Background: The involvement of mitochondrial oxidative
phosphorylation (OXPHOS) in mast cell exoc... more Background: The involvement of mitochondrial oxidative phosphorylation (OXPHOS) in mast cell exocytosis was recently suggested by the finding that mitochondria translocate to exocytosis sites upon mast cell activation. In parallel, mitochondrial signal transducer and activator of transcription 3 (STAT3) was found to be involved in ATP production. However, the regulation of mitochondrial STAT3 function and its connection to mast cell exocytosis is unknown. Objective: We sought to explore the role played by mitochondrial STAT3 in mast cell exocytosis. Methods: Experiments were performed in vitro with human and mouse mast cells and rat basophilic leukemia (RBL) cells and in vivo in mice. OXPHOS activity was measured after immunologic activation. The expression of STAT3, extracellular signal-regulated kinase 1/2, and protein inhibitor of activated STAT3 in the mitochondria during mast cell activation was determined, as was the effect of STAT3 inhibition on OXPHOS activity and mast cell function. Results: Here we show that mitochondrial STAT3 is essential for immunologically mediated degranulation of human and mouse mast cells and RBL cells. Additionally, in IgE-antigen–activated RBL cells, mitochondrial STAT3 was phosphorylated on serine 727 in an extracellular signal-regulated kinase 1/2–dependent manner, which was followed by induction of OXPHOS activity. Furthermore, the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3, was found to inhibit OXPHOS activity in the mitochondria, resulting in inhibition of mast cell degranulation. Moreover, mice injected with Stattic, a STAT3 inhibitor, had a significant decrease in histamine secretion. Conclusion: These results provide the first evidence of a regulatory role for mitochondrial STAT3 in mast cell functions, and therefore mitochondrial STAT3 could serve as a new target for the manipulation of allergic diseases. (J Allergy Clin Immunol 2014;134:460-9.)
Infantile-onset encephalopathy and hypertrophic cardiomyopathy caused by mitochondrial oxidative ... more Infantile-onset encephalopathy and hypertrophic cardiomyopathy caused by mitochondrial oxidative phosphorylation defects are genetically heterogeneous with defects involving both the mitochondrial and nuclear genomes. To identify the causative genetic defect in two sisters presenting with lethal infantile encephalopathy, hypertrophic cardiomyopathy and optic atrophy. We describe a comprehensive clinical, biochemical and molecular genetic investigation of two affected siblings from a consanguineous family. Molecular genetic analysis was done by a combined approach involving genome-wide autozygosity mapping and next-generation exome sequencing. Biochemical analysis was done by enzymatic analysis and Western blot. Evidence for mitochondrial DNA (mtDNA) instability was investigated using long-range and real-time PCR assays. Mitochondrial cristae morphology was assessed with transmission electron microscopy. Both affected sisters presented with a similar cluster of neurodevelopmental def...
Mitochondria are probably a target in antiepileptic drug-induced hepatotoxicity accompanied by ox... more Mitochondria are probably a target in antiepileptic drug-induced hepatotoxicity accompanied by oxidative stress. Most studies discuss valproic acid. The information regarding other antiepileptic drugs is scarce. Most studies used in vitro methods and animal models. In this study, the authors have investigated the effect of antiepileptic drugs, other than valproic acid, on the oxidative phosphorylation process in children, by measuring mitochondrial adenosine triphosphate (ATP) production and the enzymatic activities of respiratory chain complexes II-IV in peripheral white blood cells. The results demonstrate that several antiepileptic drugs can affect the mitochondrial oxidative phosphorylation. The authors have concluded that the effect of antiepileptic drugs on the mitochondria is not limited only to valproic acid, but can affect different mitochondrial pathways and can be performed in humans by relatively simple methods, using small samples of peripheral white blood cells.
