RESUMEN
Antibody-mediated autoimmune pathologies like membranous nephropathy are difficult to model, particularly in the absence of local target antigen expression in model organisms such as mice and rats; as is the case for phospholipase A2 receptor 1 (PLA2R1), the major autoantigen in membranous nephropathy. Here, we generated a transgenic mouse line expressing the full-length human PLA2R1 in podocytes, which has no kidney impairment after birth. Beginning from the age of three weeks, these mice spontaneously developed anti-human PLA2R1 antibodies, a nephrotic syndrome with progressive albuminuria and hyperlipidemia, and the typical morphological signs of membranous nephropathy with granular glomerular deposition of murine IgG in immunofluorescence and subepithelial electron-dense deposits by electron microscopy. Importantly, human PLA2R1-expressing Rag2-/- mice, which lack mature and functioning B and T lymphocytes, developed neither anti-PLA2R1 antibodies nor proteinuria. Thus, our work demonstrates that podocyte expression of human PLA2R1 can induce membranous nephropathy with an underlying antibody-mediated pathogenesis in mice. Importantly, this antibody-mediated model enables proof-of-concept evaluations of antigen-specific treatment strategies, e.g., targeting autoantibodies or autoantibody-producing cells, and may further help understand the autoimmune pathogenesis of membranous nephropathy.
Asunto(s)
Glomerulonefritis Membranosa , Podocitos , Animales , Humanos , Ratones , Ratas , Autoanticuerpos , Autoantígenos/genética , Glomerulonefritis Membranosa/diagnóstico , Glomérulos Renales/patología , Podocitos/patología , Receptores de Fosfolipasa A2/genética , Ratones TransgénicosRESUMEN
The phospholipase A2 receptor 1 (PLA2R1) is the major target antigen in patients with membranous nephropathy (MN), an antibody-mediated autoimmune glomerular disease. Investigation of MN pathogenesis has been hampered by the lack of reliable animal models. Here, we overcome this issue by generating a transgenic mouse line expressing a chimeric PLA2R1 (chPLA2R1) consisting of three human PLA2R1 domains (cysteine-rich, fibronectin type-II and CTLD1) and seven murine PLA2R1 domains (CTLD2-8) specifically in podocytes. Mice expressing the chPLA2R1 were healthy at birth and showed no major glomerular alterations when compared to mice with a wild-type PLA2R1 status. Upon active immunization with human PLA2R1 (hPLA2R1), chPLA2R1-positive mice developed anti-hPLA2R1 antibodies, a nephrotic syndrome, and all major histological features of MN, including granular deposition of mouse IgG and complement components in immunofluorescence and subepithelial electron-dense deposits and podocyte foot process effacement in electron microscopy. In order to investigate the role of the complement system in this model, we further crossed chPLA2R1-positive mice with mice lacking the central complement component C3 (C3-/- mice). Upon immunization with hPLA2R1, chPLA2R1-positive C3-/- mice had substantially less severe albuminuria and nephrotic syndrome when compared to chPLA2R1-positive mice with a wild-type C3 status. In conclusion, we introduce a novel active immunization model of PLA2R1-associated MN and demonstrate a pathogenic role of the complement system in this model.
Asunto(s)
Enfermedades Autoinmunes , Glomerulonefritis Membranosa , Síndrome Nefrótico , Humanos , Ratones , Animales , Receptores de Fosfolipasa A2/genética , Autoanticuerpos , Ratones Transgénicos , Vacunación , Complemento C3 , Modelos Animales de EnfermedadRESUMEN
Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.
