RESUMEN
BACKGROUND & AIMS: Putative anion transporter-1 (PAT1, SLC26A6) plays a key role in intestinal oxalate and bicarbonate secretion. PAT1 knockout (PKO) mice exhibit hyperoxaluria and nephrolithiasis. Notably, diseases such as inflammatory bowel disease are also associated with higher risk of hyperoxaluria and nephrolithiasis. However, the potential role of PAT1 deficiency in gut-barrier integrity and susceptibility to colitis is currently elusive. METHODS: Age-matched PKO and wild-type littermates were administered 3.5% dextran sulfate sodium in drinking water for 6 days. Ileum and colon of control and treated mice were harvested. Messenger RNA and protein expression of tight junction proteins were determined by reverse transcription polymerase chain reaction and western blotting. Severity of inflammation was assessed by measuring diarrheal phenotype, cytokine expression, and hematoxylin and eosin staining. Gut microbiome and associated metabolome were analyzed by 16S ribosomal RNA sequencing and mass spectrometry, respectively. RESULTS: PKO mice exhibited significantly higher loss of body weight, gut permeability, colonic inflammation, and diarrhea in response to dextran sulfate sodium treatment. In addition, PKO mice showed microbial dysbiosis and significantly reduced levels of butyrate and butyrate-producing microbes compared with controls. Co-housing wild-type and PKO mice for 4 weeks resulted in PKO-like signatures on the expression of tight junction proteins in the colons of wild-type mice. CONCLUSIONS: Our data demonstrate that loss of PAT1 disrupts gut microbiome and related metabolites, decreases gut-barrier integrity, and increases host susceptibility to intestinal inflammation. These findings, thus, highlight a novel role of the oxalate transporter PAT1 in promoting gut-barrier integrity, and its deficiency appears to contribute to the pathogenesis of inflammatory bowel diseases.
Asunto(s)
Colitis , Colon , Sulfato de Dextran , Disbiosis , Microbioma Gastrointestinal , Ratones Noqueados , Permeabilidad , Transportadores de Sulfato , Animales , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Sulfato de Dextran/toxicidad , Colitis/microbiología , Colitis/metabolismo , Colitis/inducido químicamente , Colitis/patología , Colitis/genética , Ratones , Colon/microbiología , Colon/patología , Colon/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Modelos Animales de Enfermedad , Íleon/patología , Íleon/microbiología , Íleon/metabolismo , Diarrea/microbiología , Diarrea/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Proteínas de Uniones Estrechas/genética , Ratones Endogámicos C57BL , Masculino , Antiportadores/genética , Antiportadores/metabolismo , Antiportadores/deficienciaRESUMEN
Aspartate-glutamate carrier isoform 1 (AGC1) is a carrier responsible for the export of mitochondrial aspartate in exchange for cytosolic glutamate and is part of the malate-aspartate shuttle, essential for the balance of reducing equivalents in the cells. In the brain, mutations in SLC25A12 gene, encoding for AGC1, cause an ultra-rare genetic disease, reported as a neurodevelopmental encephalopathy, whose symptoms include global hypomyelination, arrested psychomotor development, hypotonia and seizures. Among the biological components most affected by AGC1 deficiency are oligodendrocytes, glial cells responsible for myelination processes, and their precursors [oligodendrocyte progenitor cells (OPCs)]. The AGC1 silencing in an in vitro model of OPCs was documented to cause defects of proliferation and differentiation, mediated by alterations of histone acetylation/deacetylation. Disrupting AGC1 activity could possibly reduce the availability of acetyl groups, leading to perturbation of many biological pathways, such as histone modifications and fatty acids formation for myelin production. Here, we explore the transcriptome of mouse OPCs partially silenced for AGC1, reporting results of canonical analyses (differential expression) and pathway enrichment analyses, which highlight a disruption in fatty acids synthesis from both a regulatory and enzymatic stand. We further investigate the cellular effects of AGC1 deficiency through the identification of most affected transcriptional networks and altered alternative splicing. Transcriptional data were integrated with differential metabolite abundance analysis, showing downregulation of several amino acids, including glutamine and aspartate. Taken together, our results provide a molecular foundation for the effects of AGC1 deficiency in OPCs, highlighting the molecular mechanisms affected and providing a list of actionable targets to mitigate the effects of this pathology.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias , Enfermedades Mitocondriales , Células Precursoras de Oligodendrocitos , Trastornos Psicomotores , Ratones , Animales , Regulación hacia Abajo/genética , Células Precursoras de Oligodendrocitos/metabolismo , Ácido Aspártico/metabolismo , Isoformas de Proteínas/metabolismo , Ácidos GrasosRESUMEN
AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named "early infantile epileptic encephalopathy 39" (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.