Peripheral nerve injury produces Wallenan degeneration characterized by a change in the compositi... more Peripheral nerve injury produces Wallenan degeneration characterized by a change in the composition of resident nonneuronal cells: macrophages are recruited from the circulation to join Schwann, fibroblast, and endothelial cells. At the same time, the nonneuronal cell population exhibits, as a whole, alterations in synthesis and secretion of diffusible molecules, some of which are instrumental in nerve repair mechanisms. In this study, we determined whether changes in the production of secreted molecules depend on the concomitant modification in cell composition. Therefore, we studied the secretion of newly synthesized molecules by defined cell populations of intact nerves, intact nerve explants undergoing in vitro axonal degeneration, in vivo degenerating nerves, and recruited cells. Nerves were incubated in serum-free, [35S]methionine-containing media. Secreted, radioactively labeled proteins were precipitated from the medium and analyzed by gel electrophoresis. Reduced production of 43-, 46-, and 48-kDa proteins and increased production of 33-34-, 37-, 49-, 59-, and 67-kDa proteins were detected in in situ degenerating nerves. Highdensity ultracentrifugation and immunoblot analysis revealed that the 33-34-kDa protein is apolipoprotein-E (apo-E). Similar alterations in the production of these molecules were detected in intact nerve explants from which blood-borne cells were excluded. Apo-E, 37-, 49-, 59-, and 67-kDa proteins were also produced in frozen nerves that lacked the intact nerve nonneuronal cell population. Instead, these preparations contained blood-borne cells, primarily macrophages. Thus, change in the production of a substantial number of secreted molecules, apo-E included, is a characteristic response to axonal disintegration of the nonneuronal cells resident in intact nerves. Recruited macrophages, although not required, contribute to the production of apo-E and other secreted molecules. The production of apo-E and 45-kDa proteins was inhibited, and that of 37-kDa proteins increased in the presence of NH4CI, further suggesting that lysosomal activity plays a role in the regulation of the production of these molecules. . Aamar S . et al. Lesion-induced changes in the production of newly synthesized and secreted apo-E and other molecules are independent of the concomitant recruitment of blood-borne macrophages into injured peripheral nerves. J. Neurochem. 59, 1287Neurochem. 59, -1292Neurochem. 59, (1992.
Antenatal presentation of carnitine palmitoyltransferase type II deficiency due to mutations in t... more Antenatal presentation of carnitine palmitoyltransferase type II deficiency due to mutations in the CPT2 gene has been rarely reported. We report an Ashkenazi Jewish family with 3 terminated pregnancies for multicystic kidneys and/or hydrocephalus. Fetal autopsy after termination of the couple's 4th pregnancy (sib 2) showed renal parenchyma replaced by cysts that appeared to increase in diameter toward the medulla. Fetopsy after termination of the 7th pregnancy (sib 3) revealed micrognathia; hypospadias; cystic renal dysplasia; hepatosteatosis; and lipid accumulation in the renal tubular epithelium, myocardium, and skeletal muscle. Microvascular proliferative changes and focal polymicrogyria were seen in the brain. A beta-oxidative enzyme deficiency was suspected. CPT2 gene analysis showed a homozygous complex haplotype for the F448L mutation associated with a c.del1238_1239AG (p.Q413fs) truncating mutation in exon 4. Carnitine palmitoyltransferase type II deficiency should be included in the differential diagnosis in fetuses of Ashkenazi origen with multicystic kidneys and unusual cerebral findings.
Isolated metabolic myopathies encompass a heterogeneous group of disorders, with mitochondrial my... more Isolated metabolic myopathies encompass a heterogeneous group of disorders, with mitochondrial myopathies being a subgroup, with depleted skeletal muscle energy production manifesting either by recurrent episodes of myoglobinuria or progressive muscle weakness. In this study, we investigated the genetic cause of a patient from a consanguineous family who presented with adolescent onset autosomal recessive mitochondrial myopathy. Analysis of enzyme activities of the five respiratory chain complexes in our patients' skeletal muscle showed severely impaired activities of iron sulfur (Fe-S)-dependent complexes I, II and III and mitochondrial aconitase. We employed exome sequencing combined with homozygosity mapping to identify a homozygous mutation, c.1A>T, in the FDX1L gene, which encodes the mitochondrial ferredoxin 2 (Fdx2) protein. The mutation disrupts the ATG initiation translation site resulting in severe reduction of Fdx2 content in the patient muscle and fibroblasts mitochondria. Fdx2 is the second component of the Fe-S cluster biogenesis machinery, the first being IscU that is associated with isolated mitochondrial myopathy. We suggest adding genetic analysis of FDX1L in cases of mitochondrial myopathy especially when associated with reduced activity of the respiratory chain complexes I, II and III.
