Pyruvate dehydrogenase is a potential mitochondrial off-target for gentamicin based on in silico predictions and in vitro inhibition studies.

During the drug development process, organ toxicity leads to an estimated failure of one-third of novel chemical entities. Drug-induced toxicity is increasingly associated with mitochondrial dysfunction, but identifying the underlying molecular mechanisms remains a challenge. Computational modeling techniques have proven to be a good tool in searching for drug off-targets. Here, we aimed to identify mitochondrial off-targets of the nephrotoxic drugs tenofovir and gentamicin using different in silico approaches (KRIPO, ProBis and PDID). Dihydroorotate dehydrogenase (DHODH) and pyruvate dehydrogenase (PDH) were predicted as potential novel off-target sites for tenofovir and gentamicin, respectively. The predicted targets were evaluated in vitro, using (colorimetric) enzymatic activity measurements. Tenofovir did not inhibit DHODH activity, while gentamicin potently reduced PDH activity. In conclusion, the use of in silico methods appeared a valuable approach in predicting PDH as a mitochondrial off-target of gentamicin. Further research is required to investigate the contribution of PDH inhibition to overall renal toxicity of gentamicin.

Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells.

Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.

Time to Change: A Systems Pharmacology Approach to Disentangle Mechanisms of Drug-Induced Mitochondrial Toxicity.

An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, which is associated with almost half of all Food and Drug Administration black box warnings, a variety of drug withdrawals, and attrition of drug candidates. This can mainly be attributed to a historic lack of sensitive and specific assays to identify the mechanisms underlying mitochondrial toxicity during drug development. In the last decade, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based systems pharmacological approaches. Here, we propose the implementation of a tiered systems pharmacology approach to detect adverse mitochondrial drug effects during preclinical drug development, which is based on a toolset developed to study inherited mitochondrial disease. This includes phenotypic characterization, profiling of key metabolic alterations, mechanistic studies, and functional in vitro and in vivo studies. Combined with binding pocket similarity comparisons and bottom-up as well as top-down metabolic network modeling, this tiered approach enables identification of mechanisms underlying drug-induced mitochondrial dysfunction. After validation of these off-target mechanisms, drug candidates can be adjusted to minimize mitochondrial activity. Implementing such a tiered systems pharmacology approach could lead to a more efficient drug development trajectory due to lower drug attrition rates and ultimately contribute to the development of safer drugs. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs adversely affect mitochondrial function, which can be detected using phenotypic assays. However, these methods provide only limited insight into the underlying mechanisms. In recent years, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based system pharmacological approaches. Their implementation in preclinical drug development could reduce the number of drug failures, contributing to safer drug design.

Identifying trajectories of fatigue in patients with primary mitochondrial disease due to the m.3243A > G variant.

Severe fatigue is a common complaint in patients with primary mitochondrial disease. However, less is known about the course of fatigue over time. This longitudinal observational cohort study of patients with the mitochondrial DNA 3243 A>G variant explored trajectories of fatigue over 2 years, and characteristics of patients within these fatigue trajectories. Fifty-three adult patients treated at the Radboud University Medical Center Nijmegen were included. The majority of the patients reported consistent, severe fatigue (41%), followed by patients with a mixed pattern of severe and mild fatigue (36%). Then, 23% of patients reported stable mild fatigue levels. Patients with a stable high fatigue trajectory were characterized by higher disease manifestations scores, more clinically relevant mental health symptoms, and lower psychosocial functioning and quality of life compared to patients reporting stable low fatigue levels. Fatigue at baseline and disease manifestation scores predicted fatigue severity at the 2-year assessment (57% explained variance). This study demonstrates that severe fatigue is a common and stable complaint in the majority of patients. Clinicians should be aware of severe fatigue in patients with moderate to severe disease manifestation scores on the Newcastle Mitochondrial Disease Scale, the high prevalence of clinically relevant mental health symptoms and overall impact on quality of life in these patients. Screening of fatigue and psychosocial variables will guide suitable individualized treatment to improve the quality of life.

The decylTPP mitochondria-targeting moiety lowers electron transport chain supercomplex levels in primary human skin fibroblasts.

