dldhcri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

Measuring Sleep and Activity Patterns in Adult Zebrafish.

Sleep is an essential behavior that is still poorly understood. Sleep abnormalities accompany a variety of psychiatric and neurological disorders, and sleep can serve as a modifiable behavior in the treatment of these disorders. Zebrafish (Danio rerio) has proven to be a powerful model organism to study sleep and the interplay between sleep and these disorders due to the high conservation of the neuro-modulatory mechanisms that control sleep and wake states between zebrafish and humans. The zebrafish is a diurnal vertebrate with a relatively simple nervous system compared to mammalian models, exhibiting conservation of sleep ontogeny across different life stages. Zebrafish larvae are an established high-throughput model to assess sleep phenotypes and the biological underpinnings of sleep disturbances. To date, sleep measurement in juvenile and adult zebrafish has not been performed in a standardized and reproducible manner because of the relatively low-throughput nature in relation to their larval counterparts. This has left a gap in understanding sleep across later stages of life that are relevant to many psychiatric and neurodegenerative disorders. Several research groups have used homemade systems to address this gap. Here, we report employing commercially available equipment to track activity and sleep/wake patterns in juvenile and adult zebrafish. The equipment allows researchers to perform automated behavior assays in an isolated environment with light/dark and temperature control for multiple days. We first explain the experimental procedure to track the sleep and activity of adult zebrafish and then validate the protocol by measuring the effects of melatonin and DMSO administration. Key features • Allows an isolated and controllable environment to carry out activity and sleep assays in juvenile and adult zebrafish. • Measures activity of zebrafish in life stages later than early development, which requires feeding animals during the assay. • Requires use of a commercially available equipment system and six tanks. • The activity of zebrafish can be tracked for five days including an acclimation step.

High dose dietary vitamin D allocates surplus calories to muscle and growth instead of fat via modulation of myostatin and leptin signaling.

Obesity occurs because the body stores surplus calories as fat rather than as muscle. Fat secretes a hormone, leptin, that modulates energy balance at the brain. Changes in fat mass are mirrored by changes in serum leptin. Elevated leptin prompts the brain to decrease appetite and increase energy expenditure. In obesity, however, impaired leptin sensitivity mutes these leptin-mediated changes. We have limited understanding of what controls leptin production by fat or leptin sensitivity in the brain. Muscle produces a hormone, myostatin, that plays a role in muscle analogous to the one that leptin plays in fat. Absent myostatin leads to increased muscle mass and strength. As with leptin, we also do not know what controls myostatin production or sensitivity. Although fat mass and muscle mass are closely linked, the interplay between leptin and myostatin remains obscure. Here we describe an interplay linked thru vitamin D. Conventionally, it is thought that vitamin D improves strength via trophic effects at the muscle. However, we find here that high dose dietary vitamin D allocates excess calories to muscle and linear growth instead of storage as fat. Vitamin D mediates this allocation by decreasing myostatin production and increasing leptin production and sensitivity. That is, high dose vitamin D improves integration of organismal energy balance. Obesity, aging and other chronic inflammatory diseases are associated with increased fat mass and decreased muscle mass and function (e.g. sarcopenia). Our work provides a physiologic framework for how high-dose vitamin D would increase allocation of calories to muscle instead of fat in these pathologies. Additionally, our work reveals a novel link between the myostatin and leptin signaling whereby myostatin conveys energy needs to modulate leptin effects on calorie allocation. This result provides evidence to update the conventional model of energy stores sensing to a new model of energy balance sensing. In our proposed model, integration of leptin and myostatin signaling allows control of body composition independent of weight. Furthermore, our work reveals how physiologic seasonal variation in vitamin D may be important in controlling season-specific metabolism and calorie allocation to fat in winter and muscle and growth in summer.

Biallelic variants in GTF3C5, a regulator of RNA polymerase III-mediated transcription, cause a multisystem developmental disorder.

General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery.

Novel Development of Magnetic Resonance Imaging to Quantify the Structural Anatomic Growth of Diverse Organs in Adult and Mutant Zebrafish.

Zebrafish (Danio rerio) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1-/-, fbxl4-/-, and opa1+/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1-/- and opa1+/-, and marginally significant decreased heart and spinal cord volumes in surf1-/- mutants. This is interesting as we know neurological involvement can be seen in SURF1-/- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1+/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1-/--related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Genomic profiling informs diagnoses and treatment in vascular anomalies.

Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype-phenotype associations and guide medical therapy in individuals with vascular anomalies.

Pleiotropic effects of a high confidence Autism Spectrum Disorder gene, arid1b, on zebrafish sleep.

Sleep fulfills critical functions in neurodevelopment, such as promoting synaptic plasticity, neuronal wiring, and brain connectivity which are critical phenomena in Autism Spectrum Disorder (ASD) pathophysiology. Sleep disturbance, specifically insomnia, accompanies ASD and is associated with more severe core symptoms (e.g., social impairment). It is possible that focusing on identifying effective ways to treat sleep problems can help alleviate other ASD-related symptoms. A body of evidence indicates shared mechanisms and neurobiological substrates between sleep and ASD and investigation of these may inform therapeutic effects of improving sleep at both behavioral and molecular levels. In this study, we tested if sleep and social behavior were different in a zebrafish model with the arid1b gene mutated compared to controls. This gene was selected for study as expert curations conducted for the Simons Foundation for Autism Research Institute (SFARI) Gene database define it is as a 'high confidence' ASD gene (i.e., clearly implicated) encoding a chromatin remodeling protein. Homozygous arid1b mutants displayed increased arousability and light sleep compared to their heterozygous and wild type counterparts, based on testing a mechano-acoustic stimulus presenting different vibration frequencies of increasing intensity to detect sleep depth. In addition, decreased social preference was observed in arid1b heterozygous and homozygous mutant zebrafish. The behavioral phenotypes reported in our study are in line with findings from mouse models and human studies and demonstrate the utility of zebrafish as a vertebrate model system with high throughput phenotyping in the investigation of changes in sleep in models relevant to ASD. Furthermore, we demonstrate the importance of including assessments of arousal threshold when studying sleep using in vivo models.

Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2­dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

N-acetylcysteine and cysteamine bitartrate prevent azide-induced neuromuscular decompensation by restoring glutathione balance in two novel surf1-/- zebrafish deletion models of Leigh syndrome.

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1-/- larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1-/- larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1-/- animals. Overall, two novel surf1-/- zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy.

Dichloroacetate improves mitochondrial function, physiology, and morphology in FBXL4 disease models.

Pathogenic variants in the human F-box and leucine-rich repeat protein 4 (FBXL4) gene result in an autosomal recessive, multisystemic, mitochondrial disorder involving variable mitochondrial depletion and respiratory chain complex deficiencies with lactic acidemia. As no FDA-approved effective therapies for this disease exist, we sought to characterize translational C. elegans and zebrafish animal models, as well as human fibroblasts, to study FBXL4-/- disease mechanisms and identify preclinical therapeutic leads. Developmental delay, impaired fecundity and neurologic and/or muscular activity, mitochondrial dysfunction, and altered lactate metabolism were identified in fbxl-1(ok3741) C. elegans. Detailed studies of a PDHc activator, dichloroacetate (DCA), in fbxl-1(ok3741) C. elegans demonstrated its beneficial effects on fecundity, neuromotor activity, and mitochondrial function. Validation studies were performed in fbxl4sa12470 zebrafish larvae and in FBXL4-/- human fibroblasts; they showed DCA efficacy in preventing brain death, impairment of neurologic and/or muscular function, mitochondrial biochemical dysfunction, and stress-induced morphologic and ultrastructural mitochondrial defects. These data demonstrate that fbxl-1(ok3741) C. elegans and fbxl4sa12470 zebrafish provide robust translational models to study mechanisms and identify preclinical therapeutic candidates for FBXL4-/- disease. Furthermore, DCA is a lead therapeutic candidate with therapeutic benefit on diverse aspects of survival, neurologic and/or muscular function, and mitochondrial physiology that warrants rigorous clinical trial study in humans with FBXL4-/- disease.

The FusX TALE Base Editor (FusXTBE) for Rapid Mitochondrial DNA Programming of Human Cells In Vitro and Zebrafish Disease Models In Vivo.

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo. Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

Pathogenic variants in CDH11 impair cell adhesion and cause Teebi hypertelorism syndrome.

