Proton-coupled electron transfer dynamics in the alternative oxidase.

The alternative oxidase (AOX) is a membrane-bound di-iron enzyme that catalyzes O2-driven quinol oxidation in the respiratory chains of plants, fungi, and several pathogenic protists of biomedical and industrial interest. Yet, despite significant biochemical and structural efforts over the last decades, the catalytic principles of AOX remain poorly understood. We develop here multi-scale quantum and classical molecular simulations in combination with biochemical experiments to address the proton-coupled electron transfer (PCET) reactions responsible for catalysis in AOX from Trypanosoma brucei, the causative agent of sleeping sickness. We show that AOX activates and splits dioxygen via a water-mediated PCET reaction, resulting in a high-valent ferryl/ferric species and tyrosyl radical (Tyr220˙) that drives the oxidation of the quinol via electric field effects. We identify conserved carboxylates (Glu215, Asp100) within a buried cluster of ion-pairs that act as a transient proton-loading site in the quinol oxidation process, and validate their function experimentally with point mutations that result in drastic activity reduction and pK a-shifts. Our findings provide a key mechanistic understanding of the catalytic machinery of AOX, as well as a molecular basis for rational drug design against energy transduction chains of parasites. On a general level, our findings illustrate how redox-triggered conformational changes in ion-paired networks control the catalysis via electric field effects.

THE FIGHT INHERITED RETINAL BLINDNESS! PROJECT: A NEW TREATMENT OUTCOME AND NATURAL HISTORY REGISTRY FOR INHERITED RETINAL DISEASE.

PURPOSE: To design and build a new disease registry to track the natural history and outcomes of approved gene therapy in patients with inherited retinal diseases (IRDs). METHODS: A core committee of 6 members was convened to oversee the construction of the FIRB! module. A further 11 experts formed a steering committee, which discussed disease classification and variables to form minimum datasets via a consensus approach. RESULTS: The web-based FIRB! registry records baseline demographic, clinical and genetic data together with follow-up data. The Human Phenotype Ontology and Monarch Disease Ontology nomenclature were incorporated within the FIRB! architecture to standardise nomenclature. The registry software assigns individual diagnoses to one of 7 broad phenotypic groups, with minimum datasets dependent upon the broad phenotypic group. Additionally, minimum datasets were agreed upon for patients undergoing approved gene therapy with voretigene neparvovec (Luxturna). New patient entries can be completed in 5 minutes, and follow-up data can be entered in 2 minutes. CONCLUSIONS: Fight Inherited Retinal Blindness! (FIRB!) is an organized, web-based system that uses observational study methods to collect uniform data from IRD patients to track natural history and (uniquely) treatment outcomes. It is free to Users, who have control over their data.

A Combination of Low Doses of Lithium and Valproate Improves Cognitive Outcomes after Mild Traumatic Brain Injury.

The prevalence of mild traumatic brain injury (mTBI) is high compared with moderate and severe TBI, comprising almost 80% of all brain injuries. mTBI activates a complex cascade of biochemical, molecular, structural, and pathological changes that can result in neurological and cognitive impairments. These impairments can manifest even in the absence of overt brain damage. Given the complexity of changes triggered by mTBI, a combination of drugs that target multiple TBI-activated cascades may be required to improve mTBI outcomes. It has been previously demonstrated that cotreatment with the U.S. Food and Drug Administration (FDA)-approved drugs lithium plus valproate (Li + VPA) for 3 weeks after a moderate-to-severe controlled cortical impact injury reduced cortical tissue loss and improved motor function. Since both lithium and valproate can exhibit toxicity at high doses, it would be beneficial to determine if this combination treatment is effective when administered at low doses and for a shorter duration, and if it can improve cognitive function, after a mild diffuse TBI. In the present study, we tested if the combination of low doses of lithium (1 mEq/kg or 0.5 mEq/kg) plus valproate (20 mg/kg) administered for 3 days after a mild fluid percussion injury can improve hippocampal-dependent learning and memory. Our data show that the combination of low-dose Li + VPA improved spatial learning and memory, effects not seen when either drug was administered alone. In addition, postinjury Li + VPA treatment improved recognition memory and sociability and reduced fear generalization. Postinjury Li + VPA also reduced the number of anti-ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia counted using a convolutional neural network, indicating a reduction in neuroinflammation. These findings indicate that low-dose Li + VPA administered acutely after mTBI may have translational utility to reduce pathology and improve cognitive function.