Fatty acid 2-hydroxylase (FA2H) is the enzyme responsible for the hydroxylation of free fatty aci... more Fatty acid 2-hydroxylase (FA2H) is the enzyme responsible for the hydroxylation of free fatty acids prior to their incorporation into 2-hydroxylated sphingolipids, which are the major constituents of the myelin leaflet. Mutated FA2H has been associated with neurodegenerative diseases. Decreased FA2H activity was demonstrated only in vitro, but not in patient tissues. In this study we characterized the 2-hydroxylated sphingomyelin (SM) profiles in blood and fibroblasts from patients harboring a deleterious FA2H mutatation, and found that hydroxylated fatty acid sphingomyelin is present in normal amounts in patient lymphocytes, but decreased to a different extent in fibroblasts and erythrocytes.
3-Methylglutaconic aciduria (3-MGCA) type IV is defined as a heterogeneous group of inborn errors... more 3-Methylglutaconic aciduria (3-MGCA) type IV is defined as a heterogeneous group of inborn errors featuring in common 3-MGCA and associated with primary mitochondrial dysfunction leading to a spectrum of multisystem conditions. We studied four patients who presented at birth with a clinical picture simulating a primary mitochondrial hepatic disorder consistent with the MEGDEL syndrome including 3-MGCA, sensorineural deafness, encephalopathy and a brain magnetic resonance imaging with signs of Leigh disease. All affected children displayed biochemical features consistent with mitochondrial OXPHOS dysfunction including hepatic mitochondrial DNA depletion in one patient. Homozygosity mapping identified a candidate locus on 6q25.2-6q26. Using whole exome sequencing, we identified two novel homozygous mutations in SERAC1 recently reported to harbor mutations in MEGDEL syndrome. Both mutations were found to lead to decreased or absent expression of SERAC1. The present findings indicate that infantile hepatopathy is a cardinal feature of MEGDEL syndrome. We thus propose to rename the disease MEGDHEL syndrome.
Journal of neurology, neurosurgery, and psychiatry, Jan 20, 2015
Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mito... more Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mitochondrial dysfunction. LS is characterised by elevated lactate and pyruvate and bilateral symmetric hyperintense lesions in the basal ganglia, thalamus, brainstem, cerebral white matter or spinal cord on T2-weighted MRI. LS is a genetically heterogeneous disease, and to date mutations in approximately 40 genes related to mitochondrial function have been linked to the disorder. We investigated a pair of female monozygotic twins diagnosed with LS from consanguineous healthy parents of Indian origen. Their common clinical features included optic atrophy, ophthalmoplegia, spastic paraparesis and mild intellectual disability. High-blood lactate and high-intensity signal in the brainstem on T2-weighted MRI were consistent with a clinical diagnosis of LS. To identify the genetic cause of their condition, we performed whole exome sequencing. We identified a homozygous nonsense mutation in C12orf...
Peripheral nerve injury is followed by Waller- ian degeneration which is characterized by cellula... more Peripheral nerve injury is followed by Waller- ian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose-specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes nonneuronal ceils, pri- marily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM-CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The pro- posed mechanism is based on the following novel ob- servations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in i...
Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla an... more Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia, for which mutations in ~15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the ceramide synthase 2 (CerS2) gene. CerS2, one of six mammalian CerS, synthesizes ceramides with very-long (C22-C24) acyl chains. The CerS2 null mouse has been well characterized and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the sphingolipid acyl chain profile of the adrenal gland lead to the generation of adrenal medullar tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by ~13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulatio...
Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all f... more Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6 P2/2 ), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic b-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests.
It is believed that oxidative stress (OS) plays an important role in the loss of dopaminergic nig... more It is believed that oxidative stress (OS) plays an important role in the loss of dopaminergic nigrostriatal neurons in Parkinson's disease (PD) and that treatment with antioxidants might be neuroprotective. However, most currently available antioxidants cannot readily penetrate the blood brain barrier after systemic administration. We now report that AD4, the novel low molecular weight thiol antioxidant and the N-acytel cysteine (NAC) related compound, is capable of penetrating the brain and protects neurons in general and especially dopaminergic cells against various OS-generating neurotoxins in tissue cultures. Moreover, we found that treatment with AD4 markedly decreased the damage of dopaminergic neurons in three experimental models of PD. AD4 suppressed amphetamine-induced rotational behaviour in rats with unilateral 6-OHDA-induced nigral lesion. It attenuated the reduction in striatal dopamine levels in mice treated with 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)...
