TDP-43 and PINK1 mediate CHCHD10S59L mutation-induced defects in Drosophila and in vitro.

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) can cause amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, the underlying mechanisms are unclear. Here, we generate CHCH10S59L-mutant Drosophila melanogaster and HeLa cell lines to model CHCHD10-associated ALS-FTD. The CHCHD10S59L mutation results in cell toxicity in several tissues and mitochondrial defects. CHCHD10S59L independently affects the TDP-43 and PINK1 pathways. CHCHD10S59L expression increases TDP-43 insolubility and mitochondrial translocation. Blocking TDP-43 mitochondrial translocation with a peptide inhibitor reduced CHCHD10S59L-mediated toxicity. While genetic and pharmacological modulation of PINK1 expression and activity of its substrates rescues and mitigates the CHCHD10S59L-induced phenotypes and mitochondrial defects, respectively, in both Drosophila and HeLa cells. Our findings suggest that CHCHD10S59L-induced TDP-43 mitochondrial translocation and chronic activation of PINK1-mediated pathways result in dominant toxicity, providing a mechanistic insight into the CHCHD10 mutations associated with ALS-FTD.

Accumulation of amyloid precursor protein C-terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer's disease models and human brains.

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer's disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the ß-secretase-derived APP-CTF fragment (C99) combined with ß- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aß triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aß to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD.

A novel variant m.8561C>T in the overlapping region of MT-ATP6 and MT-ATP8 in a child with early-onset severe neurological signs.

Among mitochondrial diseases, isolated complex V (CV) deficiency represents a rare cause of respiratory chain (RC) dysfunction. In mammalian mitochondrial DNA (mtDNA), MT-ATP6 partly overlaps with MT-ATP8 making double mutations possible, yet extremely rarely reported principally in patients with cardiomyopathy. Here, we report a novel m.8561 C>T substitution in the overlapping region of MT-ATP6 and MT-ATP8 in a child with early-onset ataxia, psychomotor delay and microcephaly, enlarging the clinical manifestations spectrum associated with CV deficiency.

GAPDH Expression Predicts the Response to R-CHOP, the Tumor Metabolic Status, and the Response of DLBCL Patients to Metabolic Inhibitors.

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease treated with anti-CD20-based immuno-chemotherapy (R-CHOP). We identified that low levels of GAPDH predict a poor response to R-CHOP treatment. Importantly, we demonstrated that GAPDHlow lymphomas use OxPhos metabolism and rely on mTORC1 signaling and glutaminolysis. Consistently, disruptors of OxPhos metabolism (phenformin) or glutaminolysis (L-asparaginase) induce cytotoxic responses in GAPDHlow B cells and improve GAPDHlow B cell-lymphoma-bearing mice survival, while they are low or not efficient on GAPDHhigh B cell lymphomas. Ultimately, we selected four GAPDHlow DLBCL patients, who were refractory to all anti-CD20-based therapies, and targeted DLBCL metabolism using L-asparaginase (K), mTOR inhibitor (T), and metformin (M) (called KTM therapy). Three out of the four patients presented a complete response upon one cycle of KTM. These findings establish that the GAPDH expression level predicts DLBCL patients' response to R-CHOP treatment and their sensitivity to specific metabolic inhibitors.

The OncoAge Consortium: Linking Aging and Oncology from Bench to Bedside and Back Again.

It is generally accepted that carcinogenesis and aging are two biological processes, which are known to be associated. Notably, the frequency of certain cancers (including lung cancer), increases significantly with the age of patients and there is now a wealth of data showing that multiple mechanisms leading to malignant transformation and to aging are interconnected, defining the so-called common biology of aging and cancer. OncoAge, a consortium launched in 2015, brings together the multidisciplinary expertise of leading public hospital services and academic laboratories to foster the transfer of scientific knowledge rapidly acquired in the fields of cancer biology and aging into innovative medical practice and silver economy development. This is achieved through the development of shared technical platforms (for research on genome stability, (epi)genetics, biobanking, immunology, metabolism, and artificial intelligence), clinical research projects, clinical trials, and education. OncoAge focuses mainly on two pilot pathologies, which benefit from the expertise of several members, namely lung and head and neck cancers. This review outlines the broad strategic directions and key advances of OncoAge and summarizes some of the issues faced by this consortium, as well as the short- and long-term perspectives.