The evolving pathophysiology of TBI and the advantages of temporally-guided combination therapies.

Several clinical and experimental studies have demonstrated that traumatic brain injury (TBI) activates cascades of biochemical, molecular, structural, and pathological changes in the brain. These changes combine to contribute to the various outcomes observed after TBI. Given the breadth and complexity of changes, combination treatments may be an effective approach for targeting multiple detrimental pathways to yield meaningful improvements. In order to identify targets for therapy development, the temporally evolving pathophysiology of TBI needs to be elucidated in detail at both the cellular and molecular levels, as it has been shown that the mechanisms contributing to cognitive dysfunction change over time. Thus, a combination of individual mechanism-based therapies is likely to be effective when maintained based on the time courses of the cellular and molecular changes being targeted. In this review, we will discuss the temporal changes of some of the key clinical pathologies of human TBI, the underlying cellular and molecular mechanisms, and the results from preclinical and clinical studies aimed at mitigating their consequences. As most of the pathological events that occur after TBI are likely to have subsided in the chronic stage of the disease, combination treatments aimed at attenuating chronic conditions such as cognitive dysfunction may not require the initiation of individual treatments at a specific time. We propose that a combination of acute, subacute, and chronic interventions may be necessary to maximally improve health-related quality of life (HRQoL) for persons who have sustained a TBI.

Enhanced Learning and Memory in Patients with CRB1 Retinopathy.

Mutations in the CRB1 gene are associated with a diverse spectrum of retinopathies with phenotypic variability causing severe visual impairment. The CRB1 gene has a role in retinal development and is expressed in the cerebral cortex and hippocampus, but its role in cognition has not been described before. This study compares cognitive function in CRB1 retinopathy individuals with subjects with other retinopathies and the normal population. METHODS: Neuropsychological tests of cognitive function were used to test individuals with CRB1 and non-CRB1 retinopathies and compare results with a standardised normative dataset. RESULTS: CRB1 retinopathy subjects significantly outperformed those with non-CRB1 retinopathy in list learning tasks of immediate (p = 0.001) and delayed memory (p = 0.007), tests of semantic verbal fluency (p = 0.017), verbal IQ digit span subtest (p = 0.037), and estimation test of higher execution function (p = 0.020) but not in the remaining tests of cognitive function (p > 0.05). CRB1 retinopathy subjects scored significantly higher than the normal population in all areas of memory testing (p < 0.05) and overall verbal IQ tests (p = 0.0012). Non-CRB1 retinopathy subjects scored significantly higher than the normal population in story recall, verbal fluency, and overall verbal IQ tests (p = 0.0016). CONCLUSIONS: Subjects with CRB1 retinopathy may have enhanced cognitive function in areas of memory and learning. Further work is required to understand the role of CRB1 in cognition.

Neuropathy target esterase activity defines phenotypes among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism and hair anomalies. PNPLA6 encodes neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a systematic evidence-based review of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 36 variants as pathogenic and 10 variants as likely pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship, and the generation of a preclinical animal model, pave the way for therapeutic trials, using NTE as a biomarker.

Substrate Specificities of DDX1: A Human DEAD-box protein.

DDX1 is a human protein which belongs to the DEAD-box protein family of enzymes and is involved in various stages of RNA metabolism from transcription to decay. Many members of the DEAD-box family of enzymes use the energy of ATP binding and hydrolysis to perform their cellular functions. On the other hand, a few members of the DEAD-box family of enzymes bind and/or hydrolyze other nucleotides in addition to ATP. Furthermore, the ATPase activity of DEAD-box family members is stimulated differently by nucleic acids of various structures. The identity of the nucleotides that the DDX1 hydrolyzes and the structure of the nucleic acids upon which it acts in the cell remain largely unknown. Identifying the DDX1 protein's in vitro substrates is important for deciphering the molecular roles of DDX1 in cells. Here we identify the nucleic acid sequences and structures supporting the nucleotide hydrolysis activity of DDX1 and its nucleotide specificity. Our data demonstrate that the DDX1 protein hydrolyzes only ATP and deoxy-ATP in the presence of RNA. The ATP hydrolysis activity of DDX1 is stimulated by multiple molecules: single-stranded RNA molecules as short as ten nucleotides, a blunt-ended double-stranded RNA molecule, a hybrid of a double-stranded DNA-RNA molecule, and a single-stranded DNA molecule. Under our experimental conditions, the single-stranded DNA molecule stimulates the ATPase activity of DDX1 at a significantly reduced extent when compared to the other investigated RNA constructs or the hybrid double-stranded DNA/RNA molecule.