Teebi hypertelorism syndrome (THS; OMIM 145420) is a rare craniofacial disorder characterized by hypertelorism, prominent forehead, short nose with broad or depressed nasal root. Some cases of THS have been attributed to SPECC1L variants. Homozygous variants in CDH11 truncating the transmembrane and intracellular domains have been implicated in Elsahy-Waters syndrome (EWS; OMIM 211380) with hypertelorism. We report THS due to CDH11 heterozygous missense variants on 19 subjects from 9 families. All affected residues in the extracellular region of Cadherin-11 (CHD11) are highly conserved across vertebrate species and classical cadherins. Six of the variants that cluster around the EC2-EC3 and EC3-EC4 linker regions are predicted to affect Ca2+ binding that is required for cadherin stability. Two of the additional variants [c.164G > C, p.(Trp55Ser) and c.418G > A, p.(Glu140Lys)] are also notable as they are predicted to directly affect trans-homodimer formation. Immunohistochemical study demonstrates that CDH11 is strongly expressed in human facial mesenchyme. Using multiple functional assays, we show that five variants from the EC1, EC2-EC3 linker, and EC3 regions significantly reduced the cell-substrate trans adhesion activity and one variant from EC3-EC4 linker results in changes in cell morphology, focal adhesion, and migration, suggesting dominant negative effect. Characteristic features in this cohort included depressed nasal root, cardiac and umbilical defects. These features distinguished this phenotype from that seen in SPECC1L-related hypertelorism syndrome and CDH11-related EWS. Our results demonstrate heterozygous variants in CDH11, which decrease cell-cell adhesion and increase cell migratory behavior, cause a form of THS, as termed CDH11-related THS.

Combinatorial glucose, nicotinic acid and N-acetylcysteine therapy has synergistic effect in preclinical C. elegans and zebrafish models of mitochondrial complex I disease.

Mitochondrial respiratory chain disorders are empirically managed with variable antioxidant, cofactor and vitamin 'cocktails'. However, clinical trial validated and approved compounds, or doses, do not exist for any single or combinatorial mitochondrial disease therapy. Here, we sought to pre-clinically evaluate whether rationally designed mitochondrial medicine combinatorial regimens might synergistically improve survival, health and physiology in translational animal models of respiratory chain complex I disease. Having previously demonstrated that gas-1(fc21) complex I subunit ndufs2-/-C. elegans have short lifespan that can be significantly rescued with 17 different metabolic modifiers, signaling modifiers or antioxidants, here we evaluated 11 random combinations of these three treatment classes on gas-1(fc21) lifespan. Synergistic rescue occurred only with glucose, nicotinic acid and N-acetylcysteine (Glu + NA + NAC), yielding improved mitochondrial membrane potential that reflects integrated respiratory chain function, without exacerbating oxidative stress, and while reducing mitochondrial stress (UPRmt) and improving intermediary metabolic disruptions at the levels of the transcriptome, steady-state metabolites and intermediary metabolic flux. Equimolar Glu + NA + NAC dosing in a zebrafish vertebrate model of rotenone-based complex I inhibition synergistically rescued larval activity, brain death, lactate, ATP and glutathione levels. Overall, these data provide objective preclinical evidence in two evolutionary-divergent animal models of mitochondrial complex I disease to demonstrate that combinatorial Glu + NA + NAC therapy significantly improved animal resiliency, even in the face of stressors that cause severe metabolic deficiency, thereby preventing acute neurologic and biochemical decompensation. Clinical trials are warranted to evaluate the efficacy of this lead combinatorial therapy regimen to improve resiliency and health outcomes in human subjects with mitochondrial disease.

Primary Open Versus Closed Implantation Strategy for Totally Implantable Venous Access Ports: The Multicentre Randomized Controlled PORTAS-3 Trial (DRKS 00004900).

OBJECTIVES: PORTAS-3 was designed to compare the frequency of pneumothorax or haemothorax in a primary open versus closed strategy for port implantation. BACKGROUND DATA: The implantation strategy for totally implantable venous access ports with the optimal benefit/risk ratio remains unclear. METHODS: PORTAS-3 was a multicentre, randomized, controlled, parallel-group superiority trial. Adult patients with oncological disease scheduled for elective port implantation were randomized to a primary open or closed strategy. Primary endpoint was the rate of pneumothorax or haemothorax. Assuming a difference of 2.5% between the 2 groups, a sample size of 1154 patients was needed to prove superiority of the open group. A logistic regression model after the intention-to-treat principle was applied for analysis of the primary endpoint. RESULTS: Between November 9, 2014 and September 5, 2016, 1205 patients were randomized. Of these, 1159 (open n = 583; closed n = 576) were finally analyzed. The rate of pneumothorax or haemothorax was significantly reduced with the open strategy [odds ratio 0.27, 95% confidence interval (CI) 0.09-0.88; P = 0.029]. Operation time was shorter for the closed strategy. Primary success rates, tolerability, morbidity, dose rate of radiation, and 30-day mortality did not differ significantly between the groups. CONCLUSION: A primary open strategy by cut-down of the cephalic vein, if necessary enhanced by a modified Seldinger technique, reduces the frequency of pneumothorax or haemothorax after central venous port implantation significantly compared with a closed strategy by primary puncture of the subclavian vein without routine sonographic guidance. Therefore, open surgical cut-down should be the reference standard for port implantation in comparable cohorts. TRIAL REGISTRATION: German Clinical Trials Register DRKS 00004900.

