RESUMEN

Many genes, including odd-skipped related 1 (Osr1), are involved in regulation of mammalian kidney development. We describe here a new recessive mutation (kidney adysplasia and variable hydronephrosis, kavh) in the mouse that leads to downregulation of Osr1 transcript, causing several kidney defects: agenesis, hypoplasia, and hydronephrosis with variable age of onset. The mutation is closely associated with a reciprocal translocation, T(12;17)4Rk, whose Chromosome 12 breakpoint is upstream from Osr1. The kavh/kavh mutant provides a model to study kidney development and test therapies for hydronephrosis.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/genética , Hidronefrosis/etiología , Riñón/anomalías , Mutación/genética , Organogénesis/fisiología , Factores de Transcripción/genética , Anomalías Urogenitales/metabolismo , Animales , Hidronefrosis/genética , Riñón/metabolismo , Ratones Endogámicos C57BL , Organogénesis/genética , Factores de Transcripción/metabolismo

RESUMEN

Folding of transmembrane and secretory proteins occurs in the lumen of the endoplasmic reticulum (ER) before transportation to the cell surface and is monitored by the unfolded protein response (UPR) signaling pathway. The accumulation of unfolded proteins in the ER activates the UPR that restores ER homeostasis by regulating gene expression that leads to an increase in the protein-folding capacity of the ER and a decrease in the ER protein-folding load. However, prolonged UPR activity has been associated with cell death in multiple pathological conditions, including neurodegeneration. Here, we report a spontaneous recessive mouse mutation that causes progressive cerebellar granule cell death and peripheral motor axon degeneration. By positional cloning, we identify the mutation in this strain as a retrotransposon insertion in the Clcc1 gene, which encodes a putative chloride channel localized to the ER. Furthermore, we demonstrate that the C3H/HeSnJ inbred strain has late onset cerebellar degeneration due to this mutation. Interestingly, acute knockdown of Clcc1 expression in cultured cells increases sensitivity to ER stress. In agreement, GRP78, the major HSP70 family chaperone in the ER, is upregulated in Clcc1-deficient granule cells in vivo, and ubiquitinated proteins accumulate in these neurons before their degeneration. These data suggest that disruption of chloride homeostasis in the ER disrupts the protein-folding capacity of the ER, leading to eventual neuron death.

Asunto(s)

Canales de Cloruro/deficiencia , Estrés del Retículo Endoplásmico/genética , Enfermedades Neurodegenerativas , Pliegue de Proteína , Animales , Cerebelo/patología , Canales de Cloruro/genética , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Células HEK293 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Mutación/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/metabolismo , Neuronas/patología , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/patología , ARN Mensajero/metabolismo , Transfección

RESUMEN

A spontaneous mutation termed bilateral wasting kidneys (bwk) was identified in a colony of NONcNZO recombinant inbred mice. These mice exhibit a rapid increase of urinary albumin at an early age associated with glomerulosclerosis, interstitial nephritis, and tubular atrophy. The mutation was mapped to a location on chromosome 1 containing the Col4a3 and Col4a4 genes, for which mutations in the human orthologs cause the hereditary nephritis Alport syndrome. DNA sequencing identified a G-to-A mutation in the conserved GT splice donor of Col4a4 intron 30, resulting in skipping of exon 30 but maintaining the mRNA reading frame. Protein analyses showed that mutant collagen α3α4α5(IV) trimers were secreted and incorporated into the glomerular basement membrane (GBM), but levels were low, and GBM lesions typical of Alport syndrome were observed. Moving the mutation into the more renal damage-prone DBA/2J and 129S1/SvImJ backgrounds revealed differences in albuminuria and its rate of increase, suggesting an interaction between the Col4a4 mutation and modifier genes. This novel mouse model of Alport syndrome is the only one shown to accumulate abnormal collagen α3α4α5(IV) in the GBM, as also found in a subset of Alport patients. These mice will be valuable for testing potential therapies, for understanding abnormal collagen IV structure and assembly, and for gaining better insights into the mechanisms leading to Alport syndrome, and to the variability in the age of onset and associated phenotypes.