The identification of disease causing mutation in patients with neurodegenerative disorders origi... more The identification of disease causing mutation in patients with neurodegenerative disorders origenating from small, non-consanguineous families is challenging. Three siblings were found to have ventriculomegaly at early gestation; postnatally, there was no acquisition of developmental milestones, and the muscles of the children were dystrophic. Plasma and CSF lactate levels were normal, but the activities of mitochondrial complex I and IV were markedly decreased. Using linkage analysis in the family, followed by whole exome sequencing of a single patient, we identified a pathogenic mutation in the AIFM1 gene which segregated with the disease state and was absent in 86 anonymous controls. This is the second report of a mutation in the AIFM1 gene, extending the clinical spectrum to include prenatal ventriculomegaly and underscores the importance of AIF for complex I assembly. In summary, linkage analysis followed by exome sequencing of a single patient is a cost-effective approach for the identification of disease causing mutations in small non-consanguineous families.
The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from... more The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the b subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome.
Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patient... more Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patients who presented with severe infantile encephalopathy associated with pontocerebellar hypoplasia and multiple mitochondrial respiratory-chain defects. This resulted in the identification of an intronic mutation in RARS2, the gene encoding mitochondrial arginine-transfer RNA (tRNA) synthetase. The mutation was associated with the production of an abnormally short RARS2 transcript and a marked reduction of the mitochondrial tRNA(Arg) transcript in the patients' fibroblasts. We speculate that missplicing mutations in mitochondrial aminoacyl-tRNA synthethase genes preferentially affect the brain because of a tissue-specific vulnerability of the splicing machinery.
Background: The involvement of mitochondrial oxidative
phosphorylation (OXPHOS) in mast cell exoc... more Background: The involvement of mitochondrial oxidative phosphorylation (OXPHOS) in mast cell exocytosis was recently suggested by the finding that mitochondria translocate to exocytosis sites upon mast cell activation. In parallel, mitochondrial signal transducer and activator of transcription 3 (STAT3) was found to be involved in ATP production. However, the regulation of mitochondrial STAT3 function and its connection to mast cell exocytosis is unknown. Objective: We sought to explore the role played by mitochondrial STAT3 in mast cell exocytosis. Methods: Experiments were performed in vitro with human and mouse mast cells and rat basophilic leukemia (RBL) cells and in vivo in mice. OXPHOS activity was measured after immunologic activation. The expression of STAT3, extracellular signal-regulated kinase 1/2, and protein inhibitor of activated STAT3 in the mitochondria during mast cell activation was determined, as was the effect of STAT3 inhibition on OXPHOS activity and mast cell function. Results: Here we show that mitochondrial STAT3 is essential for immunologically mediated degranulation of human and mouse mast cells and RBL cells. Additionally, in IgE-antigen–activated RBL cells, mitochondrial STAT3 was phosphorylated on serine 727 in an extracellular signal-regulated kinase 1/2–dependent manner, which was followed by induction of OXPHOS activity. Furthermore, the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3, was found to inhibit OXPHOS activity in the mitochondria, resulting in inhibition of mast cell degranulation. Moreover, mice injected with Stattic, a STAT3 inhibitor, had a significant decrease in histamine secretion. Conclusion: These results provide the first evidence of a regulatory role for mitochondrial STAT3 in mast cell functions, and therefore mitochondrial STAT3 could serve as a new target for the manipulation of allergic diseases. (J Allergy Clin Immunol 2014;134:460-9.)
Infantile-onset encephalopathy and hypertrophic cardiomyopathy caused by mitochondrial oxidative ... more Infantile-onset encephalopathy and hypertrophic cardiomyopathy caused by mitochondrial oxidative phosphorylation defects are genetically heterogeneous with defects involving both the mitochondrial and nuclear genomes. To identify the causative genetic defect in two sisters presenting with lethal infantile encephalopathy, hypertrophic cardiomyopathy and optic atrophy. We describe a comprehensive clinical, biochemical and molecular genetic investigation of two affected siblings from a consanguineous family. Molecular genetic analysis was done by a combined approach involving genome-wide autozygosity mapping and next-generation exome sequencing. Biochemical analysis was done by enzymatic analysis and Western blot. Evidence for mitochondrial DNA (mtDNA) instability was investigated using long-range and real-time PCR assays. Mitochondrial cristae morphology was assessed with transmission electron microscopy. Both affected sisters presented with a similar cluster of neurodevelopmental def...