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP+) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C10-TPP+). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7-V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety.

Restoring cellular NAD(P)H levels by PPARα and LXRα stimulation to improve mitochondrial complex I deficiency.

Mitochondrial complex I (CI), the first multiprotein enzyme complex of the oxidative phosphorylation system, plays a crucial role in cellular energy production. CI deficiency is associated with a variety of clinical phenotypes, including Leigh syndrome. At the cellular level, an increased NAD(P)H concentration is one of the hallmarks in CI-deficiency. AIMS: Here, we aimed to attenuate increased NAD(P)H levels by stimulation of ATP-dependent cassette (ABC)A1 and ABCG1-mediated cellular cholesterol efflux with various PPARα and LXRα agonists. MAIN METHODS: Mitochondrial CI-deficient fibroblasts and chemically-induced CI-deficient HeLa cells were used to study the dose-dependent effects of various PPARα and LXRα agonists on cellular NAD(P)H levels and cholesterol efflux. KEY FINDINGS: In patient-derived mitochondrial CI-deficient fibroblasts, GW590735, astaxanthin, oleoylethanolamide, and GW3965 significantly reduced the enhanced NAD(P)H levels in CI-deficient fibroblasts. Similar effects were observed in chemically-induced CI-impaired HeLa cells, in which BMS-687453, Wy14643, GW7647, T0901317, DMHCA also demonstrated a beneficial effect. Surprisingly, no effect on ABCA1- and ABCG1-mediated cholesterol efflux in HeLa cells and fibroblasts was found after treatment with these compounds. The reduction in NAD(P)H levels by GW590735 could be partially reversed by inhibition of fatty acid synthase and ß-oxidation, which suggests that its beneficial effects are possibly mediated via stimulation of fatty acid metabolism rather than cholesterol efflux. SIGNIFICANCE: Collectively, PPARα and LXRα stimulation resulted in attenuated cellular NAD(P)H levels in CI-impaired HeLa cells and patient-derived fibroblasts and could eventually have a therapeutic potential in CI deficiency.

Dissecting Drug-Induced Cytotoxicity and Metabolic Dysfunction in Conditionally Immortalized Human Proximal Tubule Cells.

Fourteen to 26 percent of all hospitalized cases of acute kidney injury are explained by drug-induced toxicity, emphasizing the importance of proper strategies to pre-clinically assess renal toxicity. The MTT assay is widely used as a measure of cell viability, but largely depends on cellular metabolic activity. Consequently, MTT as a single assay may not be the best way to assess cytotoxicity of compounds that reduce mitochondrial function and cellular metabolic activity without directly affecting cell viability. Accordingly, we aim to highlight the limitations of MTT alone in assessing renal toxicity of compounds that interfere with metabolic activity. Therefore, we compared toxic effects observed by MTT with a fluorescent assay that determines compromised plasma membrane permeability. Exposure of proximal tubule epithelial cells to nephrotoxic compounds reduced cellular metabolic activity concentration- and time-dependently. We show that compared to our fluorescence-based approach, assessment of cellular metabolic activity by means of MTT provides a composite readout of cell death and metabolic impairment. An approach independent of cellular metabolism is thus preferable when assessing cytotoxicity of compounds that induce metabolic dysfunction. Moreover, combining both assays during drug development enables a first discrimination between compounds having a direct or indirect mitochondrial toxic potential.

Natural history, outcome measures and trial readiness in LAMA2-related muscular dystrophy and SELENON-related myopathy in children and adults: protocol of the LAST STRONG study.