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Agrecanos/genética , Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Predisposición Genética a la Enfermedad , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/etiología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/metabolismo , Trastornos Psicomotores/etiología , Trastornos Psicomotores/metabolismo , Agrecanos/deficiencia , Agrecanos/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Antiportadores/metabolismo , Biomarcadores , Encéfalo/metabolismo , Terapia Combinada , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Metabolismo Energético , Estudios de Asociación Genética , Ácido Glutámico/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/terapia , Humanos , Malatos/metabolismo , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/terapia , Vaina de Mielina/metabolismo , Oxidación-Reducción , Fenotipo , Trastornos Psicomotores/diagnóstico , Trastornos Psicomotores/terapiaRESUMEN
TMEM165 deficiency leads to skeletal disorder characterized by major skeletal dysplasia and pronounced dwarfism. However, the molecular mechanisms involved have not been fully understood. Here, we uncover that TMEM165 deficiency impairs the synthesis of proteoglycans by producing a blockage in the elongation of chondroitin-and heparan-sulfate glycosaminoglycan chains leading to the synthesis of proteoglycans with shorter glycosaminoglycan chains. We demonstrated that the blockage in elongation of glycosaminoglycan chains is not due to defect in the Golgi elongating enzymes but rather to availability of the co-factor Mn2+. Supplementation of cell with Mn2+ rescue the elongation process, confirming a role of TMEM165 in Mn2+ Golgi homeostasis. Additionally, we showed that TMEM165 deficiency functionally impairs TGFß and BMP signaling pathways in chondrocytes and in fibroblast cells of TMEM165 deficient patients. Finally, we found that loss of TMEM165 impairs chondrogenic differentiation by accelerating the timing of Ihh expression and promoting early chondrocyte maturation and hypertrophy. Collectively, our results indicate that TMEM165 plays an important role in proteoglycan synthesis and underline the critical role of glycosaminoglycan chains structure in the regulation of chondrogenesis. Our data also suggest that Mn2+ supplementation may be a promising therapeutic strategy in the treatment of TMEM165 deficient patients.
Asunto(s)
Antiportadores/deficiencia , Antiportadores/metabolismo , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular/genética , Condrocitos/metabolismo , Condrocitos/patología , Sulfatos de Condroitina/biosíntesis , Enanismo/metabolismo , Proteoglicanos de Heparán Sulfato/biosíntesis , Transducción de Señal/genética , Animales , Antiportadores/genética , Estudios de Casos y Controles , Proteínas de Transporte de Catión/genética , Línea Celular Tumoral , Condrogénesis/genética , Enanismo/patología , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes/métodos , Glicosilación , Células HEK293 , Humanos , Hipertrofia/metabolismo , Ratones , TransfecciónRESUMEN
Mutations in TUBB4A are associated with a spectrum of neurologic disorders categorized as TUBB4A-related leukoencephalopathy. Affected children can present with global developmental delay or normal early development, followed by a variable loss of skills over time. Further research is needed to characterize the factors associated with the divergent developmental trajectories in this rare monogenic disorder because this phenotypic spectrum is not fully explained by genotype alone.To characterize early psychomotor features, developmental milestones and age of disease onset were collected from medical records (n=54 individuals). Three subcohorts were identified: individuals with the common p.Asp249Asn variant vs all other genotypes with either early (<12 months of age) or late onset of presentation. Individuals with the p.Asp249Asn variant or those with non-p.Asp249Asn genotypes with later disease onset attained key milestones, including head control, sitting, and independent walking. Subjects with early-onset, non-p.Asp249Asn-associated disease were less likely to achieve developmental milestones. Next, we defined the developmental severity as the percentage of milestones attained by age 2 years. The mild form was defined as attaining at least 75% of key developmental milestones. Among cohort categorized as mild, individuals with p.Asp249Asn variant were more likely to lose acquired abilities when compared with non-p.Asp249Asn individuals.Our results suggest multiple influences on developmental trajectory, including a strong contribution from genotype and age of onset. Further studies are needed to identify additional factors that influence overall outcomes to better counsel families and to design clinical trials with appropriate clinical endpoints.