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.

A new mutation in the mitochondrial tRNAPro gene associated with early-onset neuromuscular phenotype and ragged-red fibers.

An 11-year-old boy with psychomotor delay, exercise intolerance, ptosis and growth delay had a muscle biopsy showing typical mitochondrial alterations (60% of ragged-red fibers and 90% of cytochrome-c oxidase-deficient fibers). Next-generation sequencing revealed a novel heteroplasmic mutation (m.15958A>T) in the MTTP gene that encodes tRNAPro. The mutation was not present in the accessible non-muscle tissues of the patient's asymptomatic mother. Mutations in the rarely affected MTTP gene are responsible for different clinical presentations. We report the third early-onset case associated with a mutation in this gene. The severity of myopathy is likely related to the high mutation rate (96%) found in the patient's muscle. The clinical heterogeneity associated with MTTP mutations illustrates the value of the next-generation sequencing in routine diagnosis of mitochondrial diseases.

Coenzyme Q10 defects may be associated with a deficiency of Q10-independent mitochondrial respiratory chain complexes.

BACKGROUND: Coenzyme Q10 (CoQ10 or ubiquinone) deficiency can be due either to mutations in genes involved in CoQ10 biosynthesis pathway, or to mutations in genes unrelated to CoQ10 biosynthesis. CoQ10 defect is the only oxidative phosphorylation disorder that can be clinically improved after oral CoQ10 supplementation. Thus, early diagnosis, first evoked by mitochondrial respiratory chain (MRC) spectrophotometric analysis, then confirmed by direct measurement of CoQ10 levels, is of critical importance to prevent irreversible damage in organs such as the kidney and the central nervous system. It is widely reported that CoQ10 deficient patients present decreased quinone-dependent activities (segments I + III or G3P + III and II + III) while MRC activities of complexes I, II, III, IV and V are normal. We previously suggested that CoQ10 defect may be associated with a deficiency of CoQ10-independent MRC complexes. The aim of this study was to verify this hypothesis in order to improve the diagnosis of this disease. RESULTS: To determine whether CoQ10 defect could be associated with MRC deficiency, we quantified CoQ10 by LC-MSMS in a cohort of 18 patients presenting CoQ10-dependent deficiency associated with MRC defect. We found decreased levels of CoQ10 in eight patients out of 18 (45 %), thus confirming CoQ10 disease. CONCLUSIONS: Our study shows that CoQ10 defect can be associated with MRC deficiency. This could be of major importance in clinical practice for the diagnosis of a disease that can be improved by CoQ10 supplementation.

Coenzyme Q10 defects may be associated with a deficiency of Q10-independent mitochondrial respiratory chain complexes

BACKGROUND: Coenzyme Q10 (CoQ10 or ubiquinone) deficiency can be due either to mutations in genes involved in CoQ10 biosynthesis pathway, or to mutations in genes unrelated to CoQ10 biosynthesis. CoQ10 defect is the only oxidative phosphorylation disorder that can be clinically improved after oral CoQ10 supplementation. Thus, early diagnosis, first evoked by mitochondrial respiratory chain (MRC) spectrophotometric analysis, then confirmed by direct measurement of CoQ10 levels, is of critical importance to prevent irreversible damage in organs such as the kidney and the central nervous system. It is widely reported that CoQ10 deficient patients present decreased quinone-dependent activities (segments I + III or G3P + III and II + III) while MRC activities of complexes I, II, III, IV and V are normal. We previously suggested that CoQ10 defect may be associated with a deficiency of CoQ10-independent MRC complexes. The aim of this study was to verify this hypothesis in order to improve the diagnosis of this disease. RESULTS: To determine whether CoQ10 defect could be associated with MRC deficiency, we quantified CoQ10 by LC-MSMS in a cohort of 18 patients presenting CoQ10-dependent deficiency associated with MRC defect. We found decreased levels of CoQ10 in eight patients out of 18 (45 %), thus confirming CoQ10 disease. CONCLUSIONS: Our study shows that CoQ10 defect can be associated with MRC deficiency. This could be of major importance in clinical practice for the diagnosis of a disease that can be improved by CoQ10 supplementation.