Asunto(s)
Ácido Glutámico/metabolismo , Cinesinas/metabolismo , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Memoria a Corto Plazo , Neuronas/metabolismo , Espastina/deficiencia , Tubulina (Proteína)/metabolismo , Potenciales de Acción , Animales , Membrana Celular/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Potenciales Postsinápticos Excitadores , Hipocampo/patología , Hipocampo/fisiopatología , Ratones Noqueados , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Actividad Motora , Neuronas/patología , Neuronas/ultraestructura , Transporte de Proteínas , Espastina/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Vesículas Sinápticas/metabolismoRESUMEN
Calcimimetic agents allosterically increase the calcium ion sensitivity of the calcium-sensing receptor (CaSR), which is expressed in the tubular system and to a lesser extent in podocytes. Activation of this receptor can reduce glomerular proteinuria and structural damage in proteinuric animal models. However, the precise role of the podocyte CaSR remains unclear. Here, a CaSR knockdown in cultured murine podocytes and a podocyte-specific CaSR knockout in BALB/c mice were generated to study its role in proteinuria and kidney function. Podocyte CaSR knockdown abolished the calcimimetic R-568 mediated calcium ion-influx, disrupted the actin cytoskeleton, and reduced cellular attachment and migration velocity. Adriamycin-induced proteinuria enhanced glomerular CaSR expression in wild-type mice. Albuminuria, podocyte foot process effacement, podocyte loss and glomerular sclerosis were significantly more pronounced in adriamycin-treated podocyte-specific CaSR knockout mice compared to wild-type littermates. Co-treatment of wild-type mice with adriamycin and the calcimimetic cinacalcet reduced proteinuria in wild-type, but not in podocyte-specific CaSR knockout mice. Additionally, four children with nephrotic syndrome, whose parents objected to glucocorticoid therapy, were treated with cinacalcet for one to 33 days. Proteinuria declined transiently by up to 96%, serum albumin increased, and edema resolved. Thus, activation of podocyte CaSR regulates key podocyte functions in vitro and reduced toxin-induced proteinuria and glomerular damage in mice. Hence, our findings suggest a potential novel role of CaSR signaling in control of glomerular disease.
Asunto(s)
Enfermedades Renales , Podocitos , Animales , Calcio/metabolismo , Cinacalcet/farmacología , Cinacalcet/uso terapéutico , Doxorrubicina/toxicidad , Humanos , Enfermedades Renales/metabolismo , Ratones , Ratones Noqueados , Podocitos/metabolismo , Proteinuria/inducido químicamente , Proteinuria/genética , Proteinuria/metabolismo , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismoRESUMEN
L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.
Asunto(s)
Enfermedades Genéticas Ligadas al Cromosoma X/tratamiento farmacológico , Discapacidad Intelectual/tratamiento farmacológico , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Peptidomiméticos/uso terapéutico , Paraplejía Espástica Hereditaria/tratamiento farmacológico , Animales , Células Cultivadas , Cerebelo/citología , Cerebelo/metabolismo , Cerebelo/patología , Ventrículos Cerebrales/metabolismo , Ventrículos Cerebrales/patología , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Clorhidrato de Duloxetina/farmacología , Clorhidrato de Duloxetina/uso terapéutico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Locomoción , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Molécula L1 de Adhesión de Célula Nerviosa/genética , Neurogénesis , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Peptidomiméticos/farmacología , Fenotipo , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/patología , Toluidinas/farmacología , Toluidinas/uso terapéutico , Trimebutino/farmacología , Trimebutino/uso terapéuticoRESUMEN
The important functions of cell adhesion molecule L1 in the nervous system depend on diverse proteolytic enzymes which generate different L1 fragments. It has been reported that cleavage in the third fibronectin type III (FNIII) homologous domain generates the fragments L1-80 and L1-140, while cleavage in the first FNIII domain yields the fragments L1-70 and L1-135. These results raised questions concerning the L1 cleavage sites. We thus generated gene-edited mice expressing L1 with mutations of the cleavage sites either in the first or third FNIII domain. By immunoprecipitations and immunoblot analyses using brain homogenates and different L1 antibodies, we show that L1-70 and L1-135 are generated in wild-type mice, but not or only to a low extent in L1 mutant mice. L1-80 and L1-140 were not detected in wild-type or mutant mice. Mass spectrometry confirmed the results from immunoprecipitations and immunoblot analyses. Based on these observations, we propose that L1-70 and L1-135 are the predominant fragments in the mouse nervous system and that the third FNIII domain is decisive for generating these fragments. Treatment of cultured cerebellar neurons with trypsin or plasmin, which were both proposed to generate L1-80 and L1-140 by cleaving in the third FNIII domain, showed by immunoprecipitations and immunoblot analyses that both proteases lead to the generation of L1-70 and L1-135, but not L1-80 and L1-140. We discuss previous observations on the basis of our new results and propose a novel view on the molecular features that render previous and present observations compatible.