Asunto(s)
Ganglios Basales/patología , Cerebelo/patología , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/genética , Leucoencefalopatías/complicaciones , Leucoencefalopatías/genética , Adolescente , Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiportadores/deficiencia , Antiportadores/genética , Atrofia , Niño , Desarrollo Infantil , Preescolar , Estudios de Cohortes , Discapacidades del Desarrollo/patología , Femenino , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Lactante , Recién Nacido , Leucoencefalopatías/patología , Masculino , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Mutación , Trastornos Psicomotores/complicaciones , Trastornos Psicomotores/genética , Trastornos Psicomotores/patología , Estudios Retrospectivos , Tubulina (Proteína)/genéticaRESUMEN
Intraventricular hemorrhage (IVH) results in periventricular inflammation, hypomyelination of the white matter, and hydrocephalus in premature infants. No effective therapy exists to prevent these disorders. Peroxisome proliferator activated receptor-γ (PPAR-γ) agonists reduce inflammation, alleviate free radical generation, and enhance microglial phagocytosis, promoting clearance of debris and red blood cells. We hypothesized that activation of PPAR-γ would enhance myelination, reduce hydrocephalus, and promote neurological recovery in newborns with IVH. These hypotheses were tested in a preterm rabbit model of IVH; autopsy brain samples from premature infants with and without IVH were analyzed. We found that IVH augmented PPAR-γ expression in microglia of both preterm human infants and rabbit kits. The treatment with PPAR-γ agonist or PPAR-γ overexpression by adenovirus delivery further elevated PPAR-γ levels in microglia, reduced proinflammatory cytokines, increased microglial phagocytosis, and improved oligodendrocyte progenitor cell (OPC) maturation in kits with IVH. Transcriptomic analyses of OPCs identified previously unrecognized PPAR-γ-induced genes for purinergic signaling, cyclic adenosine monophosphate generation, and antioxidant production, which would reprogram these progenitors toward promoting myelination. RNA-sequencing analyses of microglia revealed PPAR-γ-triggered down-regulation of several proinflammatory genes and transcripts having roles in Parkinson's disease and amyotrophic lateral sclerosis, contributing to neurological recovery in kits with IVH. Accordingly, PPAR-γ activation enhanced myelination and neurological function in kits with IVH. This also enhanced microglial phagocytosis of red blood cells but did not reduce hydrocephalus. Treatment with PPAR-γ agonist might enhance myelination and neurological recovery in premature infants with IVH.