Missense Mutations in NKAP Cause a Disorder of Transcriptional Regulation Characterized by Marfanoid Habitus and Cognitive Impairment.

NKAP is a ubiquitously expressed nucleoplasmic protein that is currently known as a transcriptional regulatory molecule via its interaction with HDAC3 and spliceosomal proteins. Here, we report a disorder of transcriptional regulation due to missense mutations in the X chromosome gene, NKAP. These mutations are clustered in the C-terminal region of NKAP where NKAP interacts with HDAC3 and post-catalytic spliceosomal complex proteins. Consistent with a role for the C-terminal region of NKAP in embryogenesis, nkap mutant zebrafish with a C-terminally truncated NKAP demonstrate severe developmental defects. The clinical features of affected individuals are highly conserved and include developmental delay, hypotonia, joint contractures, behavioral abnormalities, Marfanoid habitus, and scoliosis. In affected cases, transcriptome analysis revealed the presence of a unique transcriptome signature, which is characterized by the downregulation of long genes with higher exon numbers. These observations indicate the critical role of NKAP in transcriptional regulation and demonstrate that perturbations of the C-terminal region lead to developmental defects in both humans and zebrafish.

ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitor.

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations1. Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation2. Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient's lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.

Pre-clinical evaluation of cysteamine bitartrate as a therapeutic agent for mitochondrial respiratory chain disease.

Cysteamine bitartrate is a US Food and Drug Administration-approved therapy for nephropathic cystinosis also postulated to enhance glutathione biosynthesis. We hypothesized this antioxidant effect may reduce oxidative stress in primary mitochondrial respiratory chain (RC) disease, improving cellular viability and organismal health. Here, we systematically evaluated the therapeutic potential of cysteamine bitartrate in RC disease models spanning three evolutionarily distinct species. These pre-clinical studies demonstrated the narrow therapeutic window of cysteamine bitartrate, with toxicity at millimolar levels directly correlating with marked induction of hydrogen peroxide production. Micromolar range cysteamine bitartrate treatment in Caenorhabditis elegans gas-1(fc21) RC complex I (NDUFS2-/-) disease invertebrate worms significantly improved mitochondrial membrane potential and oxidative stress, with corresponding modest improvement in fecundity but not lifespan. At 10 to 100 µm concentrations, cysteamine bitartrate improved multiple RC complex disease FBXL4 human fibroblast survival, and protected both complex I (rotenone) and complex IV (azide) Danio rerio vertebrate zebrafish disease models from brain death. Mechanistic profiling of cysteamine bitartrate effects showed it increases aspartate levels and flux, without increasing total glutathione levels. Transcriptional normalization of broadly dysregulated intermediary metabolic, glutathione, cell defense, DNA, and immune pathways was greater in RC disease human cells than in C. elegans, with similar rescue in both models of downregulated ribosomal and proteasomal pathway expression. Overall, these data suggest cysteamine bitartrate may hold therapeutic potential in RC disease, although not through obvious modulation of total glutathione levels. Careful consideration is required to determine safe and effective cysteamine bitartrate concentrations to further evaluate in clinical trials of human subjects with primary mitochondrial RC disease.

Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly.

Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knock-in of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.

N-acetylcysteine and vitamin E rescue animal longevity and cellular oxidative stress in pre-clinical models of mitochondrial complex I disease.

