RESUMEN
Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1-/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.
Asunto(s)
Disfunción Cognitiva/metabolismo , Proteínas de la Membrana/deficiencia , Sinapsis/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cognición/fisiología , Disfunción Cognitiva/genética , Homólogo 4 de la Proteína Discs Large/genética , Homólogo 4 de la Proteína Discs Large/metabolismo , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Hipocampo/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Transducción de Señal , Sinapsis/genética , Transmisión SinápticaRESUMEN
Intellectual disability (ID) is a major health problem in our society. Genetic causes of ID remain unknown because of its vast heterogeneity. Here we report two Finnish families and one Dutch family with affected individuals presenting with mild to moderate ID, neuropsychiatric symptoms and delayed speech development. By utilizing whole exome sequencing (WES), we identified a founder missense variant c.983T>C (p.Leu328Pro) in seven affected individuals from two Finnish consanguineous families and a deletion c.799_1034-429delinsTTATGA (p.Gln267fs) in one affected individual from a consanguineous Dutch family in the C12orf4 gene on chromosome 12. Both the variants co-segregated in the respective families as an autosomal recessive trait. Screening of the p.Leu328Pro variant showed enrichment in the North Eastern sub-isolate of Finland among anonymous local blood donors with a carrier frequency of 1:53, similar to other disease mutations with a founder effect in that region. To date, only one Arab family with a three affected individuals with a frameshift insertion variant in C12orf4 has been reported. In summary, we expand and establish the clinical and mutational spectrum of C12orf4 variants. Our findings implicate C12orf4 as a causative gene for autosomal recessive ID.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Discapacidad Intelectual/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Consanguinidad , Exoma/genética , Salud de la Familia , Femenino , Finlandia , Efecto Fundador , Genes Recesivos , Genotipo , Geografía , Humanos , Masculino , Países Bajos , Linaje , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de AminoácidoRESUMEN
The molecular mechanisms that promote excitatory synapse development have been extensively studied. However, the molecular events preventing precocious excitatory synapse development so that synapses form at the correct time and place are less well understood. Here, we report the functional characterization of ARHGAP12, a previously uncharacterized Rho GTPase-activating protein (RhoGAP) in the brain. ARHGAP12 is specifically expressed in the CA1 region of the hippocampus, where it localizes to the postsynaptic compartment of excitatory synapses. ARHGAP12 negatively controls spine size via its RhoGAP activity and promotes, by interacting with CIP4, postsynaptic AMPA receptor endocytosis. Arhgap12 knockdown results in precocious maturation of excitatory synapses, as indicated by a reduction in the proportion of silent synapses. Collectively, our data show that ARHGAP12 is a synaptic RhoGAP that regulates excitatory synaptic structure and function during development.
Asunto(s)
Proteínas Activadoras de GTPasa/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Antígenos de Histocompatibilidad Menor/genética , Células Piramidales/metabolismo , Receptores AMPA/genética , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Espinas Dendríticas/fisiología , Espinas Dendríticas/ultraestructura , Embrión de Mamíferos , Endocitosis , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Técnicas de Placa-Clamp , Cultivo Primario de Células , Células Piramidales/citología , Ratas , Ratas Wistar , Receptores AMPA/metabolismo , Análisis de la Célula Individual , Sinapsis/ultraestructura , Transmisión Sináptica , Técnicas de Cultivo de TejidosAsunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Ojo/aislamiento & purificación , Retina/metabolismo , Retinitis Pigmentosa/genética , ADN Complementario , Proteínas del Ojo/metabolismo , Plásmidos , Empalme del ARN , Saccharomyces cerevisiae/genética , Técnicas del Sistema de Dos Híbridos , beta-Galactosidasa/genéticaRESUMEN
The Ran-binding protein 2 (RanBP2) is a giant scaffold and mosaic cyclophilin-related nucleoporin implicated in the Ran-GTPase cycle. There are no orthologs of the RanBP2 gene in yeast and Drosophila genomes. In humans, this bona fide gene is partially duplicated in a RanBP2 gene cluster and lies in a hot spot for recombination on Chromosome (Chr) 2q. This genetic heterogeneity renders further significance of this genomic region in human disease due to its possible involvement in genetically linked disorders such as juvenile nephronophthisis, congenital hepatic fibrosis, and chorioretinal dysplasia. Structure-function studies on bovine RanBP2 indicate that this protein is involved in integrating nucleocytoplasmic transport pathways with protein biogenesis such as production of functional opsin. To gain further insight into the complex functions of RanBP2 in the development and function of the neuroretina and other tissues, and proceed towards the functional analysis of RanBP2 and its molecular partners in vivo, we have determined the complete genomic organization of the murine RanBP2 gene. The gene consists of 29 exons spread over 50 kb and contains a mega-exon of 4663 bp that encompasses the variable Zn-finger-rich domain of RanBP2. This may account, in part, for a predisposition of recombination of this locus and variability of the number of Zn-fingers across mammalian species. The RanBP2 promoter contains tissue-specific elements. A CpG island encompasses this region up to the first intron, making RanBP2 gene expression susceptible of epigenetic regulation. This murine RanBP2 transcript has a tissue-restricted expression profile, and the conceptual protein is 82% identical to human RanBP2. The gene maps to mouse Chr 10, 30 cM proximal of the centromere.