Asunto(s)
Hemorragia Cerebral Intraventricular/metabolismo , Proteínas de la Mielina/biosíntesis , PPAR gamma/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Animales Recién Nacidos , Antiportadores/deficiencia , Antiportadores/metabolismo , Hemorragia Cerebral Intraventricular/patología , Modelos Animales de Enfermedad , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Recien Nacido Prematuro , Microglía/metabolismo , Enfermedades Mitocondriales/metabolismo , Oligodendroglía/patología , PPAR gamma/agonistas , Trastornos Psicomotores/metabolismo , Conejos , Rosiglitazona/farmacología , Análisis de Secuencia de ARN/métodosRESUMEN
PYCR2 pathogenic variants lead to an autosomal recessive hypomyelinating leukodystrophy 10 (HLD10), characterized by global developmental delay, microcephaly, facial dysmorphism, movement disorder, and hypomyelination. This study identified the first two unrelated Thai patients with HLD10. Patient 1 harbored the novel compound heterozygous variants, c.257T>G (p.Val86Gly) and c.400G>A (p.Val134Met), whereas patient 2 possessed the homozygous variant, c.400G>A (p.Val134Met), in PYCR2. Haplotype analysis revealed that the two families' members shared a 2.3 Mb region covering the c.400G>A variant, indicating a common ancestry. The variant was estimated to age 1450 years ago. Since the c.400G>A was detected in three out of four mutant alleles and with a common ancestry, this variant might be common in Thai patients. We also reviewed the phenotype and genotype of all 35 previously reported PYCR2 patients and found that majorities of cases were homozygous with a consanguineous family history, except patient 1 and another reported case who were compound heterozygous. All patients had microcephaly and developmental delay. Hypotonia and peripheral spasticity were common. Hypomyelination or delayed myelination was a typical radiographic feature. Here, we report the first two Thai patients with HLD10 with the novel PYCR2 variants expanding the genotypic spectrum and suggest that the c.400G>A might be a common mutation in Thai patients.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Discapacidades del Desarrollo/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Microcefalia/genética , Enfermedades Mitocondriales/genética , Trastornos del Movimiento/genética , Trastornos Psicomotores/genética , Pirrolina Carboxilato Reductasas/genética , Adolescente , Alelos , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiportadores/genética , Niño , Preescolar , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/patología , Femenino , Genotipo , Haplotipos/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Homocigoto , Humanos , Masculino , Microcefalia/complicaciones , Microcefalia/patología , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/patología , Trastornos del Movimiento/complicaciones , Trastornos del Movimiento/patología , Mutación , Linaje , Fenotipo , Trastornos Psicomotores/complicaciones , Trastornos Psicomotores/patología , Adulto JovenRESUMEN
BACKGROUND & AIMS: The down-regulated in adenoma (DRA) protein, encoded by SLC26A3, a key intestinal chloride anion exchanger, has recently been identified as a novel susceptibility gene for inflammatory bowel disease (IBD). However, the mechanisms underlying the increased susceptibility to inflammation induced by the loss of DRA remain elusive. Compromised barrier is a key event in IBD pathogenesis. The current studies were undertaken to elucidate the impact of DRA deficiency on epithelial barrier integrity and to define underlying mechanisms. METHODS: Wild-type and DRA-knockout (KO) mice and crypt-derived colonoids were used as models for intestinal epithelial response. Paracellular permeability was measured by using fluorescein isothiocyanate-dextran flux. Immunoblotting, immunofluorescence, immunohistochemistry, and ribonucleoprotein immunoprecipitation assays were performed. Gut microbiome analysis was conducted to investigate the impact of DRA deficiency on gut microbial communities. RESULTS: DRA-KO mice exhibited an increased colonic paracellular permeability with significantly decreased levels of tight junction/adherens junction proteins, including ZO-1, occludin, and E-cadherin. A similar expression pattern of occludin and E-cadherin was observed in colonoids derived from DRA-KO mice and short hairpin RNA-mediated DRA knockdown in Caco-2 cells. Microbial analysis showed gut dysbiosis in DRA-KO mice. However, cohousing studies showed that dysbiosis played only a partial role in maintaining tight junction protein expression. Furthermore, our results showed increased binding of RNA-binding protein CUGBP1 with occludin and E-cadherin genes in DRA-KO mouse colon, suggesting that posttranscriptional mechanisms play a key role in gut barrier dysfunction. CONCLUSIONS: To our knowledge, our studies demonstrate a novel role of DRA in maintaining the intestinal epithelial barrier function and potential implications of its dysregulation in IBD pathogenesis.