Asunto(s)

Autoantígenos/genética , Autoantígenos/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Membrana Basal Glomerular/metabolismo , Mutación , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Albuminuria/genética , Albuminuria/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Membrana Basal Glomerular/patología , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos DBA , Nefritis Hereditaria/patología , Fenotipo , Multimerización de Proteína , ARN Mensajero/metabolismo , Factores de Tiempo

RESUMEN

PURPOSE: Cilia, complex structures found ubiquitously in most vertebrate cells, serve a variety of functions ranging from cell and fluid movement, cell signaling, tissue homeostasis, to sensory perception. Meckelin is a component of ciliary and cell membranes and is encoded by Tmem67 (Mks3). In this study, the retinal morphology and ciliary function in a mouse model for Meckel Syndrome Type 3 (MKS3) throughout the course of photoreceptor development was examined. METHODS: To study the effects of a disruption in the Mks3 gene on the retina, the authors introduced a functional allele of Pde6b into B6C3Fe a/a-bpck/J mice and evaluated their retinas by ophthalmoscopic, histologic, and ultrastructural examination. In addition, immunofluorescence microscopy was used to assess protein trafficking through the connecting cilium and to examine the localization of ciliary and synaptic proteins in Tmem67(bpck) mice and controls. RESULTS: Photoreceptors degenerate early and rapidly in bpck/bpck mutant mice. In addition, phototransduction proteins, such as rhodopsin, arrestin, and transducin, are mislocalized. Ultrastructural examination of photoreceptors reveal morphologically intact connecting cilia but dysmorphic and misoriented outer segment (OS) discs, at the earliest time point examined. CONCLUSIONS: These findings underscore the important role for meckelin in intraciliary transport of phototransduction molecules and their effects on subsequent OS morphogenesis and maintenance.

Asunto(s)

Trastornos de la Motilidad Ciliar/genética , ADN/genética , Encefalocele/genética , Proteínas de la Membrana/genética , Morfogénesis/efectos de los fármacos , Mutación , Enfermedades Renales Poliquísticas/genética , Segmento Externo de la Célula en Bastón/fisiología , Animales , Cilios/genética , Cilios/metabolismo , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/metabolismo , Trastornos de la Motilidad Ciliar/patología , Modelos Animales de Enfermedad , Encefalocele/metabolismo , Encefalocele/patología , Genotipo , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , Reacción en Cadena de la Polimerasa , Transporte de Proteínas , Segmento Externo de la Célula en Bastón/ultraestructura

RESUMEN

Meckel-Gruber syndrome type 3 (MKS3; OMIM 607361) is a severe autosomal recessive disorder characterized by bilateral polycystic kidney disease. Other malformations associated with MKS3 include cystic changes in the liver, polydactyly, and brain abnormalities (occipital encephalocele, hydrocephalus, and Dandy Walker-type cerebellar anomalies). The disorder is hypothesized to be caused by defects in primary cilia. In humans, the underlying mutated gene, TMEM67, encodes transmembrane protein 67, also called meckelin (OMIM 609884), which is an integral protein of the renal epithelial cell and membrane of the primary cilium. Here, we describe a spontaneous deletion of the mouse ortholog, Tmem67, which results in polycystic kidney disease and death by 3 wk after birth. Hydrocephalus also occurs in some mutants. We verified the mutated gene by transgenic rescue and characterized the phenotype with microcomputed tomography, histology, scanning electron microscopy, and immunohistochemistry. This mutant provides a mouse model for MKS3 and adds to the growing set of mammalian models essential for studying the role of the primary cilium in kidney function.

Asunto(s)

Proteínas de la Membrana/genética , Enfermedades Renales Poliquísticas/genética , Anomalías Múltiples/genética , Anomalías Múltiples/fisiopatología , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Hidrocefalia/genética , Hidrocefalia/fisiopatología , Riñón/patología , Ratones , Ratones Mutantes , Mutación , Enfermedades Renales Poliquísticas/epidemiología , Enfermedades Renales Poliquísticas/patología , Enfermedades Renales Poliquísticas/fisiopatología , Estados Unidos/epidemiología

RESUMEN

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.

