Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies

NMNAT1 Mutations Cause Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss. Two-thirds of LCA cases are caused by mutations in 17 known disease genes (RetNet Retinal Information Network). Using exome sequencing, we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 as likely disease-causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in their kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide $(NAD^+)$ biosynthesis. Functional studies showed the p.Val9Met mutation decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated LCA families identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease and indicate that NMNAT1 mutations cause LCA

Inverting the model of genomics data sharing with the NHGRI Genomic Data Science Analysis, Visualization, and Informatics Lab-space

The NHGRI Genomic Data Science Analysis, Visualization, and Informatics Lab-space (AnVIL; https://anvilproject.org) was developed to address a widespread community need for a unified computing environment for genomics data storage, management, and analysis. In this perspective, we present AnVIL, describe its ecosystem and interoperability with other platforms, and highlight how this platform and associated initiatives contribute to improved genomic data sharing efforts. The AnVIL is a federated cloud platform designed to manage and store genomics and related data, enable population-scale analysis, and facilitate collaboration through the sharing of data, code, and analysis results. By inverting the traditional model of data sharing, the AnVIL eliminates the need for data movement while also adding security measures for active threat detection and monitoring and provides scalable, shared computing resources for any researcher. We describe the core data management and analysis components of the AnVIL, which currently consists of Terra, Gen3, Galaxy, RStudio/Bioconductor, Dockstore, and Jupyter, and describe several flagship genomics datasets available within the AnVIL. We continue to extend and innovate the AnVIL ecosystem by implementing new capabilities, including mechanisms for interoperability and responsible data sharing, while streamlining access management. The AnVIL opens many new opportunities for analysis, collaboration, and data sharing that are needed to drive research and to make discoveries through the joint analysis of hundreds of thousands to millions of genomes along with associated clinical and molecular data types

A mitochondrial DNA variant of the COX-1 subunit of C. elegnas’ complex IV significantly alters mitochondrial energy metabolism of geographically divergent wild isolates.

Mitochondrial DNA (mtDNA) sequence variation is increasingly recognized to influence the penetrance of complex diseases and climatic adaptation in mammals, although little is known about its influence on invertebrate species’ adaptation to unique geographic niches. We investigated whether natural variation in mtDNA-encoded respiratory chain subunits alters the inherent mitochondrial energy capacity of wild C. elegans isolates to match local environmental energy demands. We found that relative to the classic N2 Bristol (England) wild-type strain, CB4856 wild isolates from a warmer and more equatorial native climate (Hawaii) had a unique A12S amino acid substitution in the N-terminal string of the COX-1 core catalytic subunit of complex IV. In silico modeling predicted that the A12S substitution increased MAPK-1 kinase binding affinity, which would increase COX-1 subunit phosphorylation in CB4856. Indeed, the CB4856 worms had significantly increased mitochondrial complex IV enzyme activity relative to N2 Bristol. While CB4856 had equivalent amounts of complex IV, mitochondria, and respiratory chain supercomplexes, its integrated mitochondrial respiratory capacity and membrane potential was significantly reduced when grown at 20°C. CB4856 also had significantly reduced lifespan and increased oxidative stress when grown at 20°C. Interestingly, the mitochondrial membrane potential of CB4856 was significantly increased relative to that of N2 Bristol when grown at its native temperature of 25°C degrees. To determine the effects of only the COX-1 sequence variant without possible contribution from the CB4856 nuclear genome background, we generated a transmitochondrial cybrid worm strain, chpIR(M,N2\u3eCB4856), containing the CB4856 mtDNA in the N2 Bristol wild-type nuclear background. This strain also had increased CIV activity, which supports that the A12S mtDNA variant is causative of the increased CIV activity of CB4856 relative to N2 Bristol. Differences in comparative functional analyses among the three strains further suggest their nuclear background also modulates mitochondrial function. The cybrid C. elegans strain also had reduced lifespan relative to CB4856, highlighting the importance of precise co-evolution of mitochondrial and nuclear genomes. Overall, these data show that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA-encoded sequence alterations that modulate critical aspects of mitochondrial energy metabolism

Inhibiting cytosolic translation and autophagy improves health in mitochondrial disease

Corner of Marguerite Street and A Z Berman Drive, Lentegeur, Mitchell's Plain, Cape Town. In an area developed in the 1970s as a coloured area, to provide housing to those removed from their homes by the Group Areas Act. Many families from District Six relocated here when their community was razed.distant view, southwest elevation of mosque under construction, with school at right, 198

Probucol ameliorates renal and metabolic sequelae of primary CoQ deficiency in Pdss2 mutant mice.

Therapy of mitochondrial respiratory chain diseases is complicated by limited understanding of cellular mechanisms that cause the widely variable clinical findings. Here, we show that focal segmental glomerulopathy-like kidney disease in Pdss2 mutant animals with primary coenzyme Q (CoQ) deficiency is significantly ameliorated by oral treatment with probucol (1% w/w). Preventative effects in missense mutant mice are similar whether fed probucol from weaning or for 3 weeks prior to typical nephritis onset. Furthermore, treating symptomatic animals for 2 weeks with probucol significantly reduces albuminuria. Probucol has a more pronounced health benefit than high-dose CoQ(10) supplementation and uniquely restores CoQ(9) content in mutant kidney. Probucol substantially mitigates transcriptional alterations across many intermediary metabolic domains, including peroxisome proliferator-activated receptor (PPAR) pathway signaling. Probucol's beneficial effects on the renal and metabolic manifestations of Pdss2 disease occur despite modest induction of oxidant stress and appear independent of its hypolipidemic effects. Rather, decreased CoQ(9) content and altered PPAR pathway signaling appear, respectively, to orchestrate the glomerular and global metabolic consequences of primary CoQ deficiency, which are both preventable and treatable with oral probucol therapy

Mutations in FBXL4, encoding a mitochondrial protein, cause early-onset mitochondrial encephalomyopathy

Whole-exome sequencing and autozygosity mapping studies, independently performed in subjects with defective combined mitochondrial OXPHOS-enzyme deficiencies, identified a total of nine disease-segregating FBXL4 mutations in seven unrelated mitochondrial disease families, composed of six singletons and three siblings. All subjects manifested early-onset lactic acidemia, hypotonia, and developmental delay caused by severe encephalomyopathy consistently associated with progressive cerebral atrophy and variable involvement of the white matter, deep gray nuclei, and brainstem structures. A wide range of other multisystem features were variably seen, including dysmorphism, skeletal abnormalities, poor growth, gastrointestinal dysmotility, renal tubular acidosis, seizures, and episodic metabolic failure. Mitochondrial respiratory chain deficiency was present in muscle or fibroblasts of all tested individuals, together with markedly reduced oxygen consumption rate and hyperfragmentation of the mitochondrial network in cultured cells. In muscle and fibroblasts from several subjects, substantially decreased mtDNA content was observed. FBXL4 is a member of the F-box family of proteins, some of which are involved in phosphorylation-dependent ubiquitination and/or G protein receptor coupling. We also demonstrate that FBXL4 is targeted to mitochondria and localizes in the intermembrane space, where it participates in an approximately 400 kDa protein complex. These data strongly support a role for FBXL4 in controlling bioenergetic homeostasis and mtDNA maintenance. FBXL4 mutations are a recurrent cause of mitochondrial encephalomyopathy onset in early infancy