Asunto(s)
Encéfalo/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Proteolisis , Animales , Ratones , Ratones MutantesRESUMEN
The high-mobility-group (HMG)-domain protein Sox9 is one of few transcription factors implicated in gliogenesis in the vertebrate central nervous system. To further study the role of Sox9 in early spinal cord development, we generated a mouse that allows expression of Sox9 in a temporally and spatially controlled manner. Using this mouse, we show that premature Sox9 expression in neural precursor cells disrupted the neuroepithelium of the ventricular zone. Sox9 also compromised development and survival of neuronal precursors and neurons. Additionally, we observed in these mice substantial increases in oligodendroglial and astroglial cells. Reversing the normal order of appearance of essential transcriptional regulators during oligodendrogenesis, Sox10 preceded Olig2. Our study reinforces the notion that Sox9 has a strong gliogenic activity. It also argues that Sox9 expression has to be tightly controlled to prevent negative effects on early spinal cord structure and neuronal development.
Asunto(s)
Astrocitos/metabolismo , Oligodendroglía/metabolismo , Factor de Transcripción SOX9/metabolismo , Médula Espinal/metabolismo , Animales , Línea Celular Tumoral , Ratones , Ratones Transgénicos , Factor de Transcripción 2 de los Oligodendrocitos/genética , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Factor de Transcripción SOX9/genética , Médula Espinal/crecimiento & desarrolloRESUMEN
The phospholipase A2 receptor 1 (PLA2R1) is the major autoantigen in patients suffering from membranous nephropathy. To date, the lack of endogenous glomerular expression of PLA2R1 in mice and rats has impeded the establishment of PLA2R1-dependent animal models of this disease. Here, we generated a transgenic mouse line expressing murine full-length PLA2R1 in podocytes. Furthermore, expression of murine PLA2R1 did not result in any morphological disturbance as high-resolution confocal microscopy demonstrated an intact nephrin distribution with normal foot processes. Transfer of rabbit anti-mPLA2R1 antibodies to these mice induced nephrotic range proteinuria, hypercholesterolemia, and histomorphological signs of membranous nephropathy. Immunohistochemical and immunofluorescence analyses revealed enhanced staining for murine PLA2R1 in the presence of unaffected staining for murine thrombospondin type-1 domain-containing 7A in the diseased mice, resembling what is classically found in patients with PLA2R1-associated membranous nephropathy Thus, our mouse model of membranous nephropathy will allow investigation of PLA2R1-specific pathomechanisms and may help to develop and assess antigen-specific treatments in vivo.
Asunto(s)
Modelos Animales de Enfermedad , Glomerulonefritis Membranosa , Podocitos , Animales , Autoanticuerpos , Autoantígenos , Humanos , Ratones , Ratones Transgénicos , Receptores de Fosfolipasa A2/genéticaRESUMEN
Salt taste is one of the 5 basic taste qualities. Depending on the concentration, table salt is perceived either as appetitive or aversive, suggesting the contribution of several mechanisms to salt taste, distinguishable by their sensitivity to the epithelial sodium channel (ENaC) blocker amiloride. A taste-specific knockout of the α-subunit of the ENaC revealed the relevance of this polypeptide for low-salt transduction, whereas the response to other taste qualities remained normal. The fully functional ENaC is composed of α-, ß-, and γ-subunits. In taste tissue, however, the precise constitution of the channel and the cell population responsible for detecting table salt remain uncertain. In order to examine the cells and subunits building the ENaC, we generated mice carrying modified alleles allowing the synthesis of green and red fluorescent proteins in cells expressing the α- and ß-subunit, respectively. Fluorescence signals were detected in all types of taste papillae and in taste buds of the soft palate and naso-incisor duct. However, the lingual expression patterns of the reporters differed depending on tongue topography. Additionally, immunohistochemistry for the γ-subunit of the ENaC revealed a lack of overlap between all potential subunits. The data suggest that amiloride-sensitive recognition of table salt is unlikely to depend on the classical ENaCs formed by α-, ß-, and γ-subunits and ask for a careful investigation of the channel composition.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Papilas Gustativas/metabolismo , Amilorida/metabolismo , Animales , Clonación Molecular , Técnicas de Sustitución del Gen , Técnicas de Genotipaje , Humanos , Riñón , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Conformación Proteica , Gusto , Papilas Gustativas/citología , Percepción del Gusto , Distribución TisularRESUMEN
Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.