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Animales , Secuencia de Bases , Southern Blotting , Núcleo Celular/metabolismo , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Islas de CpG , Citoplasma/metabolismo , ADN Complementario/metabolismo , Proteínas de Unión al ADN/química , Exones , Femenino , Biblioteca de Genes , Humanos , Intrones , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Mapeo Físico de Cromosoma , Unión Proteica , Estructura Terciaria de Proteína , Recombinación Genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Distribución TisularRESUMEN
The combination of the Ran-binding domain 4 and cyclophilin domains of Ran-binding protein 2 selectively associate with a subset of G protein-coupled receptors, red/green opsins, upon cis-trans prolyl isomerase-dependent and direct modification of opsin followed by association of the modified opsin isoform to Ran-binding domain 4. This effect enhances in vivo the production of functional receptor and generates an opsin isoform with no propensity to self-aggregate in vitro. We now show that another domain of Ran-binding protein 2, cyclophilin-like domain, specifically associates with the 112-kDa subunit, P112, and other subunits of the 19 S regulatory complex of the 26 S proteasome in the neuroretina. This association possibly mediates Ran-binding protein 2 limited proteolysis into a smaller and stable isoform. Also, the interaction of Ran-binding protein 2 with P112 regulatory subunit of the 26 S proteasome involves still another protein, a putative kinesin-like protein. Our results indicate that Ran-binding protein 2 is a key component of a macro-assembly complex selectively linking protein biogenesis with the proteasome pathway and, thus, with potential implications for the presentation of misfolded and ubiquitin-like modified proteins to this proteolytic machinery.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Isomerasa de Peptidilprolil/química , Complejo de la Endopetidasa Proteasomal , Retina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Clonación Molecular , Proteínas de Unión al ADN/aislamiento & purificación , Humanos , Cinética , Leucina Zippers , Sustancias Macromoleculares , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/aislamiento & purificación , Péptido Hidrolasas/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The Ran-binding protein 2 (RanBP2) is a large scaffold cyclophilin-related protein expressed in photoreceptor cells. Red/green opsin, Ran-GTPase, and the 19 S regulatory complex of the proteasome associate with specific RanBP2 structural modules. Some of these play a role in chaperoning the functional expression of opsin. RanBP2 localization at cytoplasmic fibrils emanating from the nuclear pore complex and interaction with the Ran-GTPase support also its role in nucleocytoplasmic transport processes. The degenerate nucleoporin repeat motifs FXFG, GLFG, and XXFG have been proposed to mediate the movement of nucleocytoplasmic transport factors. In particular, RanBP2 has been implicated in nuclear import processes. Here, we show the zinc fingers of RanBP2 associate with high specificity to the nuclear export factor, exportin-1 (CRM1). The bovine RanBP2 transcript contained only five of the eight zinc fingers reported in the human counterpart and are sufficient for exportin-1 association with RanBP2. In contrast to Ran interaction with RanBP2-exportin-1 complex, exportin-1 binding to the zinc finger cluster domain of RanBP2 is insensitive to leptomycin B and nucleotide-bound state of Ran-GTPase. Our results indicate that the zinc finger-rich domain of RanBP2 constitutes a docking site for exportin-1 during nuclear export. Thus, RanBP2 emerges as a key component of the nuclear export pathway.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Carioferinas , Proteínas de Complejo Poro Nuclear , Proteínas Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Bovinos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Ácido Edético/farmacología , Ácidos Grasos Insaturados/farmacología , Humanos , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/aislamiento & purificación , Retinaldehído/metabolismo , Zinc/farmacología , Proteína Exportina 1RESUMEN
Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause X-linked retinitis pigmentosa type 3 (RP3), a severe, progressive and degenerative retinal dystrophy eventually leading to complete blindness. RPGR is ubiquitously expressed, yet mutations in the RPGR gene lead to a retina-restricted phenotype. To date, all RP3 associated missense mutations that have been identified are located in the RCC1-homologous domain (RHD) of RPGR. To investigate the molecular pathogenesis of RP3, we screened retinal yeast two-hybrid libraries with the RHD of RPGR. We identified several alternatively spliced gene products, some with retina-restricted expression, that interact specifically with RPGR in vivo and in vitro. Thus, these proteins were named RPGR-interacting protein 1 (RPGRIP1) isoforms. They contain a C-terminal RPGR-interacting domain and stretches of variable coiled-coil domains homologous to proteins involved in vesicular trafficking. The interaction between RPGR and RPGRIP1 isoforms was impaired in vivo by RP3-associated mutations in RPGR. Moreover, RPGR and RPGRIP1 co-localize in the outer segment of rod photoreceptors, which is in full agreement with the retinitis pigmentosa phenotype observed in RP3 patients. The localization of RPGRIP1 at 14q11 makes it a strong candidate gene for RP16. These results provide a clue for the retina-specific pathogenesis in RP3, and hint towards the involvement of RPGR and RPGRIP1 in mediating vesicular transport-associated processes.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas del Ojo , Mutación , Proteínas/genética , Células Fotorreceptoras Retinianas Bastones/metabolismo , Regiones no Traducidas 3' , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Proteínas Portadoras/química , Bovinos , Sistema Libre de Células , Cromosomas Humanos Par 14 , Proteínas del Citoesqueleto , Glutatión Transferasa/metabolismo , Humanos , Inmunohistoquímica , Modelos Genéticos , Datos de Secuencia Molecular , Mutación Missense , Fenotipo , Plásmidos/metabolismo , Pruebas de Precipitina , Biosíntesis de Proteínas , Isoformas de Proteínas , Estructura Terciaria de Proteína , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Temperatura , Distribución Tisular , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.
Asunto(s)
Eliminación de Gen , Retinitis Pigmentosa/genética , Línea Celular , Cromosomas Artificiales de Levadura , Cósmidos , Exones , Humanos , Reacción en Cadena de la Polimerasa , Cromosoma XRESUMEN
X-linked retinitis pigmentosa (XLRP) is a genetically heterogeneous group of progressive retinal degenerations. The disease process is initiated by premature apoptosis of rod photoreceptor cells in the retina, which leads to reduced visual acuity and, eventually, complete blindness. Mutations in the retinitis pigmentosa GTPase regulator ( RPGR ), a ubiquitously expressed gene at the RP3 locus in Xp21.1, account for approximately 20% of all X-linked cases. We have analysed the expression of this gene by northern blot hybridization, cDNA library screening and RT-PCR in various organs from mouse and man. These studies revealed at least 12 alternatively spliced isoforms. Some of the transcripts are tissue specific and contain novel exons, which elongate or truncate the previously reported open reading frame of the mouse and human RPGR gene. One of the newly identified exons is expressed exclusively in the human retina and mouse eye and contains a premature stop codon. The deduced polypeptide lacks 169 amino acids from the C-terminus of the ubiquitously expressed variant, including an isoprenylation site. Moreover, this exon was found to be deleted in a family with XLRP. Our results indicate tissue-dependent regulation of alternative splicing of RPGR in mouse and man. The discovery of a retina-specific transcript may explain why phenotypic abberations in RP3 are confined to the eye.
Asunto(s)
Proteínas Portadoras/genética , Proteínas del Ojo , Isoformas de Proteínas/genética , Retina/metabolismo , Retinitis Pigmentosa/genética , Cromosoma X/genética , Adulto , Animales , Animales Recién Nacidos , Secuencia de Bases , Ceguera/genética , Proteínas Portadoras/metabolismo , Línea Celular , ADN Complementario/química , ADN Complementario/genética , Exones/genética , Femenino , Estudios de Seguimiento , Expresión Génica , Genes/genética , Ligamiento Genético , Humanos , Intrones/genética , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , ARN/genética , ARN/metabolismo , Retina/patología , Retinitis Pigmentosa/patología , Análisis de Secuencia de ADN , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Distribución Tisular , Transcripción Genética , Pruebas de VisiónRESUMEN
The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X-linked RP (XLRP), has been mapped previously to a chromosome interval of less than 1000 kbp between the DXS1110 marker and the OTC locus at Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization (YRH)', we have recently identified a small XLRP associated microdeletion in this interval, as well as several putative exons including the 3' end of a gene that was truncated by the deletion. cDNA library screening and sequencing of a cosmid centromeric to the deletion has now enabled us to identify numerous additional exons and to detect several point mutations in patients with XLRP. The predicted gene product shows homology to RCC1, the guanine-nucleotide-exchange factor (GEF) of the Ras-like GTPase Ran. Our findings suggest that we have cloned the long-sought RP3 gene, and that it may encode the GEF of a retina-specific GTP-binding protein.