Asunto(s)
Antiportadores/deficiencia , Antiportadores de Cloruro-Bicarbonato/deficiencia , Disbiosis/inmunología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/patología , Transportadores de Sulfato/deficiencia , Animales , Antiportadores/genética , Proteínas CELF1/metabolismo , Células CACO-2 , Cadherinas/metabolismo , Antiportadores de Cloruro-Bicarbonato/genética , Modelos Animales de Enfermedad , Disbiosis/microbiología , Disbiosis/patología , Técnicas de Silenciamiento del Gen , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Masculino , Ratones , Ratones Noqueados , Ocludina/metabolismo , Permeabilidad , Transportadores de Sulfato/genética , Uniones Estrechas/patologíaRESUMEN
CASE: We are reporting the third unrelated case of cerebral aspartate-glutamate carrier isoform 1 (AGC1) deficiency. Patient is a 21-month-old Yemeni male who presented with refractory seizure disorder and developmental arrest. Neuroimaging showed cerebral volume loss and diminished N-acetylaspartate (NAA) peak. Whole exome sequencing revealed a homozygous novel missense variant in the SLC25A12 gene. Patient's seizure frequency abated drastically following initiation of ketogenic diet. DISCUSSION AND CONCLUSION: Cerebral AGC1 deficiency results in dysfunction of mitochondrial malate aspartate shuttle, thereby prohibiting myelin synthesis. There are significant phenotypic commonalities between our patient and previously reported cases including intractable epilepsy, psychomotor delay, cerebral atrophy, and diminished NAA peak. Our report also provides evidence regarding beneficial effect of ketogenic diet in this rare neurometabolic epilepsy.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Dieta Cetogénica , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Enfermedades Mitocondriales/diagnóstico , Proteínas de Transporte de Membrana Mitocondrial/genética , Trastornos Psicomotores/diagnóstico , Adulto , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiportadores/genética , Epilepsia Refractaria/dietoterapia , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/dietoterapia , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/fisiopatología , Humanos , Masculino , Enfermedades Mitocondriales/dietoterapia , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/fisiopatología , Mutación Missense , Isoformas de Proteínas , Trastornos Psicomotores/dietoterapia , Trastornos Psicomotores/genética , Trastornos Psicomotores/fisiopatología , Secuenciación del Exoma , Adulto JovenAsunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Deleción Cromosómica , Cromosomas Humanos Par 18/genética , Variaciones en el Número de Copia de ADN , Exoma/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Enfermedades Mitocondriales/patología , Mosaicismo , Trastornos Psicomotores/patología , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiportadores/genética , Niño , Femenino , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Humanos , Enfermedades Mitocondriales/genética , Prevalencia , Trastornos Psicomotores/genética , Secuenciación del ExomaRESUMEN
The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in HCO3- transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on HCO3- while, in contrast, fluid secretion by Slc26a6-/- mice was independent of HCO3-, suggesting that Slc26a6 mediates the HCO3--dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced HCO3- secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in HCO3--dependent fluid secretion but that another major HCO3--independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.
Asunto(s)
Células Acinares/metabolismo , Antiportadores/genética , Bicarbonatos/metabolismo , Líquidos Corporales/metabolismo , Páncreas Exocrino/metabolismo , Transportadores de Sulfato/genética , Células Acinares/citología , Animales , Anoctamina-1/genética , Anoctamina-1/metabolismo , Antiportadores/deficiencia , Acuaporina 5/genética , Acuaporina 5/metabolismo , Peso Corporal , Línea Celular , Membrana Celular/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Transporte Iónico , Masculino , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Tamaño de los Órganos , Páncreas Exocrino/citología , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Transportadores de Sulfato/deficienciaRESUMEN
Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate-aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor α (PDGFα) and Transforming Growth Factor ßs (TGFßs). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency.
Asunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Mitocondrias/metabolismo , Células Precursoras de Oligodendrocitos/citología , Células Precursoras de Oligodendrocitos/metabolismo , Adenosina Trifosfato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Antiportadores/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Regulación hacia Abajo , Silenciador del Gen , Lactatos/metabolismo , Ventrículos Laterales/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Neuronas/metabolismo , Factor de Crecimiento Derivado de Plaquetas , Especies Reactivas de Oxígeno/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy (lde/lde). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.