Asunto(s)
Enzimas/metabolismo , Lisosomas/metabolismo , Mucolipidosis/metabolismo , Mucolipidosis/patología , Proteoma/metabolismo , Animales , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Marcaje Isotópico , Manosafosfatos/metabolismo , Ratones Noqueados , Subunidades de Proteína/metabolismo , Proteolisis , Especificidad por SustratoRESUMEN
Oligodendrocytes are the myelinating glia of the central nervous system and ensure rapid saltatory conduction. Shortage or loss of these cells leads to severe malfunctions as observed in human leukodystrophies and multiple sclerosis, and their replenishment by reprogramming or cell conversion strategies is an important research aim. Using a transgenic approach we increased levels of the transcription factor Sox10 throughout the mouse embryo and thereby prompted Fabp7-positive glial cells in dorsal root ganglia of the peripheral nervous system to convert into cells with oligodendrocyte characteristics including myelin gene expression. These rarely studied and poorly characterized satellite glia did not go through a classic oligodendrocyte precursor cell stage. Instead, Sox10 directly induced key elements of the regulatory network of differentiating oligodendrocytes, including Olig2, Olig1, Nkx2.2 and Myrf. An upstream enhancer mediated the direct induction of the Olig2 gene. Unlike Sox10, Olig2 was not capable of generating oligodendrocyte-like cells in dorsal root ganglia. Our findings provide proof-of-concept that Sox10 can convert conducive cells into oligodendrocyte-like cells in vivo and delineates options for future therapeutic strategies.
Asunto(s)
Diferenciación Celular/genética , Sistema Nervioso Central/metabolismo , Esclerosis Múltiple/genética , Factores de Transcripción SOXE/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistema Nervioso Central/patología , Embrión de Mamíferos , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Humanos , Ratones , Esclerosis Múltiple/patología , Proteínas del Tejido Nervioso/genética , Neuroglía , Proteínas Nucleares , Factor de Transcripción 2 de los Oligodendrocitos , Oligodendroglía/metabolismo , Factores de Transcripción SOXE/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Factores de Transcripción/genética , Proteínas de Pez CebraRESUMEN
Hypothalamic tanycytes are glial-like glucosensitive cells that contact the cerebrospinal fluid of the third ventricle, and send processes into the hypothalamic nuclei that control food intake and body weight. The mechanism of tanycyte glucosensing remains undetermined. While tanycytes express the components associated with the glucosensing of the pancreatic ß cell, they respond to nonmetabolisable glucose analogues via an ATP receptor-dependent mechanism. Here, we show that tanycytes in rodents respond to non-nutritive sweeteners known to be ligands of the sweet taste (Tas1r2/Tas1r3) receptor. The initial sweet tastant-evoked response, which requires the presence of extracellular Ca2+ , leads to release of ATP and a larger propagating Ca2+ response mediated by P2Y1 receptors. In Tas1r2 null mice the proportion of glucose nonresponsive tanycytes was greatly increased in these mice, but a subset of tanycytes retained an undiminished sensitivity to glucose. Our data demonstrate that the sweet taste receptor mediates glucosensing in about 60% of glucosensitive tanycytes while the remaining 40% of glucosensitive tanycytes use some other, as yet unknown mechanism.