BACKGROUND: SELENON (SEPN1)-related myopathy (SELENON-RM) is a rare congenital myopathy characterized by slowly progressive proximal muscle weakness, early onset spine rigidity and respiratory insufficiency. A muscular dystrophy caused by mutations in the LAMA2 gene (LAMA2-related muscular dystrophy, LAMA2-MD) has a similar clinical phenotype, with either a severe, early-onset due to complete Laminin subunit α2 deficiency (merosin-deficient congenital muscular dystrophy type 1A (MDC1A)), or a mild, childhood- or adult-onset due to partial Laminin subunit α2 deficiency. For both muscle diseases, no curative treatment options exist, yet promising preclinical studies are ongoing. Currently, there is a paucity on natural history data and appropriate clinical and functional outcome measures are needed to reach trial readiness. METHODS: LAST STRONG is a natural history study in Dutch-speaking patients of all ages diagnosed with SELENON-RM or LAMA2-MD, starting August 2020. Patients have four visits at our hospital over a period of 1.5 year. At all visits, they undergo standardized neurological examination, hand-held dynamometry (age ≥ 5 years), functional measurements, questionnaires (patient report and/or parent proxy; age ≥ 2 years), muscle ultrasound including diaphragm, pulmonary function tests (spirometry, maximal inspiratory and expiratory pressure, sniff nasal inspiratory pressure; age ≥ 5 years), and accelerometry for 8 days (age ≥ 2 years); at visit one and three, they undergo cardiac evaluation (electrocardiogram, echocardiography; age ≥ 2 years), spine X-ray (age ≥ 2 years), dual-energy X-ray absorptiometry (DEXA-)scan (age ≥ 2 years) and full body magnetic resonance imaging (MRI) (age ≥ 10 years). All examinations are adapted to the patient's age and functional abilities. Correlation between key parameters within and between subsequent visits will be assessed. DISCUSSION: Our study will describe the natural history of patients diagnosed with SELENON-RM or LAMA2-MD, enabling us to select relevant clinical and functional outcome measures for reaching clinical trial-readiness. Moreover, our detailed description (deep phenotyping) of the clinical features will optimize clinical management and will establish a well-characterized baseline cohort for prospective follow-up. CONCLUSION: Our natural history study is an essential step for reaching trial readiness in SELENON-RM and LAMA2-MD. TRIAL REGISTRATION: This study has been approved by medical ethical reviewing committee Region Arnhem-Nijmegen (NL64269.091.17, 2017-3911) and is registered at ClinicalTrial.gov ( NCT04478981 ).

The Redox Modulating Sonlicromanol Active Metabolite KH176m and the Antioxidant MPG Protect Against Short-Duration Cardiac Ischemia-Reperfusion Injury.

PURPOSE: Sonlicromanol is a phase IIB clinical stage compound developed for treatment of mitochondrial diseases. Its active component, KH176m, functions as an antioxidant, directly scavenging reactive oxygen species (ROS), and redox activator, boosting the peroxiredoxin-thioredoxin system. Here, we examined KH176m's potential to protect against acute cardiac ischemia-reperfusion injury (IRI), compare it with the classic antioxidant N-(2-mercaptopropionyl)-glycine (MPG), and determine whether protection depends on duration (severity) of ischemia. METHODS: Isolated C56Bl/6N mouse hearts were Langendorff-perfused and subjected to short (20 min) or long (30 min) ischemia, followed by reperfusion. During perfusion, hearts were treated with saline, 10 µM KH176m, or 1 mM MPG. Cardiac function, cell death (necrosis), and mitochondrial damage (cytochrome c (CytC) release) were evaluated. In additional series, the effect of KH176m treatment on the irreversible oxidative stress marker 4-hydroxy-2-nonenal (4-HNE), formed during ischemia only, was determined at 30-min reperfusion. RESULTS: During baseline conditions, both drugs reduced cardiac performance, with opposing effects on vascular resistance (increased with KH176m, decreased with MPG). For short ischemia, KH176m robustly reduced all cell death parameters: LDH release (0.2 ± 0.2 vs 0.8 ± 0.5 U/min/GWW), infarct size (15 ± 8 vs 31 ± 20%), and CytC release (168.0 ± 151.9 vs 790.8 ± 453.6 ng/min/GWW). Protection by KH176m was associated with decreased cardiac 4-HNE. MPG only reduced CytC release. Following long ischemia, IRI was doubled, and KH176m and MPG now only reduced LDH release. The reduced protection against long ischemia was associated with the inability to reduce cardiac 4-HNE. CONCLUSION: Protection against cardiac IRI by the antioxidant KH176m is critically dependent on duration of ischemia. The data suggest that with longer ischemia, the capacity of KH176m to reduce cardiac oxidative stress is rate-limiting, irreversible ischemic oxidative damage maximally accumulates, and antioxidant protection is strongly diminished.