A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement.

Mitochondrial DNA instability disorders are responsible for a large clinical spectrum, among which amyotrophic lateral sclerosis-like symptoms and frontotemporal dementia are extremely rare. We report a large family with a late-onset phenotype including motor neuron disease, cognitive decline resembling frontotemporal dementia, cerebellar ataxia and myopathy. In all patients, muscle biopsy showed ragged-red and cytochrome c oxidase-negative fibres with combined respiratory chain deficiency and abnormal assembly of complex V. The multiple mitochondrial DNA deletions found in skeletal muscle revealed a mitochondrial DNA instability disorder. Patient fibroblasts present with respiratory chain deficiency, mitochondrial ultrastructural alterations and fragmentation of the mitochondrial network. Interestingly, expression of matrix-targeted photoactivatable GFP showed that mitochondrial fusion was not inhibited in patient fibroblasts. Using whole-exome sequencing we identified a missense mutation (c.176C>T; p.Ser59Leu) in the CHCHD10 gene that encodes a coiled-coil helix coiled-coil helix protein, whose function is unknown. We show that CHCHD10 is a mitochondrial protein located in the intermembrane space and enriched at cristae junctions. Overexpression of a CHCHD10 mutant allele in HeLa cells led to fragmentation of the mitochondrial network and ultrastructural major abnormalities including loss, disorganization and dilatation of cristae. The observation of a frontotemporal dementia-amyotrophic lateral sclerosis phenotype in a mitochondrial disease led us to analyse CHCHD10 in a cohort of 21 families with pathologically proven frontotemporal dementia-amyotrophic lateral sclerosis. We identified the same missense p.Ser59Leu mutation in one of these families. This work opens a novel field to explore the pathogenesis of the frontotemporal dementia-amyotrophic lateral sclerosis clinical spectrum by showing that mitochondrial disease may be at the origin of some of these phenotypes.

Relative frequencies of inherited retinal dystrophies and optic neuropathies in Southern France: assessment of 21-year data management.

PURPOSE: Inherited retinal dystrophies (IRDs) and inherited optic neuropathies (IONs) are rare diseases defined by specific clinical and molecular features. The relative prevalence of these conditions was determined in Southern France. METHODS: Patients recruited from a specialized outpatient clinic over a 21-year period underwent extensive clinical investigations and 107 genes were screened by polymerase chain reaction/sequencing. RESULTS: There were 1957 IRD cases (1481 families) distributed in 70% of pigmentary retinopathy cases (56% non-syndromic, 14% syndromic), 20% maculopathies and 7% stationary conditions. Patients with retinitis pigmentosa were the most frequent (47%) followed by Usher syndrome (10.8%). Among non-syndromic pigmentary retinopathy patients, 84% had rod-cone dystrophy, 8% cone-rod dystrophy and 5% Leber congenital amaurosis. Macular dystrophies were encountered in 398 cases (30% had Stargardt disease and 11% had Best disease). There were 184 ION cases (127 families) distributed in 51% with dominant optic neuropathies, 33% with recessive/sporadic forms and 16% with Leber hereditary optic neuropathy. Positive molecular results were obtained in 417/609 families with IRDs (68.5%) and in 27/58 with IONs (46.5%). The sequencing of 5 genes (ABCA4, USH2A, MYO7A, RPGR and PRPH2) provided a positive molecular result in 48% of 417 families with IRDs. Except for autosomal retinitis pigmentosa, in which less than half the families had positive molecular results, about 75% of families with other forms of retinal conditions had a positive molecular diagnosis. CONCLUSIONS: Although gene discovery considerably improved molecular diagnosis in many subgroups of IRDs and IONs, retinitis pigmentosa, accounting for almost half of IRDs, remains only partly molecularly defined.