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Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Corteza Cerebral/metabolismo , Enanismo/genética , Epilepsia/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Mitocondriales/genética , Neurogénesis/genética , Trastornos Psicomotores/genética , Proteínas Supresoras de Tumor/genética , Oxidorreductasa que Contiene Dominios WW/genética , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/genética , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Antiportadores/genética , Antiportadores/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Recuento de Células , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Enanismo/metabolismo , Enanismo/patología , Epilepsia/metabolismo , Epilepsia/patología , Regulación del Desarrollo de la Expresión Génica , Mutación de Línea Germinal , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Masculino , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Neuronas/metabolismo , Neuronas/patología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Prosencéfalo/crecimiento & desarrollo , Prosencéfalo/metabolismo , Prosencéfalo/patología , Trastornos Psicomotores/metabolismo , Trastornos Psicomotores/patología , Ratas , Ratas Transgénicas , Transducción de Señal , Proteínas Supresoras de Tumor/deficiencia , Oxidorreductasa que Contiene Dominios WW/deficienciaRESUMEN
Milk is a hallmark of mammals that is critical for normal growth and development of offspring. During biosynthesis of lactose in the Golgi complex, H+ is produced as a by-product, and there is no known mechanism for maintaining luminal pH within the physiological range. Here, using conditional, tissue-specific knockout mice, immunostaining, and biochemical assays, we test whether the putative H+/Ca2+/Mn2+ exchanger known as TMEM165 (transmembrane protein 165) participates in normal milk production. We find TMEM165 is crucial in the lactating mammary gland for normal biosynthesis of lactose and for normal growth rates of nursing pups. The milk of TMEM165-deficient mice contained elevated concentrations of fat, protein, iron, and zinc, which are likely caused by decreased osmosis-mediated dilution of the milk caused by the decreased biosynthesis of lactose. When normalized to total protein levels, only calcium and manganese levels were significantly lower in the milk from TMEM165-deficient dams than control dams. These findings suggest that TMEM165 supplies Ca2+ and Mn2+ to the Golgi complex in exchange for H+ to sustain the functions of lactose synthase and potentially other glycosyl-transferases. Our findings highlight the importance of cation and pH homeostasis in the Golgi complex of professional secretory cells and the critical role of TMEM165 in this process.
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Antiportadores/metabolismo , Proteínas de Transporte de Catión/metabolismo , Aparato de Golgi/metabolismo , Leche/metabolismo , Animales , Antiportadores/deficiencia , Antiportadores/genética , Peso Corporal , Proteínas de Transporte de Catión/deficiencia , Proteínas de Transporte de Catión/genética , Femenino , Técnicas de Inactivación de Genes , Lactancia , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/fisiología , Ratones , ÓsmosisRESUMEN
The apical membrane Cl-/oxalate exchanger SLC26A6 has been demonstrated to play a role in proximal tubule NaCl transport based on studies in microperfused tubules. The present study is directed at characterizing the role of SLC26A6 in NaCl homeostasis in vivo under physiological conditions. Free-flow micropuncture studies revealed that volume and Cl- absorption were similar in surface proximal tubules of wild-type and Slc26a6-/- mice. Moreover, the increments in urine flow rate and sodium excretion following thiazide and furosemide infusion were identical in wild-type and Slc26a6-/- mice, indicating no difference in NaCl delivery out of the proximal tubule. The absence of an effect of deletion of SLC26A6 on NaCl homeostasis was further supported by the absence of lower blood pressure in Slc26a6-/- compared with wild-type mice on normal or low-salt diets. Moreover, raising plasma and urine oxalate by feeding mice a diet enriched in soluble oxalate did not affect mean blood pressure. In contrast to the lack of effect of SLC26A6 deletion on NaCl homeostasis, fractional excretion of oxalate was reduced from 1.6 in wild-type mice to 0.7 in Slc26a6-/- mice. We conclude that, although SLC26A6 is dispensable for renal NaCl homeostasis, it is required for net renal secretion of oxalate.