Cognitive functioning and mental health in mitochondrial disease: A systematic scoping review.

Mitochondrial diseases (MDs) are rare, heterogeneous, hereditary and progressive in nature. In addition to the serious somatic symptoms, patients with MD also experience problems regarding their cognitive functioning and mental health. We provide an overview of all published studies reporting on any aspect of cognitive functioning and/or mental health in patients with MD and their relatives. A total of 58 research articles and 45 case studies were included and critically reviewed. Cognitive impairments in multiple domains were reported. Mental disorders were frequently reported, especially depression and anxiety. Furthermore, most studies showed impairments in self-reported psychological functioning and high prevalence of mental health problems in (matrilineal) relatives. The included studies showed heterogeneity regarding patient samples, measurement instruments and reference groups, making comparisons cautious. Results highlight a high prevalence of cognitive impairments and mental disorders in patients with MD. Recommendations for further research as well as tailored patientcare with standardized follow-up are provided. Key gaps in the literature are identified, of which studies on natural history are of highest importance.

One mutation, three phenotypes: novel metabolic insights on MELAS, MIDD and myopathy caused by the m.3243A > G mutation.

INTRODUCTION: The m.3243A > G mitochondrial DNA mutation is one of the most common mitochondrial disease-causing mutations, with a carrier rate as high as 1:400. This point mutation affects the MT-TL1 gene, ultimately affecting the oxidative phosphorylation system and the cell's energy production. Strikingly, the m.3243A > G mutation is associated with different phenotypes, including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), maternally inherited diabetes and deafness (MIDD) and myopathy. OBJECTIVES: We investigated urine metabolomes of MELAS, MIDD and myopathy patients in order to identify affected metabolic pathways and possible treatment options. METHODS: A multiplatform metabolomics approach was used to comprehensively analyze the metabolome and compare metabolic profiles of different phenotypes caused by the m.3243A > G mutation. Our analytical array consisted of NMR spectroscopy, LC-MS/MS and GC-TOF-MS. RESULTS: The investigation revealed phenotypic specific metabolic perturbations, as well as metabolic similarities between the different phenotypes. We show that glucose metabolism is highly disturbed in the MIDD phenotype, but not in MELAS or myopathy, remodeled fatty acid oxidation is characteristic of the MELAS patients, while one-carbon metabolism is strongly modified in both MELAS and MIDD, but not in the myopathy group. Lastly we identified increased creatine in the urine of the myopathy patients, but not in MELAS or MIDD. CONCLUSION: We conclude by giving novel insight on the phenotypes of the m.3243A > G mutation from a metabolomics point of view. Directives are also given for future investigations that could lead to better treatment options for patients suffering from this debilitating disease.

Stimulation of cholesterol biosynthesis in mitochondrial complex I-deficiency lowers reductive stress and improves motor function and survival in mice.

The majority of cellular energy is produced by the mitochondrial oxidative phosphorylation (OXPHOS) system. Failure of the first OXPHOS enzyme complex, NADH:ubiquinone oxidoreductase or complex I (CI), is associated with multiple signs and symptoms presenting at variable ages of onset. There is no approved drug treatment yet to slow or reverse the progression of CI-deficient disorders. Here, we present a comprehensive human metabolic network model of genetically characterized CI-deficient patient-derived fibroblasts. Model calculations predicted that increased cholesterol production, export, and utilization can counterbalance the surplus of reducing equivalents in patient-derived fibroblasts, as these pathways consume considerable amounts of NAD(P)H. We show that fibrates attenuated increased NAD(P)H levels and improved CI-deficient fibroblast growth by stimulating the production of cholesterol via enhancement of its cellular efflux. In CI-deficient (Ndufs4-/-) mice, fibrate treatment resulted in prolonged survival and improved motor function, which was accompanied by an increased cholesterol efflux from peritoneal macrophages. Our results shine a new light on the use of compensatory biological pathways in mitochondrial dysfunction, which may lead to novel therapeutic interventions for mitochondrial diseases for which currently no cure exists.

