RESUMO
BACKGROUND: Farnesol (FOL) prevents the onset of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). OBJECTIVE: We examined the transcriptomic profile of the brains of EAE mice treated with daily oral FOL using next-generation sequencing (RNA-seq). METHODS: Transcriptomics from whole brains of treated and untreated EAE mice at the peak of EAE was performed. RESULTS: EAE-induced mice, compared to naïve, healthy mice, overall showed increased expression in pathways for immune response, as well as an increased cytokine signaling pathway, with downregulation of cellular stress proteins. FOL downregulates pro-inflammatory pathways and attenuates the immune response in EAE. FOL downregulated the expression of genes involved in misfolded protein response, MAPK activation/signaling, and pro-inflammatory response. CONCLUSION: This study provides insight into the molecular impact of FOL in the brain and identifies potential therapeutic targets of the isoprenoid pathway in MS patients.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Humanos , Camundongos , Animais , Farneseno Álcool/farmacologia , Transcriptoma , Encéfalo/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Succinic semialdehyde dehydrogenase deficiency (SSADHD) is an inherited inborn error of the γ-aminobutyric acid (GABA) metabolism pathway. It results from mutations in the ALDH5A1 gene leading to elevated GABA, γ-hydroxybutyric acid (GHB), succinic semialdehyde (SSA), decreased glutamine and alterations in several other metabolites. The phenotype includes developmental and cognitive delays, hypotonia, seizures, neuropsychiatric morbidity and other nervous system pathologies. The composition of the intestinal flora of patients with SSADHD has not been characterized, and dysbiosis of the gut microbiome may unveil novel treatment paradigms. We investigated the gut microbiome in SSADHD using 16S ribosomal DNA sequencing and unmasked evidence of dysbiosis in both aldh5a1-deficient mice and patients with SSADHD. In the murine model, there was a reduction in α-diversity measurements, and there were 4 phyla, 3 classes, 5 orders, 9 families, and 15 genera that differed, with a total of 17 predicted metabolic pathways altered. In patients, there were changes in Fusobacterium, 3 classes, 4 orders, 11 families, and a predicted alteration in genes associated with the digestive system. We believe this is the first evaluation of microbiome structure in an IEM with a neurometabolic phenotype that is not treated dietarily.
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Erros Inatos do Metabolismo dos Aminoácidos , Disbiose , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Criança , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Disbiose/genética , Humanos , Camundongos , Succinato-Semialdeído Desidrogenase/deficiênciaRESUMO
Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles. For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data. In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Deficiências do Desenvolvimento/patologia , Mutação de Sentido Incorreto , Succinato-Semialdeído Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Simulação por Computador , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Células HEK293 , Humanos , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
Upon publication, it was noted that five of the on-line supplementary figures had incorrect figure: figure legend associations. These were supplementary Figs. 6, 7, 14, 15, and 23.
RESUMO
Metabolomic characterization of post-mortem tissues (frontal and parietal cortices, pons, cerebellum, hippocampus, cerebral cortex, liver and kidney) derived from a 37 y.o. male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) was performed in conjunction with four parallel series of control tissues. Amino acids, acylcarnitines, guanidino- species (guanidinoacetic acid, creatine, creatinine) and GABA-related intermediates were quantified using UPLC and mass spectrometric methods that included isotopically labeled internal standards. Amino acid analyses revealed significant elevation of aspartic acid and depletion of glutamine in patient tissues. Evidence for disruption of short-chain fatty acid metabolism, manifest as altered C4OH, C5, C5:1, C5DC (dicarboxylic) and C12OH carnitines, was observed. Creatine and guanidinoacetic acids were decreased and elevated, respectively. GABA-associated metabolites (total GABA, γ-hydroxybutyric acid, succinic semialdehyde, 4-guanidinobutyrate, 4,5-dihydroxyhexanoic acid and homocarnosine) were significantly increased in patient tissues, including liver and kidney. The data support disruption of fat, creatine and amino acid metabolism as a component of the pathophysiology of SSADHD, and underscore the observation that metabolites measured in patient physiological fluids provide an unreliable reflection of brain metabolism.