Cataract as a phenotypic marker for a mutation in WFS1, the Wolfram syndrome gene.

PURPOSE: Wolfram syndrome (WS) or diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) (OMIM 222300) is an inherited neurodegenerative disease characterized by diabetes mellitus and optic atrophy as the 2 major criteria, followed later in life by deafness, diabetes insipidus, and various signs of neurologic impairment. The presence of a cataract has been variably mentioned in WS. METHOD: Two members of a family had thorough ophthalmic examination and their DNA was screened for mutations in mitochondrial DNA, WFS1, OPA1, and OPA3 genes. RESULTS: We report a patient who first had surgery for bilateral cataract at age 5 and who subsequently presented typical signs of WS, i.e., diabetes mellitus, optic atrophy with reduced visual acuity at 20/400 on both eyes at age 22, and mild deafness. The patient was found to be a compound heterozygote for 2 truncating mutations in WFS1, the major WS gene. She carried the previously reported c.1231_1233 delCT and a novel c.2431_2465dup35 mutation. She also was heterozygote for a novel OPA1 sequence variant, c.929A>G in exon 9, whose pathogenicity remains uncertain. The patient's mother was a heterozygous carrier of the c.2431_2465dup35 mutation. She did not have diabetes mellitus or optic atrophy but had bilateral polar cataract. She did not carry the OPA1 sequence variant. CONCLUSIONS: Cataract could be a marker for the WFS1 heterozygosity in this family, namely the c.2431_2465dup35 mutation.

EGFR immunolabeling pattern may discriminate low-grade gliomas from gliosis.

Overexpression of epidermal growth factor receptor (EGFR) is common in gliomas. Gliomas are infiltrating tumors in which neoplastic glial cells can be intermingled with reactive glial cells, particularly in diffuse low-grade gliomas. As overexpression of EGFR has also been described in gliosis, it can be difficult to evaluate EGFR immunolabeling in diffuse low-grade gliomas because of this cell mix. We compared EGFR immunolabeling between gliosis and low-grade gliomas in order to identify distinctive criteria. We studied EGFR expression in 28 cases of gliosis and 39 diffuse low-grade gliomas (23 astrocytomas and 16 oligodendrogliomas). EGFR immunohistochemistry staining was performed on paraffin-embedded sections with a mouse monoclonal antibody (clone 2-18C9; Dako). Co-expression of EGFR with Olig2, Mib-1, and p53 was assessed in seven cases of low-grade gliomas using double immunolabeling. Then, EGFR immunostaining was blindly tested on 22 small specimens of indeterminate glial lesions provided by a reference neuropathological center. Two pathologists of our local center were asked to classify the lesions into diffuse low-grade glioma or gliosis according to the pattern of EGFR expression. Weak expression of EGFR was commonly detected in gliosis (23/28 cases). Strongly-stained cells were absent. Positive cells had reactive glial cell morphology. EGFR expression in gliomas was characterized by constant strongly-stained cells (39/39 cases). All strongly-stained cells had a high nucleus-to-cytoplasm ratio, with minimal to moderate nuclear atypia. Most of the strongly EGFR-positive cells were Olig2-positive. All the cases displayed cells co-expressing EGFR and Mib-1. In three p53-positive tumors, many p53-positive cells were strongly EGFR-positive. On the basis of EGFR expression, 14 out of the 22 indeterminate cases were classified as gliomas and eight as gliosis by both pathologists. Concordance with the initial diagnosis established by the reference center and concordance between the pathologists were 100%. Our results confirm that weak EGFR expression can be detected by immunohistochemistry in gliosis. They show that strong EGFR expression may be specific for neoplastic glial cells. As all low-grade gliomas contained strongly-stained cells in our study, we believe that EGFR immunohistochemistry could be a useful tool for detection of neoplastic glial cells in case of indeterminate glial lesions.

Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS; MIM 251880) is a prevalent cause of oxidative phosphorylation disorders characterized by a reduction in mtDNA copy number. The hitherto recognized disease mechanisms alter either mtDNA replication (POLG (ref. 1)) or the salvage pathway of mitochondrial deoxyribonucleosides 5'-triphosphates (dNTPs) for mtDNA synthesis (DGUOK (ref. 2), TK2 (ref. 3) and SUCLA2 (ref. 4)). A last gene, MPV17 (ref. 5), has no known function. Yet the majority of cases remain unexplained. Studying seven cases of profound mtDNA depletion (1-2% residual mtDNA in muscle) in four unrelated families, we have found nonsense, missense and splice-site mutations and in-frame deletions of the RRM2B gene, encoding the cytosolic p53-inducible ribonucleotide reductase small subunit. Accordingly, severe mtDNA depletion was found in various tissues of the Rrm2b-/- mouse. The mtDNA depletion triggered by p53R2 alterations in both human and mouse implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis.

[Homologs of MutS and MutL during mammalian meiosis]. / Les homologues de MutS et de MutL au cours de la méiose chez les mammifères.

In eukaryotes, homologs of the Escherichia coli MutS and MutL proteins are crucial for both meiotic recombination and post-replicative DNA mismatch repair. Both pathways require the formation of a MutS homolog complex which interacts with a second heterodimer, composed of two MutL homologs. During mammalian meiosis, it is likely that chromosome synapsis requires the presence of a MSH4-MSH5 heterodimer. PMS2, a MutL homolog, seems to play an important role in this process. A MSH4-MSH5 heterodimer is also likely present later with other MutL homologs (MLH1 and MLH3) and is involved in the crossing-over process. The phenotype of msh4-/- mutant mice and MSH4 immunolocalization on meiotic chromosomes suggest that MSH4 has an early function in mammalian meiotic recombination. Both MSH4 and PMS2 directly interact with the RAD51 DNA strand exchange protein. In addition, MSH4 and RAD51 proteins co-localize on mouse meiotic chromosome cores. These results suggest that MSH4 and its partners could act, just after strand exchange promoted by RAD51, to check the homology of DNA heteroduplexes.

The DNA mismatch-repair MLH3 protein interacts with MSH4 in meiotic cells, supporting a role for this MutL homolog in mammalian meiotic recombination.

The mismatch-repair (MMR) system plays a central role in maintaining genetic stability and requires evolutionarily conserved protein factors, including MutS and MutL homologs. Since the discovery of a link between the malfunction of post-replicative MMR and human cancers, a number of works have focused on the function of MutS and MutL homologs in the correction of replication errors. However, several MutS-like and MutL-like proteins also participate in meiotic recombination. The MutL homolog MLH3 has been recently identified in mammals. Several pieces of evidence support a role for this protein in post-replicative MMR. To investigate whether MLH3 also acts during meiotic recombination, we analyzed its expression in mammalian germ cells. The MLH3 gene is expressed in mouse meiotic cells and in human testis, and, as revealed by immunoprecipitation assays, the MLH3 protein is found in mouse spermatocytes. We further demonstrate that the meiosis-specific MSH4 protein, known to participate to meiotic recombination, is co-immunoprecipitated with MLH3 from mouse meiotic cell extracts. In addition, the two MLH3 protein isoforms potentially expressed in human testis (hMLH3 and hMLH3 Delta 7) interact in vitro with the hMSH4 protein. These interaction data suggest that MLH3 is associated with MSH4 in mammalian meiotic cells, and strongly support the possibility that MLH3 plays a role in mammalian meiotic recombination.