Six-year prospective follow-up study in 151 carriers of the mitochondrial DNA 3243 A>G variant.

BACKGROUND: The mitochondrial DNA (mDNA) 3243A>G variant is the most common pathogenic variant of the mDNA. To interpret results of clinical trials in mitochondrial disease, it is important to have a clear understanding of the natural course of disease. To obtain more insight into the disease burden and the progression of disease in carriers of the mDNA 3243 A>G variant, we followed a cohort of 151 carriers from 61 families prospectively for up to 6 years. METHODS: The disease severity was scored using the Newcastle Mitochondrial Disease Adult Scale (NMDAS), including SF-36 quality of life (QoL) scores. Heteroplasmy levels were measured in urinary epithelial cells (UEC), leucocytes and saliva. The progression of the disease was studied using linear mixed model analysis. RESULTS: One hundred twenty-four carriers (out of 151) were symptomatic. Four clinical groups were identified: 1) classical mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (n=7), 2) maternally inherited diabetes deafness syndrome (n=60), 3) 'other' (n=57) and 4) dormant carriers (n=27). A yearly increase of NMDAS score of 0.47 point was measured in the total group. Heteroplasmy levels in both leucocytes and UEC were only weakly correlated with disease severity. Physical QoL declined with age. The most important determinants of QoL decline were hearing loss, speech problems, exercise intolerance, gait instability, psychiatric problems and gastrointestinal involvement. CONCLUSION: The mDNA 3243 A>G variant causes a slowly progressive disease, with a yearly increase of NMDAS score of ~0.5 point overall with the clinical phenotype being the only determinant of disease progression.

Delineation of molecular findings by whole-exome sequencing for suspected cases of paediatric-onset mitochondrial diseases in the Southern Chinese population.

BACKGROUND: Mitochondrial diseases (MDs) are a group of clinically and genetically heterogeneous disorders characterized by defects in oxidative phosphorylation. Since clinical phenotypes of MDs may be non-specific, genetic diagnosis is crucial for guiding disease management. In the current study, whole-exome sequencing (WES) was performed for our paediatric-onset MD cohort of a Southern Chinese origin, with the aim of identifying key disease-causing variants in the Chinese patients with MDs. METHODS: We recruited Chinese patients who had paediatric-onset MDs and a minimum mitochondrial disease criteria (MDC) score of 3. Patients with positive target gene or mitochondrial DNA sequencing results were excluded. WES was performed, variants with population frequency ≤ 1% were analysed for pathogenicity on the basis of the American College of Medical Genetics and Genomics guidelines. RESULTS: Sixty-six patients with pre-biopsy MDC scores of 3-8 were recruited. The overall diagnostic yield was 35% (23/66). Eleven patients (17%) were found to have mutations in MD-related genes, with COQ4 having the highest mutation rate owing to the Chinese-specific founder mutation (4/66, 6%). Twelve patients (12/66, 18%) had mutations in non-MD-related genes: ATP1A3 (n = 3, two were siblings), ALDH5A1, ARX, FA2H, KCNT1, LDHD, NEFL, NKX2-2, TBCK, and WAC. CONCLUSIONS: We confirmed that the COQ4:c.370G>A, p.(Gly124Ser) variant, was a founder mutation among the Southern Chinese population. Screening for this mutation should therefore be considered while diagnosing Chinese patients suspected to have MDs. Furthermore, WES has proven to be useful in detecting variants in patients suspected to have MDs because it helps to obtain an unbiased and precise genetic diagnosis for these diseases, which are genetically heterogeneous.

NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4-/- mice and Leigh syndrome patients: A stabilizing role for NDUFAF2.

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4-/- mouse tissues. Ndufs4-/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4-/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4-/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4-/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.

Psychological functioning in children suspected for mitochondrial disease: the need for care.

