondrial or nuclear genes leading to varied clinical phenotypes. TAMM41 encodes a mitochondrial protein with cytidine diphosphate-diacylglycerol synthase activity: an essential early step in the biosynthesis of phosphatidylglycerol and cardiolipin. Cardiolipin is a mitochondria-specific phospholipid that is important for many mitochondrial processes. We report three unrelated individuals with mitochondrial disease that share clinical features, including lethargy at birth, hypotonia, developmental delay, myopathy, and ptosis. Whole exome and genome sequencing identified compound heterozygous variants in TAMM41 in each proband. Western blot analysis in fibroblasts showed a mild oxidative phosphorylation (OXPHOS) defect in only one of the three affected individuals. In skeletal muscle samples, however, there was severe loss of subunits of complexes I-IV and a decrease in fully assembled OXPHOS complexes I-V in two subjects as well as decreased TAMM41 protein levels. Similar to the tissue-specific observations on OXPHOS, cardiolipin levels were unchanged in subject fibroblasts but significantly decreased in the skeletal muscle of affected individuals. To assess the functional impact of the TAMM41 missense variants, the equivalent mutations were modeled in yeast. All three mutants failed to rescue the growth defect of the Δtam41 strains on non-fermentable (respiratory) medium compared with wild-type TAM41, confirming the pathogenicity of the variants. We establish that TAMM41 is an additional gene involved in mitochondrial phospholipid biosynthesis and modification and that its deficiency results in a mitochondrial disorder, though unlike families with pathogenic AGK (Sengers syndrome) and TAFAZZIN (Barth syndrome) variants, there was no evidence of cardiomyopathy.

Caenorhabditis elegans, a pluricellular model organism to screen new genes involved in mitochondrial genome maintenance.

The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). A large and heterogeneous group of human disorders is associated with mitochondrial genome quantitative and qualitative anomalies. Several nuclear genes have been shown to account for these severe OXPHOS disorders. However, in several cases, the disease-causing mutations still remain unknown. Caenorhabditis elegans has been largely used for studying various biological functions because this multicellular organism has short life cycle and is easy to grow in the laboratory. Mitochondrial functions are relatively well conserved between human and C.elegans, and heteroplasmy exists in this organism as in human. C. elegans therefore represents a useful tool for studying mtDNA maintenance. Suppression by RNA interference of genes involved in mtDNA replication such as polg-1, encoding the mitochondrial DNA polymerase, results in reduced mtDNA copy number but in a normal phenotype of the F1 worms. By combining RNAi of genes involved in mtDNA maintenance and EtBr exposure, we were able to reveal a strong and specific phenotype (developmental larval arrest) associated to a severe decrease of mtDNA copy number. Moreover, we tested and validated the screen efficiency for human orthologous genes encoding mitochondrial nucleoid proteins. This allowed us to identify several genes that seem to be closely related to mtDNA maintenance in C. elegans. This work reports a first step in the further development of a large-scale screening in C. elegans that should allow to identify new genes of mtDNA maintenance whose human orthologs will obviously constitute new candidate genes for patients with quantitative or qualitative mtDNA anomalies.

Cellular localization of human p53 expressed in the yeast Saccharomyces cerevisiae: effect of NLSI deletion.

The tumor suppressor p53 plays a central role in the regulation of cellular growth and apoptosis. In Saccharomyces cerevisiae, over-expression of the human wtp53 leads to growth inhibition and cell death on minimal medium. In the present work, we showed that deletion of the nuclear localization signal (NLSI) of p53 restores the yeast growth. In this heterologous context, the level of p53∆NLSI was low and the protein mainly located in the cytoplasm while the wtp53 was observed in both the cytoplasmic and nuclear compartments. Interestingly, the wtp53 protein was observed in the mitochondria, whereas the p53∆NLSI protein failed to localize in mitochondria. Moreover, mitochondrial morphology defect and release of cytochrome c in the cytosol were noticed only in the yeast strain expressing the wtp53. In conclusion, our results provide evidence that the human wtp53 is active in S. cerevisiae probably through dependent and independent transcriptional mechanisms leading to cell death. The deletion of the NLSI sequence decreases p53 nuclear translocation as well as its mitochondrial localization and consequently its effect on yeast growth.

CABC1 gene mutations cause ubiquinone deficiency with cerebellar ataxia and seizures.

Coenzyme Q(10) (CoQ(10)) plays a pivotal role in oxidative phosphorylation (OXPHOS) in that it distributes electrons between the various dehydrogenases and the cytochrome segments of the respiratory chain. Primary coenzyme Q(10) deficiency represents a clinically heterogeneous condition suggestive of genetic heterogeneity, and several disease genes have been previously identified. The CABC1 gene, also called COQ8 or ADCK3, is the human homolog of the yeast ABC1/COQ8 gene, one of the numerous genes involved in the ubiquinone biosynthesis pathway. The exact function of the Abc1/Coq8 protein is as yet unknown, but this protein is classified as a putative protein kinase. We report here CABC1 gene mutations in four ubiquinone-deficient patients in three distinct families. These patients presented a similar progressive neurological disorder with cerebellar atrophy and seizures. In all cases, enzymological studies pointed to ubiquinone deficiency. CoQ(10) deficiency was confirmed by decreased content of ubiquinone in muscle. Various missense mutations (R213W, G272V, G272D, and E551K) modifying highly conserved amino acids of the protein and a 1 bp frameshift insertion c.[1812_1813insG] were identified. The missense mutations were introduced into the yeast ABC1/COQ8 gene and expressed in a Saccharomyces cerevisiae strain in which the ABC1/COQ8 gene was deleted. All the missense mutations resulted in a respiratory phenotype with no or decreased growth on glycerol medium and a severe reduction in ubiquinone synthesis, demonstrating that these mutations alter the protein function.

