Pathogenic G6PD variants: Different clinical pictures arise from different missense mutations in the same codon.

G6PD deficiency results from mutations in the X-linked G6PD gene. More than 200 variants are associated with enzyme deficiency: each one of them may either cause predisposition to haemolytic anaemia triggered by exogenous agents (class B variants), or may cause a chronic haemolytic disorder (class A variants). Genotype-phenotype correlations are subtle. We report a rare G6PD variant, discovered in a baby presenting with severe jaundice and haemolytic anaemia since birth: the mutation of this class A variant was found to be p.(Arg454Pro). Two variants affecting the same codon were already known: G6PD Union, p.(Arg454Cys), and G6PD Andalus, p.(Arg454His). Both these class B variants and our class A variant exhibit severe G6PD deficiency. By molecular dynamics simulations, we performed a comparative analysis of the three mutants and of the wild-type G6PD. We found that the tetrameric structure of the enzyme is not perturbed in any of the variants; instead, loss of the positively charged Arg residue causes marked variant-specific rearrangement of hydrogen bonds, and it influences interactions with the substrates G6P and NADP. These findings explain severe deficiency of enzyme activity and may account for p.(Arg454Pro) expressing a more severe clinical phenotype.

The importance of gut microbiome in the perinatal period.

This narrative review describes the settlement of the neonatal microbiome during the perinatal period and its importance on human health in the long term. Delivery methods, maternal diet, antibiotic exposure, feeding practices, and early infant contact significantly shape microbial colonization, influencing the infant's immune system, metabolism, and neurodevelopment. By summarizing two decades of research, this review highlights the microbiome's role in disease predisposition and explores interventions like maternal vaginal seeding and probiotic and prebiotic supplementation that may influence microbiome development. CONCLUSION: The perinatal period is a pivotal phase for the formation and growth of the neonatal microbiome, profoundly impacting long-term health outcomes. WHAT IS KNOWN: • The perinatal period is a critical phase for the development of the neonatal microbiome, with factors such as mode of delivery, maternal diet, antibiotic exposure, and feeding practices influencing its composition and diversity, which has significant implications for long-term health. • The neonatal microbiome plays a vital role in shaping the immune system, metabolism, and neurodevelopment of infants. WHAT IS NEW: • Recent studies have highlighted the potential of targeted interventions, such as probiotic and prebiotic supplementation, and innovative practices like maternal vaginal seeding, to optimize microbiome development during the perinatal period. • Emerging evidence suggests that specific bacterial genera and species within the neonatal microbiome are associated with reduced risks of developing chronic conditions, indicating new avenues for promoting long-term health starting from early life.

Peptides Targeting the IF1-ATP Synthase Complex Modulate the Permeability Transition Pore in Cancer HeLa Cells.

The mitochondrial protein IF1 is upregulated in many tumors and acts as a pro-oncogenic protein through its interaction with the ATP synthase and the inhibition of apoptosis. We have recently characterized the molecular nature of the IF1-Oligomycin Sensitivity Conferring Protein (OSCP) subunit interaction; however, it remains to be determined whether this interaction could be targeted for novel anti-cancer therapeutic intervention. We generated mitochondria-targeting peptides to displace IF1 from the OSCP interaction. The use of one selective peptide led to displacement of the inhibitor IF1 from ATP synthase, as shown by immunoprecipitation. NMR spectroscopy analysis, aimed at clarifying whether these peptides were able to directly bind to the OSCP protein, identified a second peptide which showed affinity for the N-terminal region of this subunit overlapping the IF1 binding region. In situ treatment with the membrane-permeable derivatives of these peptides in HeLa cells, that are silenced for the IF1 inhibitor protein, showed significant inhibition in mitochondrial permeability transition and no effects on mitochondrial respiration. These peptides mimic the effects of the IF1 inhibitor protein in cancer HeLa cells and confirm that the IF1-OSCP interaction inhibits apoptosis. A third peptide was identified which counteracts the anti-apoptotic role of IF1, showing that OSCP is a promising target for anti-cancer therapies.

Mitochondrial Dysfunction Causes Cell Death in Patients Affected by Fragile-X-Associated Disorders.