Asunto(s)
Glucosa/metabolismo , Hipotálamo/metabolismo , Gusto/fisiología , Animales , Calcio/metabolismo , Femenino , Ratones Endogámicos C57BL , Neuroglía/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Purinérgicos P2Y1/metabolismoRESUMEN
Carbonic anhydrases form an enzyme family of 16 members, which reversibly catalyze the hydration of carbon dioxide to bicarbonate and protons. In lung, kidney, and brain, presence of carbonic anhydrases is associated with protons and bicarbonate transport in capillary endothelium of lung, reabsorption of bicarbonate in proximal renal tubules, and extracellular buffering. In contrast, their role in taste is less clear. Recently, carbonic anhydrase IV expression was detected in sour-sensing presynaptic taste cells and was associated with the taste of carbonation, yet the precise role and cell population remained uncertain. To examine the role of carbonic anhydrase 4-expressing cells in taste reception, we generated a mouse strain carrying a modified allele of the carbonic anhydrase 4 gene in which the coding region of the red fluorescent protein monomeric Cherry is attached to that of carbonic anhydrase 4 via an internal ribosome entry site. Monomeric Cherry fluorescence was detected in lingual papillae as well as taste buds of soft palate and naso-incisor duct. However, expression patterns on the tongue differ between posterior and fungiform papillae. Whereas monomeric Cherry auto-fluorescence was almost always co-localized with presynaptic cell markers aromatic L-amino-acid decarboxylase, synaptosomal-associated protein 25 or glutamic acid decarboxylase 67 in fungiform papillae and taste buds of palate and naso-incisor duct, monomeric Cherry-positive cells in posterior tongue papillae represent only a subpopulation of presynaptic cells. We conclude that this model is well suited for detailed investigation into the role of carbonic anhydrase in gustation and other processes.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Papilas Gustativas/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Anhidrasas Carbónicas/genética , Técnicas de Sustitución del Gen , Ingeniería Genética , Hibridación in Situ , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Microscopía Fluorescente , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Papilas Gustativas/citología , Lengua/metabolismo , Lengua/patología , Proteína Fluorescente RojaRESUMEN
Hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is emerging as a crucial regulator in cancer, infections, and inflammation. Although its contribution in translational regulation of proline repeat-rich proteins has been sufficiently demonstrated, its biological role in higher eukaryotes remains poorly understood. To establish the hypusine modification system as a novel platform for therapeutic strategies, we aimed to investigate its functional relevance in mammals by generating and using a range of new knock-out mouse models for the hypusine-modifying enzymes deoxyhypusine synthase and deoxyhypusine hydroxylase as well as for the cancer-related isoform eIF-5A2. We discovered that homozygous depletion of deoxyhypusine synthase and/or deoxyhypusine hydroxylase causes lethality in adult mice with different penetrance compared with haploinsufficiency. Network-based bioinformatic analysis of proline repeat-rich proteins, which are putative eIF-5A targets, revealed that these proteins are organized in highly connected protein-protein interaction networks. Hypusine-dependent translational control of essential proteins (hubs) and protein complexes inside these networks might explain the lethal phenotype observed after deletion of hypusine-modifying enzymes. Remarkably, our results also demonstrate that the cancer-associated isoform eIF-5A2 is dispensable for normal development and viability. Together, our results provide the first genetic evidence that the hypusine modification in eIF-5A is crucial for homeostasis in mammals. Moreover, these findings highlight functional diversity of the hypusine system compared with lower eukaryotes and indicate eIF-5A2 as a valuable and safe target for therapeutic intervention in cancer.