Oxidative stress is a known contributing factor in mitochondrial respiratory chain (RC) disease pathogenesis. Yet, no efficient means exists to objectively evaluate the comparative therapeutic efficacy or toxicity of different antioxidant compounds empirically used in human RC disease. We postulated that pre-clinical comparative analysis of diverse antioxidant drugs having suggested utility in primary RC disease using animal and cellular models of RC dysfunction may improve understanding of their integrated effects and physiologic mechanisms, and enable prioritization of lead antioxidant molecules to pursue in human clinical trials. Here, lifespan effects of N-acetylcysteine (NAC), vitamin E, vitamin C, coenzyme Q10 (CoQ10), mitochondrial-targeted CoQ10 (MS010), lipoate, and orotate were evaluated as the primary outcome in a well-established, short-lived C. elegans gas-1(fc21) animal model of RC complex I disease. Healthspan effects were interrogated to assess potential reversal of their globally disrupted in vivo mitochondrial physiology, transcriptome profiles, and intermediary metabolic flux. NAC or vitamin E fully rescued, and coenzyme Q, lipoic acid, orotic acid, and vitamin C partially rescued gas-1(fc21) lifespan toward that of wild-type N2 Bristol worms. MS010 and CoQ10 largely reversed biochemical pathway expression changes in gas-1(fc21) worms. While nearly all drugs normalized the upregulated expression of the "cellular antioxidant pathway", they failed to rescue the mutant worms' increased in vivo mitochondrial oxidant burden. NAC and vitamin E therapeutic efficacy were validated in human fibroblast and/or zebrafish complex I disease models. Remarkably, rotenone-induced zebrafish brain death was preventable partially with NAC and fully with vitamin E. Overall, these pre-clinical model animal data demonstrate that several classical antioxidant drugs do yield significant benefit on viability and survival in primary mitochondrial disease, where their major therapeutic benefit appears to result from targeting global cellular, rather than intramitochondria-specific, oxidative stress. Clinical trials are needed to evaluate whether the two antioxidants, NAC and vitamin E, that show greatest efficacy in translational model animals significantly improve the survival, function, and feeling of human subjects with primary mitochondrial RC disease.

Pharmacologic modeling of primary mitochondrial respiratory chain dysfunction in zebrafish.

Mitochondrial respiratory chain (RC) disease is a heterogeneous and highly morbid group of energy deficiency disorders for which no proven effective therapies exist. Robust vertebrate animal models of primary RC dysfunction are needed to explore the effects of variation in RC disease subtypes, tissue-specific manifestations, and major pathogenic factors contributing to each disorder, as well as their pre-clinical response to therapeutic candidates. We have developed a series of zebrafish (Danio rerio) models that inhibit, to variable degrees, distinct aspects of RC function, and enable quantification of animal development, survival, behaviors, and organ-level treatment effects as well as effects on mitochondrial biochemistry and physiology. Here, we characterize four pharmacologic inhibitor models of mitochondrial RC dysfunction in early larval zebrafish, including rotenone (complex I inhibitor), azide (complex IV inhibitor), oligomycin (complex V inhibitor), and chloramphenicol (mitochondrial translation inhibitor that leads to multiple RC complex dysfunction). A range of concentrations and exposure times of each RC inhibitor were systematically evaluated on early larval development, animal survival, integrated behaviors (touch and startle responses), organ physiology (brain death, neurologic tone, heart rate), and fluorescence-based analyses of mitochondrial physiology in zebrafish skeletal muscle. Pharmacologic RC inhibitor effects were validated by spectrophotometric analysis of Complex I, II and IV enzyme activities, or relative quantitation of ATP levels in larvae. Outcomes were prioritized that utilize in vivo animal imaging and quantitative behavioral assessments, as may optimally inform the translational potential of pre-clinical drug screens for future clinical study in human mitochondrial disease subjects. The RC complex inhibitors each delayed early embryo development, with short-term exposures of these three agents or chloramphenicol from 5 to 7 days post fertilization also causing reduced larval survival and organ-specific defects ranging from brain death, behavioral and neurologic alterations, reduced mitochondrial membrane potential in skeletal muscle (rotenone), and/or cardiac edema with visible blood pooling (oligomycin). Remarkably, we demonstrate that treating animals with probucol, a nutrient-sensing signaling network modulating drug that has been shown to yield therapeutic effects in a range of other RC disease cellular and animal models, both prevented acute rotenone-induced brain death in zebrafish larvae, and significantly rescued early embryo developmental delay from either rotenone or oligomycin exposure. Overall, these zebrafish pharmacologic RC function inhibition models offer a unique opportunity to gain novel insights into diverse developmental, survival, organ-level, and behavioral defects of varying severity, as well as their individual response to candidate therapies, in a highly tractable and cost-effective vertebrate animal model system.