The striking differences in the bioenergetics of brain and liver mitochondria are enhanced in mitochondrial disease.

Mitochondrial disorders are hallmarked by the dysfunction of oxidative phosphorylation (OXPHOS) yet are highly heterogeneous at the clinical and genetic levels. Striking tissue-specific pathological manifestations are a poorly understood feature of these conditions, even if the disease-causing genes are ubiquitously expressed. To investigate the functional basis of this phenomenon, we analyzed several OXPHOS-related bioenergetic parameters, including oxygen consumption rates, electron transfer system (ETS)-related coenzyme Q (mtCoQ) redox state and production of reactive oxygen species (ROS) in mouse brain and liver mitochondria fueled by different substrates. In addition, we determined how these functional parameters are affected by ETS impairment in a tissue-specific manner using pathologically relevant mouse models lacking either Ndufs4 or Ttc19, leading to Complex I (CI) or Complex III (CIII) deficiency, respectively. Detailed OXPHOS analysis revealed striking differences between brain and liver mitochondria in the capacity of the different metabolic substrates to fuel the ETS, reduce the ETS-related mtCoQ, and to induce ROS production. In addition, ETS deficiency due to either CI or CIII dysfunction had a much greater impact on the intrinsic bioenergetic parameters of brain compared with liver mitochondria. These findings are discussed in terms of the still rather mysterious tissue-specific manifestations of mitochondrial disease.

Lysophospholipids Are Associated With Outcomes in Hospitalized Patients With Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) accounts for 70-90% of all TBI cases. Lipid metabolites have important roles in plasma membrane biogenesis, function, and cell signaling. As TBI can compromise plasma membrane integrity and alter brain cell function, we sought to identify circulating phospholipid alterations after mTBI, and determine if these changes were associated with clinical outcomes. Patients with mTBI (Glasgow Coma Score [GCS] ≥13 and loss of consciousness <30 min) were recruited. A total of 84 mTBI subjects were enrolled after admission to a level I trauma center, with the majority having evidence of traumatic intracranial hemorrhage on brain computed tomography (CT). Plasma samples were collected within 24 h of injury with 32 mTBI subjects returning at 3 months after injury for a second plasma sample to be collected. Thirty-five healthy volunteers were enrolled as controls and had a one-time blood draw. Lipid metabolomics was performed on plasma samples from each subject. Fold change of selected lipid metabolites was determined. Multivariable regression models were created to test associations between lipid metabolites and discharge and 6-month Glasgow Outcomes Scale-Extended (GOSE) outcomes (dichotomized between "good" [GOSE ≥7] and "bad" [GOSE ≤6] functional outcomes). Plasma levels of 31 lipid metabolites were significantly associated with discharge GOSE using univariate models; three of these metabolites were significantly increased, while 14 were significantly decreased in subjects with good outcomes compared with subjects with poor outcomes. In multivariable logistic regression models, higher circulating levels of the lysophospholipids (LPL) 1-linoleoyl-glycerophosphocholine (GPC) (18:2), 1-linoleoyl-GPE (18:2), and 1-linolenoyl-GPC (18:3) were associated with both good discharge GOSE (odds ratio [OR] 12.2 [95% CI 3.35, 58.3], p = 5.23 × 10-4; OR 9.43 [95% CI 2.87, 39.6], p = 7.26 × 10-4; and OR 5.26 [95% CI 1.99, 16.7], p = 2.04 × 10-3, respectively) and 6-month (OR 4.67 [95% CI 1.49, 17.7], p = 0.013; OR 2.93 [95% CI 1.11, 8.87], p = 0.039; and OR 2.57 [95% CI 1.08, 7.11], p = 0.046, respectively). Compared with healthy volunteers, circulating levels of these three LPLs were decreased early after injury and had normalized by 3 months after injury. Logistic regression models to predict functional outcomes were created by adding each of the described three LPLs to a baseline model that included age and sex. Including 1-linoleoyl-GPC (18:2) (8.20% improvement, p = 0.009), 1-linoleoyl-GPE (18:2) (8.85% improvement, p = 0.021), or 1-linolenoyl-GPC (18:3) (7.68% improvement, p = 0.012), significantly improved the area under the curve (AUC) for predicting discharge outcomes compared with the baseline model. Models including 1-linoleoyl-GPC (18:2) significantly improved AUC for predicting 6-month outcomes (9.35% improvement, p = 0.034). Models including principal components derived from 25 LPLs significantly improved AUC for prediction of 6-month outcomes (16.0% improvement, p = 0.020). Our results demonstrate that higher plasma levels of LPLs (1-linoleoyl-GPC, 1-linoleoyl-GPE, and 1-linolenoyl-GPC) after mTBI are associated with better functional outcomes at discharge and 6 months after injury. This class of phospholipids may represent a potential therapeutic target.