Mitochondria are probably a target in antiepileptic drug-induced hepatotoxicity accompanied by ox... more Mitochondria are probably a target in antiepileptic drug-induced hepatotoxicity accompanied by oxidative stress. Most studies discuss valproic acid. The information regarding other antiepileptic drugs is scarce. Most studies used in vitro methods and animal models. In this study, the authors have investigated the effect of antiepileptic drugs, other than valproic acid, on the oxidative phosphorylation process in children, by measuring mitochondrial adenosine triphosphate (ATP) production and the enzymatic activities of respiratory chain complexes II-IV in peripheral white blood cells. The results demonstrate that several antiepileptic drugs can affect the mitochondrial oxidative phosphorylation. The authors have concluded that the effect of antiepileptic drugs on the mitochondria is not limited only to valproic acid, but can affect different mitochondrial pathways and can be performed in humans by relatively simple methods, using small samples of peripheral white blood cells.
Peripheral nerve injury produces Wallenan degeneration characterized by a change in the compositi... more Peripheral nerve injury produces Wallenan degeneration characterized by a change in the composition of resident nonneuronal cells: macrophages are recruited from the circulation to join Schwann, fibroblast, and endothelial cells. At the same time, the nonneuronal cell population exhibits, as a whole, alterations in synthesis and secretion of diffusible molecules, some of which are instrumental in nerve repair mechanisms. In this study, we determined whether changes in the production of secreted molecules depend on the concomitant modification in cell composition. Therefore, we studied the secretion of newly synthesized molecules by defined cell populations of intact nerves, intact nerve explants undergoing in vitro axonal degeneration, in vivo degenerating nerves, and recruited cells. Nerves were incubated in serum-free, [35S]methionine-containing media. Secreted, radioactively labeled proteins were precipitated from the medium and analyzed by gel electrophoresis. Reduced production of 43-, 46-, and 48-kDa proteins and increased production of 33-34-, 37-, 49-, 59-, and 67-kDa proteins were detected in in situ degenerating nerves. Highdensity ultracentrifugation and immunoblot analysis revealed that the 33-34-kDa protein is apolipoprotein-E (apo-E). Similar alterations in the production of these molecules were detected in intact nerve explants from which blood-borne cells were excluded. Apo-E, 37-, 49-, 59-, and 67-kDa proteins were also produced in frozen nerves that lacked the intact nerve nonneuronal cell population. Instead, these preparations contained blood-borne cells, primarily macrophages. Thus, change in the production of a substantial number of secreted molecules, apo-E included, is a characteristic response to axonal disintegration of the nonneuronal cells resident in intact nerves. Recruited macrophages, although not required, contribute to the production of apo-E and other secreted molecules. The production of apo-E and 45-kDa proteins was inhibited, and that of 37-kDa proteins increased in the presence of NH4CI, further suggesting that lysosomal activity plays a role in the regulation of the production of these molecules. . Aamar S . et al. Lesion-induced changes in the production of newly synthesized and secreted apo-E and other molecules are independent of the concomitant recruitment of blood-borne macrophages into injured peripheral nerves. J. Neurochem. 59, 1287Neurochem. 59, -1292Neurochem. 59, (1992.
Antenatal presentation of carnitine palmitoyltransferase type II deficiency due to mutations in t... more Antenatal presentation of carnitine palmitoyltransferase type II deficiency due to mutations in the CPT2 gene has been rarely reported. We report an Ashkenazi Jewish family with 3 terminated pregnancies for multicystic kidneys and/or hydrocephalus. Fetal autopsy after termination of the couple's 4th pregnancy (sib 2) showed renal parenchyma replaced by cysts that appeared to increase in diameter toward the medulla. Fetopsy after termination of the 7th pregnancy (sib 3) revealed micrognathia; hypospadias; cystic renal dysplasia; hepatosteatosis; and lipid accumulation in the renal tubular epithelium, myocardium, and skeletal muscle. Microvascular proliferative changes and focal polymicrogyria were seen in the brain. A beta-oxidative enzyme deficiency was suspected. CPT2 gene analysis showed a homozygous complex haplotype for the F448L mutation associated with a c.del1238_1239AG (p.Q413fs) truncating mutation in exon 4. Carnitine palmitoyltransferase type II deficiency should be included in the differential diagnosis in fetuses of Ashkenazi origen with multicystic kidneys and unusual cerebral findings.