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Aminoácidos/metabolismo , Encéfalo/metabolismo , Carnitina/análogos & derivados , Creatina/metabolismo , Creatinina/metabolismo , Deficiências do Desenvolvimento/metabolismo , Glicina/análogos & derivados , Succinato-Semialdeído Desidrogenase/deficiência , Ácido gama-Aminobutírico/análogos & derivados , Adulto , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encéfalo/patologia , Carnitina/metabolismo , Deficiências do Desenvolvimento/patologia , Glicina/metabolismo , Humanos , Masculino , Metabolômica , Succinato-Semialdeído Desidrogenase/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Murine succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with high concentrations of γ-aminobutyric acid (GABA) and γ-hydroxybutyrate (GHB) and low glutamine in the brain. To understand the pathogenic contribution of central glutamine deficiency, we exposed aldh5a1-/- (SSADHD) mice and their genetic controls (aldh5a1+/+ ) to either a 4% (w/w) glutamine-containing diet or a glutamine-free diet from conception until postnatal day 30. Endpoints included brain, liver and blood amino acids, brain GHB, ataxia scores, and open field testing. Glutamine supplementation did not improve aldh5a1-/- brain glutamine deficiency nor brain GABA and GHB. It decreased brain glutamate but did not change the ratio of excitatory (glutamate) to inhibitory (GABA) neurotransmitters. In contrast, glutamine supplementation significantly increased brain arginine (30% for aldh5a1+/+ and 18% for aldh5a1-/- mice), and leucine (12% and 18%). Glutamine deficiency was confirmed in the liver. The test diet increased hepatic glutamate in both genotypes, decreased glutamine in aldh5a1+/+ but not in aldh5a1-/- , but had no effect on GABA. Dried bloodspot analyses showed significantly elevated GABA in mutants (approximately 800% above controls) and decreased glutamate (approximately 25%), but no glutamine difference with controls. Glutamine supplementation did not impact blood GABA but significantly increased glutamine and glutamate in both genotypes indicating systemic exposure to dietary glutamine. Ataxia and pronounced hyperactivity were observed in aldh5a1-/- mice but remained unchanged by the diet intervention. The study suggests that glutamine supplementation improves peripheral but not central glutamine deficiency in experimental SSADHD. Future studies are needed to fully understand the pathogenic role of brain glutamine deficiency in SSADHD.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Biomarcadores/sangue , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Glutamina/administração & dosagem , Succinato-Semialdeído Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Aminoácidos/metabolismo , Animais , Encéfalo/patologia , Deficiências do Desenvolvimento/sangue , Suplementos Nutricionais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Succinato-Semialdeído Desidrogenase/sangue , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
We present an update to the status of research on succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD), a rare disorder of GABA metabolism. This is an unusual disorder featuring the accumulation of both GABA and its neuromodulatory analog, gamma-hydroxybutyric acid (GHB), and recent studies have advanced the potential clinical application of NCS-382, a putative GHB receptor antagonist. Animal studies have provided proof-of-concept that enzyme replacement therapy could represent a long-term therapeutic option. The characterization of neuronal stem cells (NSCs) derived from aldehyde dehydrogenase 5a1-/- (aldh5a1-/-) mice, the murine model of SSADHD, has highlighted NSC utility as an in vitro system in which to study therapeutics and associated toxicological properties. Gene expression analyses have revealed that transcripts encoding GABAA receptors are down-regulated and may remain largely immature in aldh5a1-/- brain, characterized by excitatory as opposed to inhibitory outputs, the latter being the expected action in the mature central nervous system. This indicates that agents altering chloride channel activity may be therapeutically relevant in SSADHD. The most recent therapeutic prospects include mTOR (mechanistic target of rapamycin) inhibitors, drugs that have received attention with the elucidation of the effects of elevated GABA on autophagy. The outlook for novel therapeutic trials in SSADHD continues to improve.
