1,086 research outputs found

    Insights into molecular mechanisms of disease in Neurodegeneration with Brain Iron Accumulation; unifying theories.

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    Neurodegeneration with brain iron accumulation (NBIA) is a group of disorders characterised by dystonia, parkinsonism and spasticity. Iron accumulates in the basal ganglia and may be accompanied by Lewy bodies, axonal swellings and hyperphosphorylated tau depending on NBIA subtype. Mutations in 10 genes have been associated with NBIA that include Ceruloplasmin (Cp) and Ferritin Light Chain (FTL), both directly involved in iron homeostasis, as well as Pantothenate Kinase 2 (PANK2), Phospholipase A2 group 6 (PLA2G6), Fatty acid hydroxylase 2 (FA2H), Coenzyme A synthase (COASY), C19orf12, WDR45 and DCAF17 (C2orf37). These genes are involved in seemingly unrelated cellular pathways, such as lipid metabolism, Coenzyme A synthesis and autophagy. A greater understanding of the cellular pathways that link these genes and the disease mechanisms leading to iron dyshomeostasis is needed. Additionally, the major overlap seen between NBIA and more common neurodegenerative diseases may highlight conserved disease processes. In this review, we will discuss clinical and pathological findings for each NBIA-related gene, discuss proposed disease mechanisms such as mitochondrial health, oxidative damage, autophagy/mitophagy and iron homeostasis and speculate potential overlap between NBIA subtypes

    Pantothenate Kinase 1 Is Required to Support the Metabolic Transition from the Fed to the Fasted State

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    Coenzyme A (CoA) biosynthesis is regulated by the pantothenate kinases (PanK), of which there are four active isoforms. The PanK1 isoform is selectively expressed in liver and accounted for 40% of the total PanK activity in this organ. CoA synthesis was limited using a Pank1−/− knockout mouse model to determine whether the regulation of CoA levels was critical to liver function. The elimination of PanK1 reduced hepatic CoA levels, and fasting triggered a substantial increase in total hepatic CoA in both Pank1−/− and wild-type mice. The increase in hepatic CoA during fasting was blunted in the Pank1−/− mouse, and resulted in reduced fatty acid oxidation as evidenced by abnormally high accumulation of long-chain acyl-CoAs, acyl-carnitines, and triglycerides in the form of lipid droplets. The Pank1−/− mice became hypoglycemic during a fast due to impaired gluconeogenesis, although ketogenesis was normal. These data illustrate the importance of PanK1 and elevated liver CoA levels during fasting to support the metabolic transition from glucose utilization and fatty acid synthesis to gluconeogenesis and fatty acid oxidation. The findings also suggest that PanK1 may be a suitable target for therapeutic intervention in metabolic disorders that feature hyperglycemia and hypertriglyceridemia

    Determining the Role of Nudt7 in the Regulation of Cellular CoA Levels and Metabolism

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    Coenzyme A (CoA) is an essential cofactor required for hundreds of metabolic processes. Because it is such a critical cofactor, CoA levels are tightly regulated. In the fasted state and in diabetic mice, the concentration of CoA increases dramatically in the liver. This phenotype is associated with constitutively low CoA degradation, a process that is emerging as a potentially important mechanism for CoA regulation. Nudt7 and Nudt19 are two mammalian peroxisomal enzymes with CoA-degrading activity, which are highly expressed in the liver and kidney, respectively. Limited information is available on the biochemistry of Nudt7 and Nudt19; the structural basis for their distinct features and the extent to which Nudt7 contributes to maintaining homeostatic CoA levels in vivo are currently unknown. We used a combination of techniques including mutagenesis, molecular modeling, and enzymatic assays on purified proteins, plus metabolomics and measurement of fatty acid oxidation in whole tissue homogenates and intact hepatocytes to: 1) characterize the biochemical, structural, and regulatory properties of Nudt7 and Nudt19 and 2) determine the effects that manipulations of Nudt7 expression have on CoA levels and lipid metabolism in mouse liver. This research establishes the importance of Nudt7-dependent CoA degradation in the regulation of select acyl-CoA species and the output of peroxisomal metabolic pathways such as bile acid synthesis and peroxisomal fatty acid oxidation

    4 '-Phosphopantetheine corrects CoA, iron, and dopamine metabolic defects in mammalian models of PKAN

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    Pantothenate kinase-associated neurodegeneration (PKAN) is an inborn error of CoA metabolism causing dystonia, parkinsonism, and brain iron accumulation. Lack of a good mammalian model has impeded studies of pathogenesis and development of rational therapeutics. We took a new approach to investigating an existing mouse mutant of Pank2 and found that isolating the disease-vulnerable brain revealed regional perturbations in CoA metabolism, iron homeostasis, and dopamine metabolism and functional defects in complex I and pyruvate dehydrogenase. Feeding mice a CoA pathway intermediate, 4 '-phosphopantetheine, normalized levels of the CoA-, iron-, and dopamine-related biomarkers as well as activities of mitochondrial enzymes. Human cell changes also were recovered by 4 '-phosphopantetheine. We can mechanistically link a defect in CoA metabolism to these secondary effects via the activation of mitochondrial acyl carrier protein, which is essential to oxidative phosphorylation, iron-sulfur cluster biogenesis, and mitochondrial fatty acid synthesis. We demonstrate the fidelity of our model in recapitulating features of the human disease. Moreover, we identify pharmacodynamic biomarkers, provide insights into disease pathogenesis, and offer evidence for 4 '-phosphopantetheine as a candidate therapeutic for PKAN

    Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease

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    Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development

    Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target

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    The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells

    What is the role of caspase-2 mediated lipoapoptosis in the pathogenesis of the metabolic syndrome-associated liver disease, nonalcoholic fatty liver disease (NAFLD)?

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    Nonalcoholic fatty liver disease can be considered the hepatic manifestation of obesity and the metabolic syndrome. It is the number one cause of chronic liver disease in the Western world. Lipotoxicity in the liver induces epithelial lesion that triggers a wound healing response. In susceptible subjects the wound healing response is ineffective in repairing and regenerating the injured liver, leading to scarring, fibrogenesis and eventually hepatic cirrhosis. Though important advances in the knowledge of the pathogenesis of NAFLD have occurred since its firsts descriptions in 1980, gaps in knowledge still precludes Hepatologists to find an effective treatment for this pandemic. We first extensively characterized and compared two dietary mouse models of NAFLD, methionine-choline deficient (MCD) and Western diets. We found that MCD diet induces severe disease with significant fibrosis, whereas Western diet induces mild disease, but associates with obesity, insulin resistance and the metabolic syndrome. Afterwards, we demonstrated a pivotal role of caspase-2 in the development of the metabolic syndrome, NAFLD, progression to severe liver disease and hepatic fibrogenesis. Caspase-2 was up-regulated in human NAFLD and in in several different mouse models of NAFLD, correlating with the degree of fibrosis. Also, caspase-2 deficient mice were protected from the metabolic syndrome and liver injury/fibrosis in both MCD and Western diet mouse models. Finally, we found that in different mouse models of NAFLD, hepatic free coenzyme A content is decreased, which could potentiate caspase-2 activation. We conducted a preclinical trial in mice submitted to MCD diet, treating them with coenzyme A precursors. This approach failed to correct hepatic free coenzyme A levels and had no impact in liver histology or caspase-2 expression/activation. Our work places caspase-2 as a potential therapeutic target for obesity-associated diseases, such as type 2 diabetes mellitus and NAFLD.Fundação Calouste GulbenkianFundação CampalimaudMinistério da saúdeFundação para a Ciência e Tecnologi

    iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease

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    Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent
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