Asunto(s)

Alanina-ARNt Ligasa/genética , Alanina-ARNt Ligasa/metabolismo , Enfermedades Neurodegenerativas/enzimología , Pliegue de Proteína , Acetilación , Alanina/genética , Alanina/metabolismo , Alanina-ARNt Ligasa/química , Animales , Catálisis , Escherichia coli/genética , Fibroblastos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Enfermedades Neurodegenerativas/genética , Fenotipo , Estructura Terciaria de Proteína , Células de Purkinje/metabolismo , Células de Purkinje/patología , ARN de Transferencia de Alanina/genética , Serina/genética , Serina/metabolismo

RESUMEN

Mouse deafness mutations provide valuable models of human hearing disorders and entry points into molecular pathways important to the hearing process. A newly discovered mouse mutation named hurry-scurry (hscy) causes deafness and vestibular dysfunction. Scanning electron microscopy of cochleae from 8-day-old mutants revealed disorganized hair bundles, and by 50 days of age, many hair cells are missing. To positionally clone hscy, 1,160 F(2) mice were produced from an intercross of (C57BL/6-hscy x CAST/EiJ) F(1) hybrids, and the mutation was localized to a 182-kb region of chromosome 17. A missense mutation causing a critical cysteine to phenylalanine codon change was discovered in a previously undescribed gene within this candidate interval. The gene is predicted to encode an integral membrane protein with four transmembrane helices. A synthetic peptide designed from the predicted protein was used to produce specific polyclonal antibodies, and strong immunoreactivity was observed on hair bundles of both inner and outer hair cells in cochleae of newborn +/+ controls and +/hscy heterozygotes but was absent in hscy/hscy mutants. Accordingly, the gene was given the name "tetraspan membrane protein of hair cell stereocilia," symbol Tmhs. Two related proteins (>60% amino acid identity) are encoded by genes on mouse chromosomes 5 and 6 and, together with the Tmhs-encoded protein (TMHS), comprise a distinct tetraspan subfamily. Our localization of TMHS to the apical membrane of inner ear hair cells during the period of stereocilia formation suggests a function in hair bundle morphogenesis.

Asunto(s)

Sordera/genética , Expresión Génica , Células Ciliadas Auditivas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones/genética , Mutación Missense/genética , Animales , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Análisis por Conglomerados , Cruzamientos Genéticos , ADN Complementario/genética , Componentes del Gen , Células Ciliadas Auditivas/ultraestructura , Técnicas Histológicas , Inmunohistoquímica , Ratones Mutantes , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Análisis de Secuencia de ADN

RESUMEN

In the outcrossing of a new recessive mouse mutation causing hair loss, a new wavy-coated phenotype appeared. The two distinct phenotypes were shown to be alternative manifestations of the same gene mutation and attributable to a single modifier locus. The new mutation, curly bare (cub), was mapped to distal Chr 11 and the modifier (mcub) was mapped to Chr 5. When homozygous for the recessive mcub allele, cub/cub mice appear hairless. A single copy of the dominant Mcub allele confers a full, curly coat in cub/cub mice. Reciprocal transfer of full-thickness skin grafts between mutant and control animals showed that the skin phenotype was tissue autonomous. The hairless cub/cub mcub/mcub mice show normal contact sensitivity responses to oxazolone. The similarity of the wavy coat phenotype to those of Tgfa and Egfr mutations and the map positions of cub and mcub suggest candidate genes that interact in the EGF receptor signal transduction pathway.

Asunto(s)

Mapeo Cromosómico , Genes Reguladores/genética , Cabello/anomalías , Animales , Cruzamientos Genéticos , Cabello/fisiología , Hipersensibilidad/genética , Ratones , Trasplante de Piel