Mitochondria are involved in multiple aspects of neurodevelopmental processes and play a major role in the pathogenetic mechanisms leading to neuro-degenerative diseases. Fragile-X-related disorders (FXDs) are genetic conditions that occur due to the dynamic expansion of CGG repeats of the FMR1 gene encoding for the RNA-binding protein FMRP, particularly expressed in the brain. This gene expansion can lead to premutation (PM, 56-200 CGGs), full mutation (FM, >200 CGGs), or unmethylated FM (UFM), resulting in neurodegeneration, neurodevelopmental disorders, or no apparent intellectual disability, respectively. To investigate the mitochondrial mechanisms that are involved in the FXD patients, we analyzed mitochondrial morphology and bioenergetics in fibroblasts derived from patients. Donut-shaped mitochondrial morphology and excessive synthesis of critical mitochondrial proteins were detected in FM, PM, and UFM cells. Analysis of mitochondrial oxidative phosphorylation in situ reveals lower respiration in PM fibroblasts. Importantly, mitochondrial permeability transition-dependent apoptosis is sensitized to reactive oxygen species in FM, PM, and UFM models. This study elucidated the mitochondrial mechanisms that are involved in the FXD phenotypes, and indicated altered mitochondrial function and morphology. Importantly, a sensitization to permeability transition and apoptosis was revealed in FXD cells. Overall, our data suggest that mitochondria are novel drug targets to relieve the FXD symptoms.

Cross-cultural adaptation and validation of the Italian version of the Montreal Children's Hospital Feeding Scale in a special healthcare needs population.

BACKGROUND: The Montreal Children's Hospital Feeding Scale (MCH-FS) allows paediatricians and other health care professionals to identify feeding difficulties among children. AIM: To translate and adapt the MCH-FS into Italian, and to evaluate the validity and reliability of this Italian version of the Montreal Children's Hospital Feeding Scale (I-MCH-FS). METHODS & PROCEDURES: A total of 150 children with special healthcare needs were admitted to the Rare Disease Unit of the Paediatrics Department at the Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy, between March 2021 and March 2022 (74 males; mean age = 3.85 ± 1.96 years; median age = 4 years; age range = 6 months-6 years and 11 months) and 150 healthy participants (83 males; mean age = 3.5 ± 1.98 years; median age = 3 years; age range = 6 months-6 years and 11 months) were included in the study, which was approved by the local ethics committee. The original version of the MCH-FS was translated and cross-cultural adapted through five stages: (1) initial translation, (2) synthesis of the translations, (3) back translation, (4) expert committee and (5) test of the prefinal version. Test-retest reliability and internal consistency were assessed using Pearson r, Spearman r and Cronbach's alpha, respectively. Construct validity was established by comparing data obtained from patients with those of healthy participants using the Mann-Whitney U-test. OUTCOMES & RESULTS: A Pearson r of 0.98, a Spearman r of 0.95 and Cronbach's alpha value of 0.86 were obtained. In the clinical group, 40.6% children were classified as having feeding disorders (n = 61), while in the normative group 4.7% were diagnosed with feeding problems (n = 7). Mean total score of the clinical group was significatively different from the normative's. CONCLUSIONS & IMPLICATIONS: The I-MCH-FS is a valid and reliable one-page, quick screening tool used to identify feeding disorders among children with special needs in outpatient paediatric setting. WHAT THIS PAPER ADDS: What is already known on the subject The MCH-FS is a valid and reliable parent-report measure aimed at discriminating between children presenting or not feeding disorders. What this paper adds to existing knowledge This paper presents the translation and cross-cultural adaptation of the scale into the Italian language. What are the potential or actual clinical implications of this work? The Italian version of the MCH-FS can be used in the special healthcare needs population.

Validation and cross-cultural adaptation of the Italian version of the paediatric eating assessment tool (I-PEDI-EAT-10) in genetic syndromes.