Asunto(s)
Antiportadores/metabolismo , Túbulos Renales Proximales/metabolismo , Ácido Oxálico/orina , Eliminación Renal , Cloruro de Sodio Dietético/orina , Transportadores de Sulfato/metabolismo , Animales , Antiportadores/deficiencia , Antiportadores/genética , Presión Sanguínea , Dieta Hiposódica , Femenino , Genotipo , Homeostasis , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Fenotipo , Transportadores de Sulfato/deficiencia , Transportadores de Sulfato/genéticaRESUMEN
Folate metabolism in the brain is critically important and serves a number of vital roles in nucleotide synthesis, single carbon metabolism/methylation, amino acid metabolism, and mitochondrial translation. Genetic defects in almost every enzyme of folate metabolism have been reported to date, and most have neurological sequelae. We report 2 patients presenting with a neurometabolic disorder associated with biallelic variants in the MTHFS gene, encoding 5,10-methenyltetrahydrofolate synthetase. Both patients presented with microcephaly, short stature, severe global developmental delay, progressive spasticity, epilepsy, and cerebral hypomyelination. Baseline CSF 5-methyltetrahydrolate (5-MTHF) levels were in the low-normal range. The first patient was treated with folinic acid, which resulted in worsening cerebral folate deficiency. Treatment in this patient with a combination of oral L-5-methyltetrahydrofolate and intramuscular methylcobalamin was able to increase CSF 5-MTHF levels, was well tolerated over a 4â¯month period, and resulted in subjective mild improvements in functioning. Measurement of MTHFS enzyme activity in fibroblasts confirmed reduced activity. The direct substrate of the MTHFS reaction, 5-formyl-THF, was elevated 30-fold in patient fibroblasts compared to control, supporting the hypothesis that the pathophysiology of this disorder is a manifestation of toxicity from this metabolite.
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Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Ligasas de Carbono-Nitrógeno/genética , Epilepsia/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Microcefalia/genética , Enfermedades Mitocondriales/genética , Trastornos Psicomotores/genética , Sistemas de Transporte de Aminoácidos Acídicos/líquido cefalorraquídeo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiportadores/líquido cefalorraquídeo , Antiportadores/genética , Antiportadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Ligasas de Carbono-Nitrógeno/líquido cefalorraquídeo , Ligasas de Carbono-Nitrógeno/deficiencia , Ligasas de Carbono-Nitrógeno/metabolismo , Epilepsia/líquido cefalorraquídeo , Epilepsia/complicaciones , Epilepsia/patología , Femenino , Receptor 1 de Folato/deficiencia , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/líquido cefalorraquídeo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/complicaciones , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Masculino , Enfermedades Metabólicas/líquido cefalorraquídeo , Enfermedades Metabólicas/complicaciones , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/patología , Microcefalia/líquido cefalorraquídeo , Microcefalia/complicaciones , Microcefalia/patología , Enfermedades Mitocondriales/líquido cefalorraquídeo , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/metabolismo , Malformaciones del Sistema Nervioso/líquido cefalorraquídeo , Malformaciones del Sistema Nervioso/complicaciones , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Distrofias Neuroaxonales , Trastornos Psicomotores/líquido cefalorraquídeo , Trastornos Psicomotores/complicaciones , Trastornos Psicomotores/metabolismo , Tetrahidrofolatos/líquido cefalorraquídeo , Tetrahidrofolatos/metabolismoRESUMEN
BACKGROUND AND AIMS: Glycogen storage disease type Ib (GSD1b) is a rare metabolic and immune disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT) and characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenomas. Despite maximal therapy, based on a strict diet and on granulocyte colony-stimulating factor treatment, long-term severe complications still develop. Understanding the pathophysiology of GSD1b is a prerequisite to develop new therapeutic strategies and depends on the availability of animal models. The G6PT-KO mouse mimics the human disease but is very fragile and rarely survives weaning. We generated a conditional G6PT-deficient mouse as an alternative model for studying the long-term pathophysiology of the disease. We utilized this conditional mouse to develop an inducible G6PT-KO model to allow temporally regulated G6PT deletion by the administration of tamoxifen (TM). METHODS: We generated a conditional G6PT-deficient mouse utilizing the CRElox strategy. Histology, histochemistry, and phenotype analyses were performed at different times after TM-induced G6PT inactivation. Neutrophils and monocytes were isolated and analyzed for functional activity with standard techniques. RESULTS: The G6PT-inducible KO mice display the expected disturbances of G6P metabolism and myeloid dysfunctions of the human disorder, even though with a milder intensity. CONCLUSIONS: TM-induced inactivation of G6PT in these mice leads to a phenotype which mimics that of human GSD1b patients. The conditional mice we have generated represent an excellent tool to study the tissue-specific role of the G6PT gene and the mechanism of long-term complications in GSD1b.
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Antiportadores/deficiencia , Modelos Animales de Enfermedad , Glucosa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/genética , Homeostasis , Proteínas de Transporte de Monosacáridos/deficiencia , Animales , Antiportadores/genética , Enfermedad del Almacenamiento de Glucógeno Tipo I/etiología , Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Ratones , Ratones Noqueados , Proteínas de Transporte de Monosacáridos/genética , Neutropenia/etiología , Tamoxifeno/administración & dosificaciónRESUMEN
Early-onset epileptic encephalopathies (EOEEs) are a genetically heterogeneous collection of severe epilepsies often associated with psychomotor regression. Mutations in SZT2, a known seizure threshold regulator gene, are a newly identified cause of EOEE. We present an individual with EOEE, macrocephaly, and developmental regression with compound heterozygous mutations in SZT2 as identified by whole exome sequencing. Serial imaging characterized the novel finding of progressive loss of central myelination. This case expands our clinical understanding of the SZT2-phenotype and emphasizes the role of this gene in the diagnostic investigation for EOEE and leukoencephalopathies.
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Leucoencefalopatías/genética , Mutación , Proteínas del Tejido Nervioso/genética , Espasmos Infantiles/genética , Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiportadores/deficiencia , Antiportadores/genética , Preescolar , Discapacidades del Desarrollo/genética , Femenino , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico por imagen , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/etiología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Heterocigoto , Humanos , Lactante , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/etiología , Imagen por Resonancia Magnética , Megalencefalia/diagnóstico por imagen , Megalencefalia/genética , Enfermedades Mitocondriales/diagnóstico por imagen , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/genética , Trastornos Psicomotores/diagnóstico por imagen , Trastornos Psicomotores/etiología , Trastornos Psicomotores/genética , Espasmos Infantiles/diagnóstico por imagen , Espasmos Infantiles/etiologíaAsunto(s)
Sistemas de Transporte de Aminoácidos Acídicos/deficiencia , Antiportadores/deficiencia , Distonía/etiología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/fisiopatología , Enfermedades Mitocondriales/fisiopatología , Trastornos Psicomotores/fisiopatología , Síndrome de Sturge-Weber/complicaciones , Niño , Distonía/diagnóstico por imagen , Distonía/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico por imagen , Humanos , Enfermedades Mitocondriales/diagnóstico por imagen , Trastornos Psicomotores/diagnóstico por imagen , Síndrome de Sturge-Weber/diagnóstico por imagen , Síndrome de Sturge-Weber/genética , Tomografía Computarizada por Rayos X , Tubulina (Proteína)/genéticaRESUMEN
Patients with short QT syndrome (SQTS) may present with syncope, ventricular fibrillation or sudden cardiac death. Six SQTS susceptibility genes, encoding cation channels, explain <25% of SQTS cases. Here we identify a missense mutation in the anion exchanger (AE3)-encoding SLC4A3 gene in two unrelated families with SQTS. The mutation causes reduced surface expression of AE3 and reduced membrane bicarbonate transport. Slc4a3 knockdown in zebrafish causes increased cardiac pHi, short QTc, and reduced systolic duration, which is rescued by wildtype but not mutated SLC4A3. Mechanistic analyses suggest that an increase in pHi and decrease in [Cl-]i shortened the action potential duration. However, other mechanisms may also play a role. Altered anion transport represents a mechanism for development of arrhythmia and may provide new therapeutic possibilities.