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Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Benzocicloeptenos/uso terapêutico , Deficiências do Desenvolvimento/tratamento farmacológico , Terapia de Reposição de Enzimas , Antagonistas GABAérgicos/uso terapêutico , Succinato-Semialdeído Desidrogenase/deficiência , Serina-Treonina Quinases TOR/antagonistas & inibidores , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Animais , Deficiências do Desenvolvimento/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
This study has extended previous metabolic measures in postmortem tissues (frontal and parietal lobes, pons, cerebellum, hippocampus, and cerebral cortex) obtained from a 37-year-old male patient with succinic semialdehyde dehydrogenase deficiency (SSADHD) who expired from SUDEP (sudden unexplained death in epilepsy). Histopathologic characterization of fixed cortex and hippocampus revealed mild to moderate astrogliosis, especially in white matter. Analysis of total phospholipid mass in all sections of the patient revealed a 61% increase in cortex and 51% decrease in hippocampus as compared to (n = 2-4) approximately age-matched controls. Examination of mass and molar composition of major phospholipid classes showed decreases in phospholipids enriched in myelin, such as phosphatidylserine, sphingomyelin, and ethanolamine plasmalogen. Evaluation of gene expression (RT2 Profiler PCR Arrays, GABA, glutamate; Qiagen) revealed dysregulation in 14/15 GABAA receptor subunits in cerebellum, parietal, and frontal lobes with the most significant downregulation in ∊, θ, ρ1, and ρ2 subunits (7.7-9.9-fold). GABAB receptor subunits were largely unaffected, as were ionotropic glutamate receptors. The metabotropic glutamate receptor 6 was consistently downregulated (maximum 5.9-fold) as was the neurotransmitter transporter (GABA), member 13 (maximum 7.3-fold). For other genes, consistent dysregulation was seen for interleukin 1ß (maximum downregulation 9.9-fold) and synuclein α (maximal upregulation 6.5-fold). Our data provide unique insight into SSADHD brain function, confirming astrogliosis and lipid abnormalities previously observed in the null mouse model while highlighting long-term effects on GABAergic/glutamatergic gene expression in this disorder.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encéfalo/patologia , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Expressão Gênica/genética , Lipídeos/análise , Succinato-Semialdeído Desidrogenase/deficiência , Adulto , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Autopsia , Deficiências do Desenvolvimento/metabolismo , Humanos , Masculino , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
((S)-(+)/(R)-(-)) vigabatrin (SabrilR; γ-vinyl GABA), an antiepileptic irreversibly inactivating GABA-transaminase, was administered to male C57Bl6 J mice via continuous infusion (0, 40, 80 mg/kg/d) for 12 days. Our study design pooled retina, eye (minus retina), whole brain and plasma from n = 24 animals for each dose to provide n = 8 triplicates per treatment group. Hypothesizing that (S)-(+) VGB (active isomer) would preferentially accumulate in retina, we determined VGB isomers, comprehensive amino acids, and pharmacokinetic parameters. In brain, eye and plasma, the ((S)-(+)/(R)-(-)) ratio varied from 0.73 to 1.29 and 13.3 in retina, accompanied by a partition coefficient (tissue/plasma, ((S)-(+);(R)-(-))) of 5.8;0.34, 0.63;0.49, and 0.51;0.34 in retina, eye and brain, respectively. Racemic VGB (nmol/g; plasma, nmol/mL, range of means for dose) content was: retina, 25-36; eye (minus retina), 4.8-8.0; brain, 3.1-6.8 and plasma, 8.7-14.9. GABA tissue content (nmol/g) was 1246-3335, 18-64 and 2615-3200 as a function of VGB dose for retina, eye (minus retina) and brain, respectively. The retinal glial cell toxin 2-aminoadipic acid also increased with VGB dose (76-96 nmol/g). Partitioning of active (S)-(+) VGB to retina suggests the involvement of a stereospecific transporter, the identification of which could reveal new therapeutic paradigms that might mitigate VGB's well-known retinal toxicity and expand its clinical utility.