BACKGROUND: The Pediatric Eating Assessment Tool (PEDI-EAT-10) is a reliable and valid tool for rapid identification of dysphagia in patients aged 18 months to 18 years. AIMS: To translate and adapt the PEDI-EAT-10 into the Italian language and evaluate its validity and reliability. METHODS & PROCEDURES: The translation and cross-cultural adaptation of the tool consisted of five stages: initial translation, synthesis of the translations, back translation, expert committee evaluation and test of the prefinal version. The internal consistency of the translated tool was analysed in a clinical group composed of 200 patients with special healthcare needs aged between 18 months and 18 years. They were consecutively enrolled at the Rare Disease Unit, Paediatrics Department, Fondazione Policlinico Agostino Gemelli-IRCCS, Rome. For test-retest reliability, 50 caregivers filled in the PEDI-EAT-10 questionnaire for a second time after a 2-week period. Construct validity was established by comparing data obtained from patients with data from healthy participants (n = 200). The study was approved by the local ethics committee. OUTCOMES & RESULTS: Psychometric data obtained from patients (104 M; mean age = 8.08 ± 4.85 years; median age = 7 years) showed satisfactory internal consistency (Cronbach's α = 0.89) and test-retest reliability (Pearson r = 0.99; Spearman r = 0.96). A total of 30% of children were classified as having a high risk of penetration/aspiration. The Italian PEDI-EAT-10 mean total score of the clinical group was significantly different from that resulting from healthy participants. CONCLUSIONS & IMPLICATIONS: The PEDI-EAT-10 was successfully translated into Italian, validated and found to be a reliable one-page rapid screening tool to identify dysphagia in children and adolescents with special needs. WHAT THIS PAPER ADDS: What is already known on the subject The PEDI-EAT-10 is a valid and reliable quick discriminative paediatric tool for identifying penetration/aspiration risks. What this paper adds to the existing knowledge In the present study we successfully translated and adapted the PEDI-EAT-10 into the Italian language. What are the potential or actual clinical implications of this work? This translation and adaptation increase access to valid feeding and swallowing assessment for children of Italian-speaking families. In addition, the I-PEDI-EAT-10 can suggest further assessment of patients' swallowing abilities.

Management of nutritional and gastrointestinal issues in RASopathies: A narrative review.

Noonan, Costello, and cardio-facio-cutaneous syndrome are neurodevelopmental disorders belonging to the RASopathies, a group of syndromes caused by alterations in the RAS/MAPK pathway. They are characterized by similar clinical features, among which feeding difficulties, growth delay, and gastro-intestinal disorders are frequent, causing pain and discomfort in patients. Hereby, we describe the main nutritional and gastrointestinal issues reported in individuals with RASopathies, specifically in Noonan syndrome, Noonan syndrome-related disorders, Costello, and cardio-facio-cutaneous syndromes. Fifty percent of children with Noonan syndrome may experience feeding difficulties that usually have a spontaneous resolution by the second year of life, especially associated to genes different than PTPN11 and SOS1. More severe manifestations often require artificial enteral nutrition in infancy are observed in Costello syndrome, mostly associated to c.34G>A substitution in the HRAS gene. In cardio-facio-cutaneous syndrome feeding issues are usually present (90-100% of cases), especially in individuals carrying variants in BRAF, MAP2K1, and MAP2K2 genes, and artificial enteral intervention, even after scholar age, may be required. Moreover, disorders associated with gastrointestinal dysmotility as gastro-esophageal reflux and constipation are commonly reported in all the above-mentioned syndromes. Given the impact on growth and on the quality of life of these patients, early evaluation and prompt personalized management plans are fundamental.

Prevalence of gastrointestinal disorders in individuals with RASopathies: May RAS/MAP/ERK pathway dysfunctions be a model of neuropathic pain and visceral hypersensitivity?

RASopathies are a group of neurodevelopmental syndromes caused by germline variants in genes of the Ras/MAP/ERK pathway. Growth failure, neurological involvement, and pain represent the main features of these conditions. ERK signaling cascade plays a crucial role in nociception and visceral pain and it is likely implicated in the genesis of neuropathic pain and maintenance of altered pain states. We studied the prevalence of abdominal pain and functional gastrointestinal (GI) disorders in a large sample of individuals with RASopathies. A brief pain inventory questionnaire and semi-structured dedicated interview were used to investigate presence and localization of pain. A Rome IV questionnaire was used to screen for functional GI disorders. Eighty patients with clinical and molecular diagnoses of RASopathy were recruited (42 with Noonan syndrome; 17 with Costello Syndrome and 21 with cardio-facio-cutaneous syndrome). Overall, the prevalence of abdominal pain was 44% and prevalence of functional GI disorders was 78% with constipation, abdominal pain, and aerophagia being the most frequently detected ones. A significant association was found between pain and irritable bowel syndrome, functional constipation and aerophagia. Children with RASopathies have a high prevalence of functional gastrointestinal disorders. These children could represent a good in vivo model to study neuropathic pain, visceral hypersensitivity and gut-brain axis disorders.

Bone tissue homeostasis and risk of fractures in Costello syndrome: A 4-year follow-up study.