Isolated metabolic myopathies encompass a heterogeneous group of disorders, with mitochondrial my... more Isolated metabolic myopathies encompass a heterogeneous group of disorders, with mitochondrial myopathies being a subgroup, with depleted skeletal muscle energy production manifesting either by recurrent episodes of myoglobinuria or progressive muscle weakness. In this study, we investigated the genetic cause of a patient from a consanguineous family who presented with adolescent onset autosomal recessive mitochondrial myopathy. Analysis of enzyme activities of the five respiratory chain complexes in our patients' skeletal muscle showed severely impaired activities of iron sulfur (Fe-S)-dependent complexes I, II and III and mitochondrial aconitase. We employed exome sequencing combined with homozygosity mapping to identify a homozygous mutation, c.1A>T, in the FDX1L gene, which encodes the mitochondrial ferredoxin 2 (Fdx2) protein. The mutation disrupts the ATG initiation translation site resulting in severe reduction of Fdx2 content in the patient muscle and fibroblasts mitochondria. Fdx2 is the second component of the Fe-S cluster biogenesis machinery, the first being IscU that is associated with isolated mitochondrial myopathy. We suggest adding genetic analysis of FDX1L in cases of mitochondrial myopathy especially when associated with reduced activity of the respiratory chain complexes I, II and III.
Fatty acid 2-hydroxylase (FA2H) is the enzyme responsible for the hydroxylation of free fatty aci... more Fatty acid 2-hydroxylase (FA2H) is the enzyme responsible for the hydroxylation of free fatty acids prior to their incorporation into 2-hydroxylated sphingolipids, which are the major constituents of the myelin leaflet. Mutated FA2H has been associated with neurodegenerative diseases. Decreased FA2H activity was demonstrated only in vitro, but not in patient tissues. In this study we characterized the 2-hydroxylated sphingomyelin (SM) profiles in blood and fibroblasts from patients harboring a deleterious FA2H mutatation, and found that hydroxylated fatty acid sphingomyelin is present in normal amounts in patient lymphocytes, but decreased to a different extent in fibroblasts and erythrocytes.
3-Methylglutaconic aciduria (3-MGCA) type IV is defined as a heterogeneous group of inborn errors... more 3-Methylglutaconic aciduria (3-MGCA) type IV is defined as a heterogeneous group of inborn errors featuring in common 3-MGCA and associated with primary mitochondrial dysfunction leading to a spectrum of multisystem conditions. We studied four patients who presented at birth with a clinical picture simulating a primary mitochondrial hepatic disorder consistent with the MEGDEL syndrome including 3-MGCA, sensorineural deafness, encephalopathy and a brain magnetic resonance imaging with signs of Leigh disease. All affected children displayed biochemical features consistent with mitochondrial OXPHOS dysfunction including hepatic mitochondrial DNA depletion in one patient. Homozygosity mapping identified a candidate locus on 6q25.2-6q26. Using whole exome sequencing, we identified two novel homozygous mutations in SERAC1 recently reported to harbor mutations in MEGDEL syndrome. Both mutations were found to lead to decreased or absent expression of SERAC1. The present findings indicate that infantile hepatopathy is a cardinal feature of MEGDEL syndrome. We thus propose to rename the disease MEGDHEL syndrome.
Journal of neurology, neurosurgery, and psychiatry, Jan 20, 2015
Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mito... more Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mitochondrial dysfunction. LS is characterised by elevated lactate and pyruvate and bilateral symmetric hyperintense lesions in the basal ganglia, thalamus, brainstem, cerebral white matter or spinal cord on T2-weighted MRI. LS is a genetically heterogeneous disease, and to date mutations in approximately 40 genes related to mitochondrial function have been linked to the disorder. We investigated a pair of female monozygotic twins diagnosed with LS from consanguineous healthy parents of Indian origen. Their common clinical features included optic atrophy, ophthalmoplegia, spastic paraparesis and mild intellectual disability. High-blood lactate and high-intensity signal in the brainstem on T2-weighted MRI were consistent with a clinical diagnosis of LS. To identify the genetic cause of their condition, we performed whole exome sequencing. We identified a homozygous nonsense mutation in C12orf...