Assuntos
Retina , 4-Aminobutirato Transaminase , Animais , Anticonvulsivantes/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vigabatrina/toxicidadeRESUMO
Succinic semialdehyde dehydrogenase deficiency (SSADHD) manifests with low levels of glutamine in the brain, suggesting that central glutamine deficiency contributes to pathogenesis. Recently, we attempted to rescue the disease phenotype of aldh5a1 -/- mice, a murine model of SSADHD with dietary glutamine supplementation. No clinical rescue and no central glutamine improvement were observed. Here, we report the results of follow-up studies of the cellular and molecular basis of the resistance of the brain to glutamine supplementation. We first determined if the expression of genes involved in glutamine metabolism was impacted by glutamine feeding. We then searched for changes of brain histology in response to glutamine supplementation, with a focus on astrocytes, known regulators of glutamine synthesis in the brain. Glutamine supplementation significantly modified the expression of glutaminase (gls) (0.6-fold down), glutamine synthetase (glul) (1.5-fold up), and glutamine transporters (solute carrier family 7, member 5 [slc7a5], 2.5-fold up; slc38a2, 0.6-fold down). The number of GLUL-labeled cells was greater in the glutamine-supplemented group than in controls (P < .05). Reactive astrogliosis, a hallmark of brain inflammation in SSADHD, was confirmed. We observed a 2-fold stronger astrocyte staining in mutants than in wild-type controls (optical density/cell were 1.8 ± 0.08 in aldh5a1 -/- and 0.99 ± 0.06 in aldh5a1 +/+ ; P < .0001), and a 3-fold higher expression of gfap and vimentin. However, glutamine supplementation did not improve the histological and molecular signature of astrogliosis. Thus, glutamine supplementation impacts genes implicated in central glutamine homeostasis without improving reactive astrogliosis. The mechanisms underlying glutamine deficiency and its contribution to SSADHD pathogenesis remain unknown and should be the focus of future investigations.
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Vigabatrin (VGB; γ-vinyl-GABA) is an antiepileptic drug that elevates CNS GABA via irreversible inactivation of the GABA catabolic enzyme GABA-transaminase. VGB's clinical utility, however, can be curtailed by peripheral visual field constriction (pVFC) and thinning of the retinal nerve fiber layer (RNFL). Earlier studies from our laboratory revealed disruptions of autophagy by VGB. Here, we tested the hypothesis that VGB administration to animals would reveal alterations of gene expression in VGB-treated retina that associated with autophagy. VGB (140 mg/kg/d; subcutaneous minipump) was continuously administered to mice (n = 6 each VGB/vehicle) for 12 days, after which animals were euthanized. Retina was isolated for transcriptome (RNAseq) analysis and further validation using qRT-PCR and immunohistochemistry (IHC). For 112 differentially expressed retinal genes (RNAseq), two databases (Gene Ontology; Kyoto Encyclopedia of Genes and Genomes) were used to identify genes associated with visual function. Twenty four genes were subjected to qRT-PCR validation, and five (Gb5, Bdnf, Cplx9, Crh, Sox9) revealed significant dysregulation. IHC of fixed retinas verified significant down-regulation of Gb5 in photoreceptor cells. All of these genes have been previously shown to play a role in retinal function/circuitry signaling. Minimal impact of VGB on retinal autophagic gene expression was observed. This is the first transcriptome analysis of retinal gene expression associated with VGB intake, highlighting potential novel molecular targets potentially related to VGB's well known ocular toxicity.
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Anticonvulsivantes/farmacologia , Perfilação da Expressão Gênica/métodos , Rede Nervosa/fisiologia , Retina/fisiologia , Vigabatrina/farmacologia , Vias Visuais/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/química , Rede Nervosa/efeitos dos fármacos , Retina/química , Retina/efeitos dos fármacos , Análise de Sequência de RNA/métodos , Vias Visuais/química , Vias Visuais/efeitos dos fármacosRESUMO
BACKGROUND: Previous work has identified age-related negative correlations for γ-hydroxybutyric acid (GHB) and γ-aminobutyric acid (GABA) in plasma of patients with succinic semialdehyde dehydrogenase deficiency (SSADHD). Using plasma and dried blood spots (DBS) collected in an ongoing natural history study, we tested the hypothesis that other biomarkers would follow a similar age-related negative correlation as seen for GHB/GABA. Samples (mixed sex) included: patients (n = 21 unique samples, 1-39.5 yrs) and parallel controls (n = 9 unique samples, 8.4-34.8 yrs). Archival control data (DBS only; n = 171, 0.5-39.9 yrs) was also included. RESULTS: Metabolites assessed included amino acids (plasma, DBS) and acylcarnitines, creatine, creatinine, and guanidinoacetate (DBS only). Age-related negative correlations for glycine (plasma, DBS) and sarcosine (N-methylglycine, plasma) were detected, accompanied by elevated proline and decreased levels of succinylacetone, argininosuccinate, formaminoglutamate, and creatinine. Significantly low acylcarnitines were detected in patients across all chain lengths (short-, medium- and long-chain). Significant age-dependent positive correlations for selected acylcarnitines (C6-, C12DC(dicarboxylic)-, C16-, C16:1-, C18:1-, C18:2OH-carnitines) were detected in patients and absent in controls. Receiver operating characteristic (ROC) curves for all binary comparisons revealed argininosuccinate and succinylacetone to be the most discriminating biomarkers (area > 0.92). CONCLUSIONS: Age-dependent acylcarnitine correlations may represent metabolic compensation responsive to age-related changes in GHB and GABA. Our study highlights novel biomarkers in SSADHD and expands the metabolic pathophysiology of this rare disorder of GABA metabolism.