Risk Assessment of Psychotropic Drugs on Mitochondrial Function Using In Vitro Assays.

Mitochondria are potential targets responsible for some drug- and xenobiotic-induced organ toxicities. However, molecular mechanisms of drug-induced mitochondrial toxicities are mostly unknown. Here, multiple in vitro assays were used to investigate the effects of 22 psychotropic drugs on mitochondrial function. The acute extracellular flux assay identified inhibitors of the electron transport chain (ETC), i.e., aripiprazole, phenytoin, and fluoxetine, an uncoupler (reserpine), substrate inhibitors (quetiapine, carbamazepine, buspirone, and tianeptine), and cytotoxic compounds (chlorpromazine and valproic acid) in HepG2 cells. Using permeabilized HepG2 cells revealed minimum effective concentrations of 66.3, 6730, 44.5, and 72.1 µM for the inhibition of complex-I-linked respiration for quetiapine, valproic acid, buspirone, and fluoxetine, respectively. Assessing complex-II-linked respiration in isolated rat liver mitochondria revealed haloperidol is an ETC inhibitor, chlorpromazine is an uncoupler in basal respiration and an ETC inhibitor under uncoupled respiration (IC50 = 135 µM), while olanzapine causes a mild dissipation of the membrane potential at 50 µM. This research elucidates some mechanisms of drug toxicity and provides some insight into their safety profile for clinical drug decisions.

Enhanced presynaptic mitochondrial energy production is required for memory formation.

Some of the prominent features of long-term memory formation include protein synthesis, gene expression, enhanced neurotransmitter release, increased excitability, and formation of new synapses. As these processes are critically dependent on mitochondrial function, we hypothesized that increased mitochondrial respiration and dynamics would play a prominent role in memory formation. To address this possibility, we measured mitochondrial oxygen consumption (OCR) in hippocampal tissue punches from trained and untrained animals. Our results show that context fear training significantly increased basal, ATP synthesis-linked, and maximal OCR in the Shaffer collateral-CA1 synaptic region, but not in the CA1 cell body layer. These changes were recapitulated in synaptosomes isolated from the hippocampi of fear-trained animals. As dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, we examined its role in the increased mitochondrial respiration observed after fear training. Drp1 inhibitors decreased the training-associated enhancement of OCR and impaired contextual fear memory, but did not alter the number of synaptosomes containing mitochondria. Taken together, our results show context fear training increases presynaptic mitochondria respiration, and that Drp-1 mediated enhanced energy production in CA1 pre-synaptic terminals is necessary for context fear memory that does not result from an increase in the number of synaptosomes containing mitochondria or an increase in mitochondrial mass within the synaptic layer.

Caudal DMN neurons innervate the spleen and release CART peptide to regulate neuroimmune function.