Costello syndrome (CS) is a neurodevelopmental disorder with a distinctive musculoskeletal phenotype and reduced bone mineral density (BMD) caused by activating de novo mutations in the HRAS gene. Herein, we report the results of a prospective study evaluating the efficacy of a 4-year vitamin D supplementation on BMD and bone health. A cohort of 16 individuals ranging from pediatric to adult age with molecularly confirmed CS underwent dosages of bone metabolism biomarkers (serum/urine) and dual-energy X-ray absorptiometry (DXA) scans to assess bone and body composition parameters. Results were compared to age-matched control groups. At baseline evaluation, BMD was significantly reduced (p ≤ 0.05) compared to controls, as were the 25(OH)vitD levels. Following the 4-year time interval, despite vitamin D supplementation therapy at adequate dosages, no significant improvement in BMD was observed. The present data confirm that 25(OH)vitD and BMD parameters are reduced in CS, and vitamin D supplementation is not sufficient to restore proper BMD values. Based on this evidence, routine monitoring of bone homeostasis to prevent bone deterioration and possible fractures in adult patients with CS is highly recommended.

Characterization of bone homeostasis in individuals affected by cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous syndrome (CFCS) is a rare disorder characterized by distinctive craniofacial appearance, cardiac, neurologic, cutaneous, and musculoskeletal abnormalities. It is due to heterozygous mutations in BRAF, MAP2K1, MAP2K2, and KRAS genes, belonging to the RAS/MAPK pathway. The role of RAS signaling in bone homeostasis is highly recognized, but data on bone mineral density (BMD) in CFCS are lacking. In the present study we evaluated bone parameters, serum and urinary bone metabolites in 14 individuals with a molecularly confirmed diagnosis of CFCS. Bone assessment was performed through dual X-ray absorptiometry (DXA); height-adjusted results were compared to age- and sex-matched controls. Blood and urinary bone metabolites were also analyzed and compared to the reference range. Despite vitamin D supplementation and almost normal bone metabolism biomarkers, CFCS patients showed significantly decreased absolute values of DXA-assessed subtotal and lumbar BMD (p ≤ 0.05), compared to controls. BMD z-scores and t-scores (respectively collected for children and adults) were below the reference range in CFCS, while normal in healthy controls. These findings confirmed a reduction in BMD in CFCS and highlighted the importance of monitoring bone health in these affected individuals.

Body mass index in type 2 spinal muscular atrophy: a longitudinal study.

The aim of this retrospective study was to review body mass index (BMI) in a large cohort of Italian pediatric type 2 spinal muscular atrophy (SMA) patients, aged between 0 and 20 years and to establish possible differences in relation to a number of variables such as ventilation, motor function, and survival motor neuron 2 gene copies. Cross-sectional data were collected from 102 patients for a total of 344 visits. Standard growth charts for height and weight were used as reference, with age adjusted BMI calculated using the Center for Disease and Prevention Children's BMI Tool. In the 344 visits, weight ranged between 3.90 and 83 kg, and the BMI between 8.4 and 31.6 with a BMI/age z-scores < - 2SD present in 28% and BMI/age z-scores > + 2SD in 9% of the measurements. The BMI/age z-scores were relatively stable < 5 years of age with an increasing number of patients < - 2SD after the age of 5, and a wider range of BMI/age z-scores after the age of 13. A difference on the BMI/age z-scores was found among the different age subgroups (< 5, 5-12, ≥ 13 years). A multivariate analysis in 58 patients with longitudinal assessments showed that baseline BMI/age z-scores and gender were significantly contributing to the changes while other variables were not. CONCLUSION: Our results confirm that careful surveillance of weight and BMI/age z-scores is needed in type 2 SMA. Further studies, including assessments of chewing and swallowing and of lean/fat body mass, will help to better understand the possible mechanisms underlying weight issues. WHAT IS KNOWN: • Feeding difficulties have been reported in a few studies and were invariably found in patients with type 1 SMA. • Type 2 SMA patients often have low BMI with a relevant number of patients requiring tube feeding. WHAT IS NEW: • Reduction in BMI/age z-score overtime appeared to depend on baseline BMI/age z-score and gender. • Patients with a low BMI/age z-score were at higher risk of developing further reduction.

The role of mitochondrial ATP synthase in cancer.

The mitochondrial ATP synthase is a multi-subunit enzyme complex located in the inner mitochondrial membrane which is essential for oxidative phosphorylation under physiological conditions. In this review, we analyse the enzyme functions involved in cancer progression by dissecting specific conditions in which ATP synthase contributes to cancer development or metastasis. Moreover, we propose the role of ATP synthase in the formation of the permeability transition pore (PTP) as an additional mechanism which controls tumour cell death. We further describe transcriptional and translational modifications of the enzyme subunits and of the inhibitor protein IF1 that may promote adaptations leading to cancer metabolism. Finally, we outline ATP synthase gene mutations and epigenetic modifications associated with cancer development or drug resistance, with the aim of highlighting this enzyme complex as a potential novel target for future anti-cancer therapy.

Update on chronic intestinal pseudo-obstruction.

PURPOSE OF REVIEW: Chronic intestinal pseudo-obstruction (CIPO) is the most severe and disabling form of gastrointestinal dysmotility characterized by an impairment of coordinated propulsive activity in the gastrointestinal tract mimicking mechanical intestinal obstruction. Over the last few years, major advances have been made in the diagnostic and therapeutic management of this rare disorder. RECENT FINDINGS: The present narrative review aims to summarize the current literature about the management of CIPO focusing on significant novelties about definition, epidemiology, diagnosis, and therapeutic options. The most significant advancement is a consensus on classification and dedicated diagnostic criteria for CIPO in children highlighting the distinctive features between adult and pediatric forms of CIPO (hence pediatric intestinal pseudo-obstruction). Despite no single diagnostic test is pathognomonic of CIPO and no recommended drug treatment is advised to improve gastrointestinal motility, recent reports suggest promising results in both diagnostic testing and therapy that might assist the diagnosis and help the management of patients with CIPO. SUMMARY: The articles referenced in this review will help in optimizing the clinical management of this rare and severe disease in adult population.

The unique histidine in OSCP subunit of F-ATP synthase mediates inhibition of the permeability transition pore by acidic pH.

The permeability transition pore (PTP) is a Ca2+-dependent mitochondrial channel whose opening causes a permeability increase in the inner membrane to ions and solutes. The most potent inhibitors are matrix protons, with channel block at pH 6.5. Inhibition is reversible, mediated by histidyl residue(s), and prevented by their carbethoxylation by diethylpyrocarbonate (DPC), but their assignment is unsolved. We show that PTP inhibition by H+ is mediated by the highly conserved histidyl residue (H112 in the human mature protein) of oligomycin sensitivity conferral protein (OSCP) subunit of mitochondrial F1FO (F)-ATP synthase, which we also show to undergo carbethoxylation after reaction of mitochondria with DPC. Mitochondrial PTP-dependent swelling cannot be inhibited by acidic pH in H112Q and H112Y OSCP mutants, and the corresponding megachannels (the electrophysiological counterpart of the PTP) are insensitive to inhibition by acidic pH in patch-clamp recordings of mitoplasts. Cells harboring the H112Q and H112Y mutations are sensitized to anoxic cell death at acidic pH. These results demonstrate that PTP channel formation and its inhibition by H+ are mediated by the F-ATP synthase.

Genetic susceptibility of increased intestinal permeability is associated with progressive liver disease and diabetes in patients with non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Increased intestinal permeability plays a key role in the pathogenesis of fat deposition in the liver. The aim of our study was to assess whether a single nucleotide polymorphism of protein tyrosine phosphatase non-receptor type 2 (PTPN2) (rs2542151 T→G), involved in intestinal permeability, may be associated with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). METHODS AND RESULTS: We recruited a prospective cohort of NAFLD subjects and matched controls. Clinical data, PTPN2 genotype and laboratory data were collected for each patient. Results were stratified according to liver histology and diabetes. We enrolled 566 cases and 377 controls. PTPN2 genotype distribution did not significantly differ between patients and controls. In the entire population, patients with PTPN2 rs2542151 T→G (dominant model) have a higher prevalence of diabetes; 345 patients (60.9%) underwent liver biopsy: 198 (57.4%) had steatohepatitis and 75 (21.7%) had advanced fibrosis. At multiple logistic regression analysis PTPN2 rs2542151 T→G was associated with T2DM (OR 2.14, 95% CI 1.04-4.40, P = 0.03). Patients who underwent liver biopsy, rs2542151 T→G of PTPN2 was independently associated with severe steatosis (OR 2.00, 95% CI 1.17-3.43, p = 0.01) and severe fibrosis (OR 2.23, 95% CI 1.06-4.72, P = 0.03). CONCLUSION: Our study shows that NAFLD patients with rs2542151 T→G of PTPN2 have a higher severity of fatty liver disease and a higher prevalence of T2DM. These results suggest that individual genetic susceptibility to intestinal permeability could play a role in liver disease progression.

SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Pain in individuals with RASopathies: Prevalence and clinical characterization in a sample of 80 affected patients.

Pain in individuals with RASopathies is a neglected topic in literature. In this article, we assessed prevalence and profile of pain in a sample of 80 individuals affected by RASopathies. The study sample included individuals with Noonan syndrome (N = 42), Costello syndrome (N = 17), and cardio-facio-cutaneous syndrome (N = 21). A set of standardized questionnaires and scales were administered (VAS/numeric scale, r-FLACC, Wang-Baker scale, NPSI, BPI, NCCPC-R) to detect and characterize acute and chronic pain and to study the influence of pain on quality of life (PEDs-QL, SF-36) and sleeping patterns (SDSC); revision of past medical history and multisystemic evaluation was provided. Available clinical data were correlated to the presence of pain. High prevalence of acute (44%) and chronic (61%) pain was documented in the examined sample. Due to age and intellectual disability, acute pain was localized in 18/35 individuals and chronic pain in 33/49. Muscle-skeletal and abdominal pain was more frequently reported. The intensity of acute and chronic pain interfered with daily activities in 1/3 of the sample. Pain negatively impacted on QoL and sleeping patterns. This work documents that pain is highly prevalent in RASopathies. Future studies including subjective and objective measures of pain are required to discriminate a somatosensory abnormality from an abnormal elaboration of painful stimuli at a central level.

Ca2+ binding to F-ATP synthase ß subunit triggers the mitochondrial permeability transition.

F-ATP synthases convert the electrochemical energy of the H+ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F-ATP synthases can also undergo a Ca2+-dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca2+ binding site and the mechanism(s) through which Ca2+ can transform the energy-conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the "Ca2+-trigger site" of the PTP to the catalytic site of the F-ATP synthase ß subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the ß subunit, which show a selective decrease in Ca2+-ATP hydrolysis, confer resistance to Ca2+-induced, PTP-dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F-ATP synthase to a channel and of its role in cell death.

The idebenone metabolite QS10 restores electron transfer in complex I and coenzyme Q defects.

Idebenone is a hydrophilic short-chain coenzyme (Co) Q analogue, which has been used as a potential bypass of defective complex I in both Leber Hereditary Optic Neuropathy and OPA1-dependent Dominant Optic Atrophy. Based on its potential antioxidant effects, it has also been tested in degenerative disorders such as Friedreich's ataxia, Huntington's and Alzheimer's diseases. Idebenone is rapidly modified but the biological effects of its metabolites have been characterized only partially. Here we have studied the effects of quinones generated during in vivo metabolism of idebenone with specific emphasis on 6-(9-carboxynonyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (QS10). QS10 partially restored respiration in cells deficient of complex I or of CoQ without inducing the mitochondrial permeability transition, a detrimental effect of idebenone that may offset its potential benefits [Giorgio et al. (2012) Biochim. Biophys. Acta 1817: 363-369]. Remarkably, respiration was largely rotenone-insensitive in complex I deficient cells and rotenone-sensitive in CoQ deficient cells. These findings indicate that, like idebenone, QS10 can provide a bypass to defective complex I; and that, unlike idebenone, QS10 can partially replace endogenous CoQ. In zebrafish (Danio rerio) treated with rotenone, QS10 was more effective than idebenone in allowing partial recovery of respiration (to 40% and 20% of the basal respiration of untreated embryos, respectively) and allowing zebrafish survival (80% surviving embryos at 60 h post-fertilization, a time point at which all rotenone-treated embryos otherwise died). We conclude that QS10 is potentially more active than idebenone in the treatment of diseases caused by complex I defects, and that it could also be used in CoQ deficiencies of genetic and acquired origin.

High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits.

BACKGROUND/AIMS: The permeability transition pore (PTP) is an unselective, Ca2+-dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca2+-activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. METHODS: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu2+ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. RESULTS: Null mutants for the subunits e and g display dimer formation upon Cu2+ treatment and show PTP-dependent mitochondrial Ca2+ release but not swelling. Cu2+ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. CONCLUSION: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.