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos , Deficiências do Desenvolvimento , Plasma , Succinato-Semialdeído Desidrogenase/deficiência , Adolescente , Adulto , Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Biomarcadores , Criança , Pré-Escolar , Deficiências do Desenvolvimento/sangue , Deficiências do Desenvolvimento/diagnóstico , Humanos , Lactente , Succinato-Semialdeído Desidrogenase/sangue , Adulto JovemRESUMO
Purpose: Vigabatrin (VGB) is an effective antiepileptic that increases concentrations of inhibitory γ-aminobutyric acid (GABA) by inhibiting GABA transaminase. Reports of VGB-associated visual field loss limit its clinical usefulness, and retinal toxicity studies in laboratory animals have yielded conflicting results. Methods: We examined the functional and morphologic effects of VGB in C57BL/6J mice that received either VGB or saline IP from 10 to 18 weeks of age. Retinal structure and function were assessed in vivo by optical coherence tomography (OCT), ERG, and optomotor response. After euthanasia, retinas were processed for immunohistochemistry, and retinal GABA, and VGB quantified by mass spectrometry. Results: No significant differences in visual acuity or total retinal thickness were identified between groups by optomotor response or optical coherence tomography, respectively. After 4 weeks of VGB treatment, ERG b-wave amplitude was enhanced, and amplitudes of oscillatory potentials were reduced. Dramatic rod and cone bipolar and horizontal cell remodeling, with extension of dendrites into the outer nuclear layer, was observed in retinas of VGB-treated mice. VGB treatment resulted in a mean 3.3-fold increase in retinal GABA concentration relative to controls and retinal VGB concentrations that were 20-fold greater than brain. Conclusions: No evidence of significant retinal thinning or ERG a- or b-wave deficits were apparent, although we describe significant alterations in ERG b-wave and oscillatory potentials and in retinal cell morphology in VGB-treated C57BL/6J mice. The dramatic concentration of VGB in retina relative to the target tissue (brain), with a corresponding increase in retinal GABA, offers insight into the pathophysiology of VGB-associated visual field loss.
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Anticonvulsivantes/farmacologia , GABAérgicos/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Retina/efeitos dos fármacos , Vigabatrina/farmacologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Músculos Oculomotores/efeitos dos fármacos , Distribuição Aleatória , Retina/fisiopatologia , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/fisiopatologia , Tomografia de Coerência Óptica , Campos Visuais/fisiologiaRESUMO
Objective: Succinic Semialdehyde Dehydrogenase (SSADH) deficiency is a disorder of elevated gamma-amino butyric acid (GABA) and gamma hydroxybutyric acid (GHB) and a complex neuropsychiatric profile. Adult reports suggest worsening epilepsy and high SUDEP risk. Methods: Subjects with confirmed SSADH deficiency were recruited into a longitudinal study. Plasma thyroid hormone and total GABA/GHB were quantified by standard clinical chemistry methodologies and mass spectrometry, respectively. Results: A total of 133 subjects with SSADH deficiency are enrolled in the registry; 49 participated in the longitudinal study. The age range of the population is 8 weeks to 63 years (median 7.75 year; 44% male). There is a significant difference in proportions among the age groups in subjects affected with hypotonia, compulsive behavior, sleep disturbances, and seizures. Epilepsy is present in 50% of the total population, and more prevalent in subjects 12 years and older (P = 0.001). The median age of onset for absence seizures was 2 years, and 12 years for generalized tonic-clonic seizures (P < 0.01). The SUDEP rate in adults was 12% (4/33). There was a significant age-dependent negative correlation between GABA and T3 levels. Interpretation: There is an age-dependent association with worsening of epilepsy, behavioral disturbances including obsessive-compulsive behavior, and sleep disturbances with age in SSADH deficiency. There is a high risk of SUDEP. We have observed more absence seizures in younger patients, compared to tonic-clonic in the older cohort, which correlates with age-related changes in GABA and GHB concentration and thyroid function, as well as the natural history of seizures in the murine model.