A nonproteolytic proteasome activity controls organelle fission in yeast.

To understand the processes underlying organelle function, dynamics and inheritance, it is necessary to identify and characterize the regulatory components involved. Recently in yeast and mammals, proteins of the membrane fission machinery (Dnm1-Mdv1-Caf4-Fis1 in yeast and DLP1-FIS1 in human) have been shown to have a dual localization on mitochondria and peroxisomes, where they control mitochondrial fission and peroxisome division. Here, we show that whereas vacuole fusion is regulated by the proteasome degradation function, mitochondrial fission and peroxisomal division are not controlled by the proteasome activity but rather depend on a new function of the proteasomal lid subunit Rpn11. Rpn11 was found to regulate the Fis1-dependent fission machinery of both organelles. These findings indicate a unique role of the Rpn11 protein in mitochondrial fission and peroxisomal proliferation that is independent of its role in proteasome-associated deubiquitylation.

Analysis of the rpn11-m1 proteasomal mutant reveals connection between cell cycle and mitochondrial biogenesis.

The proteasomal lid subunit Rpn11 is essential for maintaining a correct cell cycle and mitochondrial morphology in Saccharomyces cerevisiae. In this paper, we show that the rpn11-m1 mutant has a peculiar cell cycle defect reminiscent of mutants defective in the FEAR pathway that delay the release of the Cdc14 protein phosphatase from the nucleolus. We analyzed the rpn11-m1 phenotypes and found that overexpression of Cdc14 suppresses all the rpn11-m1 defects, including the mitochondrial ones. Suppression by Cdc14 of the rpn11-m1 mitochondrial morphology defect reveals an uncharacterized connection between mitochondrial and cell cycle events. Interestingly, the overexpression of Cdc14 also partially restores the tubular network in an Δmmm2 strain, which lacks a mitochondrial protein belonging to the complex necessary to anchor the mitochondrion to the actin cytoskeleton. Altogether our findings indicate, for the first time, a cross-talk between the cell cycle and mitochondrial morphology.

Autophagy facilitates mitochondrial rebuilding after acute heat stress via a DRP-1-dependent process.

Acute heat stress (aHS) can induce strong developmental defects in Caenorhabditis elegans larva but not lethality or sterility. This stress results in transitory fragmentation of mitochondria, formation of aggregates in the matrix, and decrease of mitochondrial respiration. Moreover, active autophagic flux associated with mitophagy events enables the rebuilding of the mitochondrial network and developmental recovery, showing that the autophagic response is protective. This adaptation to aHS does not require Pink1/Parkin or the mitophagy receptors DCT-1/NIX and FUNDC1. We also find that mitochondria are a major site for autophagosome biogenesis in the epidermis in both standard and heat stress conditions. In addition, we report that the depletion of the dynamin-related protein 1 (DRP-1) affects autophagic processes and the adaptation to aHS. In drp-1 animals, the abnormal mitochondria tend to modify their shape upon aHS but are unable to achieve fragmentation. Autophagy is induced, but autophagosomes are abnormally elongated and clustered on mitochondria. Our data support a role for DRP-1 in coordinating mitochondrial fission and autophagosome biogenesis in stress conditions.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

LPIN1 gene mutations: a major cause of severe rhabdomyolysis in early childhood.

Autosomal recessive LPIN1 mutations have been recently described as a novel cause of rhabdomyolysis in a few families. The purpose of the study was to evaluate the prevalence of LPIN1 mutations in patients exhibiting severe episodes of rhabdomyolysis in infancy. After exclusion of primary fatty acid oxidation disorders, LPIN1 coding sequence was determined in genomic DNA and cDNA. Among the 29 patients studied, 17 (59%) carried recessive nonsense or frameshift mutations, or a large scale intragenic deletion. In these 17 patients, episodes of rhabdomyolysis occurred at a mean age of 21 months. Secondary defect of mitochondrial fatty oxidation or respiratory chain was found in skeletal muscle of two patients. The intragenic deletion, c.2295-866_2410-30del, was identified in 8/17 patients (47%), all Caucasians, and occurred on the background of a common haplotype, suggesting a founder effect. This deleted human LPIN1 form was unable to complement Delta pah1 yeast for growth on glycerol, in contrast to normal LPIN1. Since more than 50% of our series harboured LPIN1 mutations, LPIN1 should be regarded as a major cause of severe myoglobinuria in early childhood. The high frequency of the intragenic LPIN1 deletion should provide a valuable criterion for fast diagnosis, prior to muscle biopsy.