BACKGROUND: Mitochondrial diseases (MD) are generally serious and progressive, inherited metabolic diseases. There is a high comorbidity of anxiety and depression and limitations in daily functioning. The complexity and duration of the diagnostic process and lack of knowledge about prognosis leads to uncertainty. In this study, we investigated the psychological well-being of children who are suspected for MD and their parents. METHODS: In total 122 children suspected for MD and their parents, received questionnaires as part of standard clinical investigation. RESULTS: Parent proxy report revealed a lower quality of life (QoL) compared to norms and even more physical problems compared to chronically ill patients. They also reported more behavioral problems in general and more internalizing problems compared to the norms. Most frequent reported somatic complaints were tiredness and pain. Parents did not report enhanced levels of stress regarding parenting and experienced sufficient social support. At the end of the diagnostic process, 5.7% of the children received the genetically confirmed diagnosis of MD, 26% showed non-conclusive abnormalities in the muscle biopsy, 54% did not receive any diagnosis, and the remaining received other diagnoses. Strikingly, children without a diagnosis showed equally QoL and behavioral problems as children with a diagnosis, and even more internalizing problems. CONCLUSIONS: This study highlights the psychological concerns of children with a suspicion of MD. It is important to realize that as well as children with a confirmed diagnosis, children without a diagnosis are vulnerable since explanation for their complaints is still lacking.

Effects of clofibrate and KH176 on life span and motor function in mitochondrial complex I-deficient mice.

Mitochondrial complex I (CI), the first multiprotein enzyme complex of the OXPHOS system, executes a major role in cellular ATP generation. Consequently, dysfunction of this complex has been linked to inherited metabolic disorders, including Leigh disease (LD), an often fatal disease in early life. Development of clinical effective treatments for LD remains challenging due to the complex pathophysiological nature. Treatment with the peroxisome proliferation-activated receptor (PPAR) agonist bezafibrate improved disease phenotype in several mitochondrial disease mouse models mediated via enhanced mitochondrial biogenesis and fatty acid ß-oxidation. However, the therapeutic potential of this mixed PPAR (α, δ/ß, γ) agonist is severely hampered by hepatotoxicity, which is possibly caused by activation of PPARγ. Here, we aimed to investigate the effects of the PPARα-specific fibrate clofibrate in mitochondrial CI-deficient (Ndufs4-/-) mice. Clofibrate increased lifespan and motor function of Ndufs4-/- mice, while only marginal hepatotoxic effects were observed. Due to the complex clinical and cellular phenotype of CI-deficiency, we also aimed to investigate the therapeutic potential of clofibrate combined with the redox modulator KH176. As described previously, single treatment with KH176 was beneficial, however, combining clofibrate with KH176 did not result in an additive effect on disease phenotype in Ndufs4-/- mice. Overall, both drugs have promising, but independent and nonadditive, properties for the pharmacological treatment of CI-deficiency-related mitochondrial diseases.

Variants in NGLY1 lead to intellectual disability, myoclonus epilepsy, sensorimotor axonal polyneuropathy and mitochondrial dysfunction.

NGLY1 encodes the enzyme N-glycanase that is involved in the degradation of glycoproteins as part of the endoplasmatic reticulum-associated degradation pathway. Variants in this gene have been described to cause a multisystem disease characterized by neuromotor impairment, neuropathy, intellectual disability, and dysmorphic features. Here, we describe four patients with pathogenic variants in NGLY1. As the clinical features and laboratory results of the patients suggested a multisystem mitochondrial disease, a muscle biopsy had been performed. Biochemical analysis in muscle showed a strongly reduced ATP production rate in all patients, while individual OXPHOS enzyme activities varied from normal to reduced. No causative variants in any mitochondrial disease genes were found using mtDNA analysis and whole exome sequencing. In all four patients, variants in NGLY1 were identified, including two unreported variants (c.849T>G (p.(Cys283Trp)) and c.1067A>G (p.(Glu356Gly)). Western blot analysis of N-glycanase in muscle and fibroblasts showed a complete absence of N-glycanase. One patient showed a decreased basal and maximal oxygen consumption rates in fibroblasts. Mitochondrial morphofunction fibroblast analysis showed patient specific differences when compared to control cell lines. In conclusion, variants in NGLY1 affect mitochondrial energy metabolism which in turn might contribute to the clinical disease course.