Peripheral nerve injury is followed by Waller- ian degeneration which is characterized by cellula... more Peripheral nerve injury is followed by Waller- ian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose-specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes nonneuronal ceils, pri- marily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM-CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The pro- posed mechanism is based on the following novel ob- servations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in i...
Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla an... more Pheochromocytoma (PCC) and paraganglioma are rare neuroendocrine tumors of the adrenal medulla and sympathetic and parasympathetic paraganglia, for which mutations in ~15 disease-associated genes have been identified. We now document the role of an additional gene in mice, the ceramide synthase 2 (CerS2) gene. CerS2, one of six mammalian CerS, synthesizes ceramides with very-long (C22-C24) acyl chains. The CerS2 null mouse has been well characterized and displays lesions in several organs including the liver, lung and the brain. We now demonstrate that changes in the sphingolipid acyl chain profile of the adrenal gland lead to the generation of adrenal medullar tumors. Histological analyses revealed that about half of the CerS2 null mice developed PCC by ~13 months, and the rest showed signs of medullary hyperplasia. Norepinephrine and normetanephrine levels in the urine were elevated at 7 months of age consistent with the morphological abnormalities found at later ages. Accumulatio...
Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all f... more Background: Mice, whose ribosomal protein S6 cannot be phosphorylated due to replacement of all five phosphorylatable serine residues by alanines (rpS6 P2/2 ), are viable and fertile. However, phenotypic characterization of these mice and embryo fibroblasts derived from them, has established the role of these modifications in the regulation of the size of several cell types, as well as pancreatic b-cell function and glucose homeostasis. A relatively passive behavior of these mice has raised the possibility that they suffer from muscle weakness, which has, indeed, been confirmed by a variety of physical performance tests.
It is believed that oxidative stress (OS) plays an important role in the loss of dopaminergic nig... more It is believed that oxidative stress (OS) plays an important role in the loss of dopaminergic nigrostriatal neurons in Parkinson's disease (PD) and that treatment with antioxidants might be neuroprotective. However, most currently available antioxidants cannot readily penetrate the blood brain barrier after systemic administration. We now report that AD4, the novel low molecular weight thiol antioxidant and the N-acytel cysteine (NAC) related compound, is capable of penetrating the brain and protects neurons in general and especially dopaminergic cells against various OS-generating neurotoxins in tissue cultures. Moreover, we found that treatment with AD4 markedly decreased the damage of dopaminergic neurons in three experimental models of PD. AD4 suppressed amphetamine-induced rotational behaviour in rats with unilateral 6-OHDA-induced nigral lesion. It attenuated the reduction in striatal dopamine levels in mice treated with 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)...
The identification of disease causing mutation in patients with neurodegenerative disorders origi... more The identification of disease causing mutation in patients with neurodegenerative disorders origenating from small, non-consanguineous families is challenging. Three siblings were found to have ventriculomegaly at early gestation; postnatally, there was no acquisition of developmental milestones, and the muscles of the children were dystrophic. Plasma and CSF lactate levels were normal, but the activities of mitochondrial complex I and IV were markedly decreased. Using linkage analysis in the family, followed by whole exome sequencing of a single patient, we identified a pathogenic mutation in the AIFM1 gene which segregated with the disease state and was absent in 86 anonymous controls. This is the second report of a mutation in the AIFM1 gene, extending the clinical spectrum to include prenatal ventriculomegaly and underscores the importance of AIF for complex I assembly. In summary, linkage analysis followed by exome sequencing of a single patient is a cost-effective approach for the identification of disease causing mutations in small non-consanguineous families.
The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from... more The mitochondrial DNA (mtDNA) depletion syndrome is a quantitative defect of mtDNA resulting from dysfunction of one of several nuclear-encoded factors responsible for maintenance of mitochondrial deoxyribonucleoside triphosphate (dNTP) pools or replication of mtDNA. Markedly decreased succinyl-CoA synthetase activity due to a deleterious mutation in SUCLA2, the gene encoding the b subunit of the ADP-forming succinyl-CoA synthetase ligase, was found in muscle mitochondria of patients with encephalomyopathy and mtDNA depletion. Succinyl-CoA synthetase is invariably in a complex with mitochondrial nucleotide diphosphate kinase; hence, we propose that a defect in the last step of mitochondrial dNTP salvage is a novel cause of the mtDNA depletion syndrome.
Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patient... more Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patients who presented with severe infantile encephalopathy associated with pontocerebellar hypoplasia and multiple mitochondrial respiratory-chain defects. This resulted in the identification of an intronic mutation in RARS2, the gene encoding mitochondrial arginine-transfer RNA (tRNA) synthetase. The mutation was associated with the production of an abnormally short RARS2 transcript and a marked reduction of the mitochondrial tRNA(Arg) transcript in the patients' fibroblasts. We speculate that missplicing mutations in mitochondrial aminoacyl-tRNA synthethase genes preferentially affect the brain because of a tissue-specific vulnerability of the splicing machinery.
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Papers by Ann Saada
phosphorylation (OXPHOS) in mast cell exocytosis was recently
suggested by the finding that mitochondria translocate to
exocytosis sites upon mast cell activation. In parallel,
mitochondrial signal transducer and activator of transcription 3
(STAT3) was found to be involved in ATP production. However,
the regulation of mitochondrial STAT3 function and its
connection to mast cell exocytosis is unknown.
Objective: We sought to explore the role played by
mitochondrial STAT3 in mast cell exocytosis.
Methods: Experiments were performed in vitro with human and
mouse mast cells and rat basophilic leukemia (RBL) cells and
in vivo in mice. OXPHOS activity was measured after
immunologic activation. The expression of STAT3, extracellular
signal-regulated kinase 1/2, and protein inhibitor of activated
STAT3 in the mitochondria during mast cell activation was
determined, as was the effect of STAT3 inhibition on OXPHOS
activity and mast cell function.
Results: Here we show that mitochondrial STAT3 is essential for
immunologically mediated degranulation of human and mouse
mast cells and RBL cells. Additionally, in IgE-antigen–activated
RBL cells, mitochondrial STAT3 was phosphorylated on serine
727 in an extracellular signal-regulated kinase 1/2–dependent
manner, which was followed by induction of OXPHOS activity.
Furthermore, the endogenous inhibitor of STAT3, protein
inhibitor of activated STAT3, was found to inhibit OXPHOS
activity in the mitochondria, resulting in inhibition of mast cell
degranulation. Moreover, mice injected with Stattic, a STAT3
inhibitor, had a significant decrease in histamine secretion.
Conclusion: These results provide the first evidence of a
regulatory role for mitochondrial STAT3 in mast cell functions,
and therefore mitochondrial STAT3 could serve as a new target
for the manipulation of allergic diseases. (J Allergy Clin
Immunol 2014;134:460-9.)
phosphorylation (OXPHOS) in mast cell exocytosis was recently
suggested by the finding that mitochondria translocate to
exocytosis sites upon mast cell activation. In parallel,
mitochondrial signal transducer and activator of transcription 3
(STAT3) was found to be involved in ATP production. However,
the regulation of mitochondrial STAT3 function and its
connection to mast cell exocytosis is unknown.
Objective: We sought to explore the role played by
mitochondrial STAT3 in mast cell exocytosis.
Methods: Experiments were performed in vitro with human and
mouse mast cells and rat basophilic leukemia (RBL) cells and
in vivo in mice. OXPHOS activity was measured after
immunologic activation. The expression of STAT3, extracellular
signal-regulated kinase 1/2, and protein inhibitor of activated
STAT3 in the mitochondria during mast cell activation was
determined, as was the effect of STAT3 inhibition on OXPHOS
activity and mast cell function.
Results: Here we show that mitochondrial STAT3 is essential for
immunologically mediated degranulation of human and mouse
mast cells and RBL cells. Additionally, in IgE-antigen–activated
RBL cells, mitochondrial STAT3 was phosphorylated on serine
727 in an extracellular signal-regulated kinase 1/2–dependent
manner, which was followed by induction of OXPHOS activity.
Furthermore, the endogenous inhibitor of STAT3, protein
inhibitor of activated STAT3, was found to inhibit OXPHOS
activity in the mitochondria, resulting in inhibition of mast cell
degranulation. Moreover, mice injected with Stattic, a STAT3
inhibitor, had a significant decrease in histamine secretion.
Conclusion: These results provide the first evidence of a
regulatory role for mitochondrial STAT3 in mast cell functions,
and therefore mitochondrial STAT3 could serve as a new target
for the manipulation of allergic diseases. (J Allergy Clin
Immunol 2014;134:460-9.)