Asunto(s)
Lisina/análogos & derivados , Oxigenasas de Función Mixta/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Factores de Iniciación de Péptidos/metabolismo , Animales , Homeostasis/genética , Humanos , Lisina/genética , Lisina/metabolismo , Ratones , Ratones Noqueados , Oxigenasas de Función Mixta/metabolismo , Neoplasias/genética , Neoplasias/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Factores de Iniciación de Péptidos/genética , Biosíntesis de Proteínas , Mapas de Interacción de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Differentiation of oligodendrocytes and myelin production in the vertebrate central nervous system require highly concerted changes in gene expression. The transcription factors Sox10 and Myrf are both central to this process and jointly regulate expression of myelin genes. Here we show that Sox10 and Myrf also cooperate in the activation of the gene coding for the dual specificity protein phosphatase Dusp15 (also known as VHY) during this process. Activation is mediated by the Dusp15 promoter, which is also sufficient to drive oligodendroglial gene expression in vivo. It contains both a functional Sox10 and a functional Myrf binding site. Whereas Sox10 binds as a monomer, Myrf binds as a trimer. Available data furthermore indicate that cooperative activation is not a function of facilitated binding, but occurs at a later step of the activation process. shRNA-mediated knockdown of Dusp15 reduced expression of early and late differentiation markers in CG4 and primary oligodendroglial cells, whereas Dusp15 overexpression increased it transiently. This argues that Dusp15 is not only a joint target of Sox10 and Myrf in oligodendrocytes but may also mediate some of their effects during oligodendrocyte differentiation and myelin formation. GLIA 2016;64:2120-2132.
Asunto(s)
Fosfatasas de Especificidad Dual/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Factores de Transcripción SOXE/metabolismo , Factores de Transcripción/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/citología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Fosfatasas de Especificidad Dual/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , Ratas , Factores de Transcripción SOXE/genética , Factores de Transcripción/genética , TransfecciónRESUMEN
We have previously reported that the hormone calcitonin (CT) negatively regulates bone formation by inhibiting the release of sphingosine-1-phosphate from bone-resorbing osteoclasts. In the context of this study we additionally observed that CT repressed the expression of Pate4, encoding the secreted protein caltrin/Svs7, in osteoclasts from wildtype mice. To assess a possible function of Pate4 in bone remodeling, we utilized commercially available embryonic stem cells with a targeted Pate4 allele to generate Pate4-deficient mice. These were born at the expected Mendelian ratio and did not display obvious abnormalities until the age of 6 months. A bone-specific histomorphometric analysis further revealed that bone remodeling is unaffected in male and female Pate4-deficient mice. Since a subsequently performed multi-tissue expression analysis confirmed that Pate4 is primarily expressed in prostate and seminal vesicles, we additionally analyzed the respective tissues of Pate4-deficient mice, but failed to detect histological abnormalities. Most importantly, as assessed by mating with female wildtype mice, we did not observe reduced fertility associated with Pate4-deficiency. Taken together, our study was the first to generate and analyze a mouse model lacking Pate4, a gene with strong expression in prostate and seminal vesicles, yet without major function for fertility.
Asunto(s)
Anomalías Múltiples/genética , Resorción Ósea/genética , Proteínas Portadoras/genética , Ratones/genética , Proteínas de Secreción de la Vesícula Seminal/genética , Animales , Predisposición Genética a la Enfermedad/genética , Ratones Noqueados , FenotipoRESUMEN
Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.
Asunto(s)
Cromosomas de los Mamíferos/genética , Impresión Genómica , Madres , Proteínas Represoras/metabolismo , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Animales Congénicos , Blastocisto/metabolismo , Línea Celular , Pérdida del Embrión/genética , Padre , Femenino , Silenciador del Gen , Masculino , Ratones , Ratones Transgénicos , ARN Largo no Codificante , ARN no Traducido/genética , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Ubiquitina-Proteína LigasasRESUMEN
Myelin is essential for rapid saltatory conduction and is produced by Schwann cells in the peripheral nervous system and oligodendrocytes in the central nervous system. In both cell types the transcription factor Sox10 is an essential component of the myelin-specific regulatory network. Here we identify Myrf as an oligodendrocyte-specific target of Sox10 and map a Sox10 responsive enhancer to an evolutionarily conserved element in intron 1 of the Myrf gene. Once induced, Myrf cooperates with Sox10 to implement the myelination program as evident from the physical interaction between both proteins and the synergistic activation of several myelin-specific genes. This is strongly reminiscent of the situation in Schwann cells where Sox10 first induces and then cooperates with Krox20 during myelination. Our analyses indicate that the regulatory network for myelination in oligodendrocytes is organized along similar general principles as the one in Schwann cells, but is differentially implemented.