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Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/epidemiologia , Deficiências do Desenvolvimento/sangue , Deficiências do Desenvolvimento/epidemiologia , Hidroxibutiratos/sangue , Succinato-Semialdeído Desidrogenase/deficiência , Hormônios Tireóideos/sangue , Ácido gama-Aminobutírico/sangue , Adolescente , Adulto , Fatores Etários , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Biomarcadores/sangue , Criança , Pré-Escolar , Deficiências do Desenvolvimento/complicações , Feminino , Humanos , Lactente , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Succinato-Semialdeído Desidrogenase/sangue , Adulto JovemRESUMO
The anticonvulsant vigabatrin (VGB; SabrilR) irreversibly inhibits GABA transaminase to increase neural GABA, yet its mechanism of retinal toxicity remains unclear. VGB is suggested to alter several amino acids, including homocarnosine, ß-alanine, ornithine, glycine, taurine, and 2-aminoadipic acid (AADA), the latter a homologue of glutamic acid. Here, we evaluate the effect of VGB on amino acid concentrations in mice, employing a continuous VGB infusion (subcutaneously implanted osmotic minipumps), dose-escalation paradigm (35-140â¯mg/kg/d, 12 days), and amino acid quantitation in eye, visual and prefrontal cortex, total brain, liver and plasma. We hypothesized that continuous VGB dosing would reveal numerous hitherto undescribed amino acid disturbances. Consistent amino acid elevations across tissues included GABA, ß-alanine, carnosine, ornithine and AADA, as well as neuroactive aspartic and glutamic acids, serine and glycine. Maximal increase of AADA in eye occurred at 35â¯mg/kg/d (41⯱â¯2â¯nmol/g (nâ¯=â¯21, vehicle) to 60⯱â¯8.5 (nâ¯=â¯8)), and at 70â¯mg/kg/d for brain (97⯱â¯6 (nâ¯=â¯21) to 145⯱â¯6 (nâ¯=â¯6)), visual cortex (128⯱â¯6 to 215⯱â¯19) and prefrontal cortex (124⯱â¯11 to 200⯱â¯13; mean⯱â¯SEM; pâ¯<â¯0.05), the first demonstration of tissue AADA accumulation with VGB in mammal. VGB effects on basic amino acids, including guanidino-species, suggested the capacity of VGB to alter urea cycle function and nitrogen disposal. The known toxicity of AADA in retinal glial cells highlights new avenues for assessing VGB retinal toxicity and other off-target effects.
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4-Aminobutirato Transaminase/metabolismo , Aminoácidos/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Vigabatrina/farmacologia , 4-Aminobutirato Transaminase/antagonistas & inibidores , Aminoácidos/sangue , Aminoácidos/genética , Animais , Anticonvulsivantes/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Retina/efeitos dos fármacos , Retina/metabolismoRESUMO
Vigabatrin (VGB; (S)-(+)/(R)-(-) 4-aminohex-5-enoic acid), an antiepileptic irreversibly inactivating GABA transaminase (GABA-T), manifests use-limiting ocular toxicity. Hypothesizing that the active S enantiomer of VGB would preferentially accumulate in eye and visual cortex (VC) as one potential mechanism for ocular toxicity, we infused racemic VGB into mice via subcutaneous minipump at 35, 70, and 140 mg/kg/d (n = 6-8 animals/dose) for 12 days. VGB enantiomers, total GABA and ß-alanine (BALA), 4-guanidinobutyrate (4-GBA), and creatine were quantified by mass spectrometry in eye, brain, liver, prefrontal cortex (PFC), and VC. Plasma VGB concentrations increased linearly by dose (3 ± 0.76 (35 mg/kg/d); 15.1 ± 1.4 (70 mg/kg/d); 34.6 ± 3.2 µmol/L (140 mg/kg/d); mean ± SEM) with an S/R ratio of 0.74 ± 0.02 (n = 14). Steady state S/R ratios (35, 70 mg/kg/d doses) were highest in eye (5.5 ± 0.2; P < 0.0001), followed by VC (3.9 ± 0.4), PFC (3.6 ± 0.3), liver (2.9 ± 0.1), and brain (1.5 ± 0.1; n = 13-14 each). Total VGB content of eye exceeded that of brain, PFC and VC at all doses. High-dose VGB diminished endogenous metabolite production, especially in PFC and VC. GABA significantly increased in all tissues (all doses) except brain; BALA increases were confined to liver and VC; and 4-GBA was prominently increased in brain, PFC and VC (and eye at high dose). Linear correlations between enantiomers and GABA were observed in all tissues, but only in PFC/VC for BALA, 4-GBA, and creatine. Preferential accumulation of the VGB S isomer in eye and VC may provide new insight into VGB ocular toxicity.