BACKGROUND: Inflammation is a fundamental biological response to injury and infection, which if unregulated can contribute to the pathophysiology of many diseases. The vagus nerve, which primarily originates from the dorsal motor nucleus (DMN), plays an important role in rapidly dampening inflammation by regulating splenic function. However, direct vagal innervation of the spleen, which houses the majority of immune and inflammatory cells, has not been established. As an alternative to direct innervation, an anti-inflammatory reflex pathway has been proposed which involves the vagus nerve, the sympathetic celiac ganglion, and the neurotransmitter norepinephrine. Although sympathetic regulation of inflammation has been shown, the interaction of the vagus nerve and the celiac ganglia requires a unique interaction of parasympathetic and sympathetic inputs, making this putative mechanism of brain-spleen interaction controversial. BODY: As neuropeptides can be expressed at relatively high levels in neurons, we reasoned that DMN neuropeptide immunoreactivity could be used to determine their target innervation. Employing immunohistochemistry, subdiaphragmatic vagotomy, viral tract tracing, CRISPR-mediated knock-down, and functional assays, we show that cocaine and amphetamine-regulated transcript (CART) peptide-expressing projection neurons in the caudal DMN directly innervate the spleen. In response to lipopolysaccharide (LPS) stimulation, CART acts to reduce inflammation, an effect that can be augmented by intrasplenic administration of a synthetic CART peptide. These in vivo effects could be recapitulated in cultured splenocytes, suggesting that these cells express the as yet unidentified CART receptor(s). CONCLUSION: Our results provide evidence for direct connections between the caudal DMN and spleen. In addition to acetylcholine, these neurons express the neuropeptide CART that, once released, acts to suppress inflammation by acting directly upon splenocytes.

A Carrier Female Manifesting an Unusual X-Linked Retinoschisis Phenotype Associated with the Pathogenic Variant c.266delA, p.(Tyr89LeufsTer37) in RS1, and Skewed X-Inactivation.

X-linked retinoschisis (XLRS) is the most common juvenile macular degeneration in males. Unlike most other X-linked retinal dystrophies, carrier heterozygous females are very rarely reported to show clinical features of the disease. Herein, we describe unusual retinal features in a 2-year-old female infant with family history and genetic testing consistent with XLRS.

Neuropathy target esterase activity predicts retinopathy among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism, and hair anomalies. PNPLA6 encodes Neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a clinical meta-analysis of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6 -associated clinical diagnoses unambiguously reclassified 10 variants as likely pathogenic and 36 variants as pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship and the generation of a preclinical animal model pave the way for therapeutic trials, using NTE as a biomarker.

IMI-Management and Investigation of High Myopia in Infants and Young Children.

Purpose: The purpose of this study was to evaluate the epidemiology, etiology, clinical assessment, investigation, management, and visual consequences of high myopia (≤-6 diopters [D]) in infants and young children. Findings: High myopia is rare in pre-school children with a prevalence less than 1%. The etiology of myopia in such children is different than in older children, with a high rate of secondary myopia associated with prematurity or genetic causes. The priority following the diagnosis of high myopia in childhood is to determine whether there is an associated medical diagnosis that may be of greater overall importance to the health of the child through a clinical evaluation that targets the commonest features associated with syndromic forms of myopia. Biometric evaluation (including axial length and corneal curvature) is important to distinguishing axial myopia from refractive myopia associated with abnormal development of the anterior segment. Additional investigation includes ocular imaging, electrophysiological tests, genetic testing, and involvement of pediatricians and clinical geneticists is often warranted. Following investigation, optical correction is essential, but this may be more challenging and complex than in older children. Application of myopia control interventions in this group of children requires a case-by-case approach due to the lack of evidence of efficacy and clinical heterogeneity of high myopia in young children. Conclusions: High myopia in infants and young children is a rare condition with a different pattern of etiology to that seen in older children. The clinical management of such children, in terms of investigation, optical correction, and use of myopia control treatments, is a complex and often multidisciplinary process.

Prediction of drug-induced mitochondrial dysfunction using succinate-cytochrome c reductase activity, QSAR and molecular docking.

There is increasing evidence that links mitochondrial off-target effects with organ toxicities. For this reason, predictive strategies need to be developed to identify mitochondrial dysfunction early in the drug discovery process. In this study, as a major mechanism of mitochondrial toxicity, first, the inhibitory activity of 35 compounds against succinate-cytochrome c reductase (SCR) was investigated. This in vitro study led to the generation of consistent experimental data for a diverse range of compounds, including pharmaceutical drugs and fungicides. Next, molecular docking and protein-ligand interaction fingerprinting (PLIF) analysis were used to identify significant residues and protein-ligand interactions for the Qo site of complex III and Q site of complex II. Finally, this data was used for the development of QSAR models using a regression-based approach to highlight structural and chemical features that might be responsible for SCR inhibition. The statistically validated QSAR models from this work highlighted the importance of low aqueous solubility, low ionisation, fewer 6-membered rings and shorter hydrocarbon alkane chains in the molecular structure for increased inhibition of SCR, hence mitochondrial toxicity. PLIF analysis highlighted two key residues for inhibitory activity of the Qo site of complex III: His 161 as H-bond acceptor and Pro 270 for arene interactions. Currently, there are limited structure-activity models published in the scientific literature for the prediction of mitochondrial toxicity. We believe this study helps shed light on the chemical space for the inhibition of mitochondrial electron transport chain (ETC).