Asunto(s)
Diferenciación Celular/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Redes Reguladoras de Genes , Factores de Transcripción SOXE/genética , Células de Schwann/metabolismo , Factores de Transcripción/genética , Animales , Línea Celular , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Ratones , Ratones Transgénicos , Vaina de Mielina/genética , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismoRESUMEN
Puberty is a transition period of reproductive development from juvenile stages to adulthood and depends upon the activity of gonadotropin-releasing hormone (GnRH) neurons. GnRH neurons are initially activated in utero but remain quiescent throughout the juvenile period. Premature reactivation of GnRH neurons results in precocious puberty in mice and humans, but the mechanisms underlying developmental control of GnRH neuron activity remain unknown. The neuropeptide kisspeptin, a potent activator of GnRH neurons that is implicated as a critical permissive signal triggering puberty and a major regulator of the adult female hypothalamus-pituitary-gonadal axis, is paradoxically produced by neurons in the developing brain well before puberty onset. Thus, the neural circuits controlling the timing of reproductive maturation remain elusive. Here, we delineate the underlying neural circuitry using conditional genetic transsynaptic tracing in female mouse embryos. We find that kisspeptin-producing neurons in the arcuate nucleus (ARC) already communicate with a specific subset of GnRH neurons in utero. We show that ARC kisspeptin neurons are upstream of GnRH neurons, and that GnRH neuron connectivity to ARC kisspeptin neurons does not depend on their spatial position in the brain. Furthermore, we demonstrate that the neural circuits between ARC kisspeptin and GnRH neurons are fully established and operative before birth. Finally, we find that most GnRH neurons express the kisspeptin receptor GPR54 upon circuit formation, suggesting that the signaling system implicated in gatekeeping puberty becomes operative in the embryo.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Hormona Liberadora de Gonadotropina/biosíntesis , Red Nerviosa/metabolismo , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Animales , Núcleo Arqueado del Hipotálamo/embriología , Femenino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Red Nerviosa/embriología , Embarazo , Receptores de Kisspeptina-1RESUMEN
Phosphorylated creatine (Cr) serves as an energy buffer for ATP replenishment in organs with highly fluctuating energy demand. The central role of Cr in the brain and muscle is emphasized by severe neurometabolic disorders caused by Cr deficiency. Common symptoms of inborn errors of creatine synthesis or distribution include mental retardation and muscular weakness. Human mutations in l-arginine:glycine amidinotransferase (AGAT), the first enzyme of Cr synthesis, lead to severely reduced Cr and guanidinoacetate (GuA) levels. Here, we report the generation and metabolic characterization of AGAT-deficient mice that are devoid of Cr and its precursor GuA. AGAT-deficient mice exhibited decreased fat deposition, attenuated gluconeogenesis, reduced cholesterol levels and enhanced glucose tolerance. Furthermore, Cr deficiency completely protected from the development of metabolic syndrome caused by diet-induced obesity. Biochemical analyses revealed the chronic Cr-dependent activation of AMP-activated protein kinase (AMPK), which stimulates catabolic pathways in metabolically relevant tissues such as the brain, skeletal muscle, adipose tissue and liver, suggesting a mechanism underlying the metabolic phenotype. In summary, our results show marked metabolic effects of Cr deficiency via the chronic activation of AMPK in a first animal model of AGAT deficiency. In addition to insights into metabolic changes in Cr deficiency syndromes, our genetic model reveals a novel mechanism as a potential treatment option for obesity and type 2 diabetes mellitus.