Assuntos
Anticonvulsivantes/farmacocinética , Vigabatrina/farmacocinética , Transtornos da Visão/prevenção & controle , 4-Aminobutirato Transaminase/antagonistas & inibidores , Animais , Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/química , Avaliação Pré-Clínica de Medicamentos , Olho/efeitos dos fármacos , Olho/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Estereoisomerismo , Distribuição Tecidual , Vigabatrina/efeitos adversos , Vigabatrina/química , Transtornos da Visão/induzido quimicamente , Córtex Visual/efeitos dos fármacos , Córtex Visual/metabolismo , Campos Visuais/efeitos dos fármacosRESUMO
A high-throughput approach to prokaryotic differential display has been developed. A large number of reverse transcription polymerase chain reactions (RT-PCR) are performed on total RNA isolated from induced and control bacterial cultures. Each RT-PCR reaction uses a single oligonucleotide primer and constitutes an independent sampling of the mRNA population. The large number of reactions performed allows the repeated sampling of the targeted polycistronic mRNA, which is clearly identified among possible false positives.
Assuntos
Primers do DNA/química , Perfilação da Expressão Gênica/métodos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Automação , Clonagem Molecular , DNA/química , DNA Complementar , Expressão Gênica , Modelos Genéticos , Células Procarióticas/metabolismo , RNA/química , RNA Mensageiro/metabolismo , RNA Ribossômico/química , Moldes Genéticos , Transcrição GênicaRESUMO
SUMMARY The DF signal molecule regulates the production of both yellow pigments (xanthomonadins) and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris. These two bacterial products are crucial to the epiphytic survival and pathogenicity of this pathogen on its plant hosts. Previous work suggested that DF is a butyrolactone, which the Streptomyces bacteria are known to utilize as signals. pigB is one of seven transcriptional units in the X. c. pv. campestris xanthomonadin gene cluster, and its inactivation results in the loss of DF signal, xanthomonadin and EPS production. Here, determination and analysis of the pigB DNA sequence reveals the presence of two open reading frames, the first (xanB1) encoding a putative reductase/halogenase, and the second (xanB2) showing the highest level of identity to Streptomyces genes encoding putative pteridine-dependent dioxygenase-like proteins. We show that xanB2 (but not xanB1) is needed for production of the DF signal, and that some Streptomyces strains produce functional analogues of DF. A role for xanB2 in the biosynthesis of DF is proposed.
RESUMO
High-throughput mRNA differential display (DD) was used to identify genes induced by cyclohexane in Brachymonas petroleovorans CHX, a recently isolated beta-proteobacterium that grows on cyclohexane. Two metabolic gene clusters were identified multiple times in independent reverse transcription polymerase chain reactions (RT-PCR) in the course of this DD experiment. These clusters encode genes believed to be required for cyclohexane metabolism. One gene cluster (8 kb) encodes the subunits of a multicomponent hydroxylase related to the soluble butane of Pseudomonas butanovora and methane monooxygenases (sMMO) of methanotrophs. We propose that this butane monooxygenase homologue carries out the oxidation of cyclohexane into cyclohexanol during growth. A second gene cluster (11 kb) contains almost all the genes required for the oxidation of cyclohexanol to adipic acid. Real-time PCR experiments confirmed that genes from both clusters are induced by cyclohexane. The role of the Baeyer-Villiger cyclohexanone monooxygenase of the second cluster was confirmed by heterologous expression in Escherichia coli.