Retinopathy and optic atrophy in a case of COQ2-related primary coenzyme Q10 deficiency.

PURPOSE: To describe a case of primary coenzyme Q10 deficiency in a child manifesting as early-onset renal failure, retinal dystrophy, and optic atrophy leading to progressive vision loss. METHODS: Clinical presentation and workup including visual fields, electroretinogram, and optical coherence tomography are presented. Genetic testing was performed. RESULTS: An eight-year-old female with nephropathy requiring renal transplantation subsequently developed progressive cone-rod dystrophy and optic atrophy. The patient had negative results on a targeted next-generation sequencing retinal dystrophy panel but whole-exome sequencing revealed two variants in COQ2 (likely biallelic), consistent with a diagnosis of primary coenzyme Q10 deficiency. CONCLUSIONS: Primary coenzyme Q10 deficiency is a rare disorder with variable systemic and ocular findings; there is also genetic heterogeneity. Genetic testing aids in the diagnosis of this condition, and variants in the COQ2 and PDSS1 genes appear to have the strongest association with ocular manifestations. Oral supplementation of coenzyme Q10 may slow progression of disease. This case highlights the utility of whole-exome sequencing in the diagnosis of a rare syndromic form of ocular disease and reports a novel phenotypic association for this condition.

Xenotopic expression of alternative oxidase (AOX) to study mechanisms of mitochondrial disease.

The mitochondrial respiratory chain or electron transport chain (ETC) facilitates redox reactions which ultimately lead to the reduction of oxygen to water (respiration). Energy released by this process is used to establish a proton electrochemical gradient which drives ATP formation (oxidative phosphorylation, OXPHOS). It also plays an important role in vital processes beyond ATP formation and cellular metabolism, such as heat production, redox and ion homeostasis. Dysfunction of the ETC can thus impair cellular and organismal viability and is thought to be the underlying cause of a heterogeneous group of so-called mitochondrial diseases. Plants, yeasts, and many lower organisms, but not insects and vertebrates, possess an enzymatic mechanism that confers resistance to respiratory stress conditions, i.e., the alternative oxidase (AOX). Even in cells that naturally lack AOX, it is autonomously imported into the mitochondrial compartment upon xenotopic expression, where it refolds and becomes catalytically engaged when the cytochrome segment of the ETC is blocked. AOX was therefore proposed as a tool to study disease etiologies. To this end, AOX has been xenotopically expressed in mammalian cells and disease models of the fruit fly and mouse. Surprisingly, AOX showed remarkable rescue effects in some cases, whilst in others it had no effect or even exacerbated a condition. Here we summarize what has been learnt from the use of AOX in various disease models and discuss issues which still need to be addressed in order to understand the role of the ETC in health and disease.

Multidisciplinary team directed analysis of whole genome sequencing reveals pathogenic non-coding variants in molecularly undiagnosed inherited retinal dystrophies.

The purpose of this paper is to identify likely pathogenic non-coding variants in inherited retinal dystrophy (IRD) genes, using genome sequencing (GS). Patients with IRD were recruited to the study and underwent comprehensive ophthalmological evaluation and GS. The results of GS were investigated through virtual gene panel analysis, and plausible pathogenic variants and clinical phenotype evaluated by the multidisciplinary team (MDT) discussion. For unsolved patients in whom a specific gene was suspected to harbor a missed pathogenic variant, targeted re-analysis of non-coding regions was performed on GS data. Candidate variants were functionally tested by messenger RNA analysis, minigene or luciferase reporter assays. Previously unreported, likely pathogenic, non-coding variants in 7 genes (PRPF31, NDP, IFT140, CRB1, USH2A, BBS10 and GUCY2D), were identified in 11 patients. These were shown to lead to mis-splicing (PRPF31, IFT140, CRB1 and USH2A) or altered transcription levels (BBS10 and GUCY2D). MDT-led, phenotype-driven, non-coding variant re-analysis of GS is effective in identifying the missing causative alleles.

Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells.

Mitosomes are highly reduced forms of mitochondria which have lost two of the 'defining' features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite's life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells.