RESUMEN
Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.
Asunto(s)
Anilidas/farmacología , Movimiento Celular/genética , Corteza Cerebral/metabolismo , Epilepsia/genética , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Proteínas de la Membrana/genética , Neuronas/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Epilepsia/metabolismo , Silenciador del Gen , Humanos , Neuronas/citología , Neuronas/efectos de los fármacos , Ratas , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
The onset of feeding at birth is a vital step for the adaptation of the neonate to extra uterine life. Prader-Willi syndrome (PWS) is a complex neurogenetic disorder caused by the alteration of several imprinted contiguous genes including MAGEL2. PWS presents with various clinical manifestations, including poor suckling behaviour and feeding problems in neonates. Hypothalamic defects have been proposed, but the pathophysiological mechanisms remain poorly understood. Here, we report that a Magel2-deficient mouse with 50% neonatal mortality had an altered onset of suckling activity and subsequent impaired feeding, suggesting a role of MAGEL2 in the suckling deficit seen in PW newborns. The hypothalamus of Magel2 mutant neonates showed a significant reduction in oxytocin (OT). Furthermore, injection of a specific OT receptor antagonist in wild-type neonates recapitulated the feeding deficiency seen in Magel2 mutants, and a single injection of OT, 3-5 h after birth, rescued the phenotype of Magel2 mutant pups, allowing all of them to survive. Our study illustrates the crucial role of feeding onset behaviour after birth. We propose that OT supply might constitute a promising avenue for the treatment of feeding difficulties in PW neonates and potentially of other newborns with impaired feeding onset.
Asunto(s)
Antígenos de Neoplasias/genética , Conducta Alimentaria/efectos de los fármacos , Impresión Genómica/efectos de los fármacos , Oxitocina/administración & dosificación , Oxitocina/farmacología , Proteínas/genética , Animales , Animales Recién Nacidos , Animales Lactantes/metabolismo , Antígenos de Neoplasias/metabolismo , Femenino , Marcación de Gen , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Inmunohistoquímica , Inyecciones Subcutáneas , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptidos/metabolismo , Sistemas Neurosecretores/efectos de los fármacos , Sistemas Neurosecretores/fisiología , Orexinas , Fenotipo , Proteínas/metabolismo , Receptores de Oxitocina/antagonistas & inhibidores , Vasopresinas/metabolismoRESUMEN
It is a challenge to identify the molecular networks contributing to the neural basis of human speech. Mutations in transcription factor FOXP2 cause difficulties mastering fluent speech (developmental verbal dyspraxia, DVD), whereas mutations of sushi-repeat protein SRPX2 lead to epilepsy of the rolandic (sylvian) speech areas, with DVD or with bilateral perisylvian polymicrogyria. Pathophysiological mechanisms driven by SRPX2 involve modified interaction with the plasminogen activator receptor (uPAR). Independent chromatin-immunoprecipitation microarray screening has identified the uPAR gene promoter as a potential target site bound by FOXP2. Here, we directly tested for the existence of a transcriptional regulatory network between human FOXP2 and the SRPX2/uPAR complex. In silico searches followed by gel retardation assays identified specific efficient FOXP2-binding sites in each of the promoter regions of SRPX2 and uPAR. In FOXP2-transfected cells, significant decreases were observed in the amounts of both SRPX2 (43.6%) and uPAR (38.6%) native transcripts. Luciferase reporter assays demonstrated that FOXP2 expression yielded a marked inhibition of SRPX2 (80.2%) and uPAR (77.5%) promoter activity. A mutant FOXP2 that causes DVD (p.R553H) failed to bind to SRPX2 and uPAR target sites and showed impaired down-regulation of SRPX2 and uPAR promoter activity. In a patient with polymicrogyria of the left rolandic operculum, a novel FOXP2 mutation (p.M406T) was found in the leucine-zipper (dimerization) domain. p.M406T partially impaired the FOXP2 regulation of SRPX2 promoter activity, whereas that of the uPAR promoter remained unchanged. Together with recently described FOXP2-CNTNAP2 and SRPX2/uPAR links, the FOXP2-SRPX2/uPAR network provides exciting insights into molecular pathways underlying speech-related disorders.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Redes Reguladoras de Genes , Proteínas del Tejido Nervioso/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Trastornos del Habla/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso/genética , Ensayo de Cambio de Movilidad Electroforética , Femenino , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Genes Reporteros , Células HEK293 , Humanos , Luciferasas/metabolismo , Masculino , Malformaciones del Desarrollo Cortical/complicaciones , Malformaciones del Desarrollo Cortical/genética , Proteínas de la Membrana , Datos de Secuencia Molecular , Mutación Missense/genética , Proteínas de Neoplasias , Proteínas del Tejido Nervioso/genética , Linaje , Regiones Promotoras Genéticas/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Trastornos del Habla/complicacionesRESUMEN
Mutations in SRPX2 (Sushi-Repeat Protein, X-linked 2) cause rolandic epilepsy with speech impairment (RESDX syndrome) or with altered development of the speech cortex (bilateral perisylvian polymicrogyria). The physiological roles of SRPX2 remain unknown to date. One way to infer the function of SRPX2 relies on the identification of the as yet unknown SRPX2 protein partners. Using a combination of interactome approaches including yeast two-hybrid screening, co-immunoprecipitation experiments, cell surface binding and surface plasmon resonance (SPR), we show that SRPX2 is a ligand for uPAR, the urokinase-type plasminogen activator (uPA) receptor. Previous studies have shown that uPAR(-/-) knock-out mice exhibited enhanced susceptibility to epileptic seizures and had brain cortical anomalies consistent with altered neuronal migration and maturation, all features that are reminiscent to the phenotypes caused by SRPX2 mutations. SPR analysis indicated that the p.Y72S mutation associated with rolandic epilepsy and perisylvian polymicrogyria, led to a 5.8-fold gain-of-affinity of SRPX2 with uPAR. uPAR is a crucial component of the extracellular plasminogen proteolysis system; two more SRPX2 partners identified here, the cysteine protease cathepsin B (CTSB) and the metalloproteinase ADAMTS4, are also components of the extracellular proteolysis machinery and CTSB is a well-known activator of uPA. The identification of functionally related SRPX2 partners provides the first and exciting insights into the possible role of SRPX2 in the brain, and suggests that a network of SRPX2-interacting proteins classically involved in the proteolytic remodeling of the extracellular matrix and including uPAR participates in the functioning, in the development and in disorders of the speech cortex.
Asunto(s)
Corteza Cerebral/metabolismo , Epilepsia Rolándica/metabolismo , Mutación , Proteínas del Tejido Nervioso/metabolismo , Trastornos del Habla/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Epilepsia Rolándica/genética , Expresión Génica , Humanos , Proteínas de la Membrana , Proteínas de Neoplasias , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Ratas , Trastornos del Habla/genética , Técnicas del Sistema de Dos Híbridos , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
The syntaxins are proteins associated with various intracellular membrane compartments. They are major participants in a large variety of physiological processes where membrane fusion occurs, including exocytosis. We have identified a novel syntaxin isoform generated by alternative splicing of the human STX1B gene. In contrast with the canonical syntaxins, this isoform (STX1B-DeltaTMD) lacked the classical C-terminal transmembrane domain and localized to the nucleus of various tumoral and non-tumoral cell types including human brain cortical neurons in vivo. The reversible blockade of STX1B-DeltaTMD nuclear import demonstrated that nuclear import occurred via a Ran-dependent pathway. A specific and glycine-rich C-terminus of 15 amino acids served as an unconventional nuclear localization signal. STX1B-DeltaTMD colocalized with Lamin A/C and NuMA (NUclear Mitotic Apparatus protein) in interphasic nuclei, and with NuMA and gamma-tubulin in the pericentrosomal region of the mitotic spindle in dividing cells. In a series of 37 human primary brain tumors, the ratio of STX1B-DeltaTMD to Lamin A/C transcripts was a significant prognostic marker of survival, independent of tumor staging. The characterization of STX1B-DeltaTMD as the first nucleoplasmic syntaxin with no transmembrane domain, illustrates the importance of alternative splicing in the emergence of unsuspected properties of the syntaxins in human cells, in both physiological and pathological conditions.
Asunto(s)
Núcleo Celular/metabolismo , Sintaxina 1/metabolismo , Empalme Alternativo/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular , Centrosoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Lamina Tipo A/genética , Proteínas Mutantes/metabolismo , Matriz Nuclear/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sintaxina 1/química , Proteína de Unión al GTP ran/metabolismoRESUMEN
Human mesial temporal lobe epilepsies (MTLE) are the most frequent form of partial epilepsies and display frequent pharmacoresistance. The molecular alterations underlying human MTLE remain poorly understood. A two-step transcriptional analysis consisting in cDNA microarray experiments followed by quantitative RT-PCR validations was performed. Because the entorhinal cortex (EC) plays an important role in the pathophysiology of the MTLE and usually discloses no detectable or little cell loss, resected EC and each corresponding lateral temporal neocortex (LTC) of MTLE patients were used as the source of disease-associated and control RNAs, respectively. Six genes encoding (i) a serotonin receptor (HTR2A) and a neuropeptide Y receptor type 1 (NPY1R), (ii) a protein (FHL2) associating with the KCNE1 (minK) potassium channel subunit and with presenilin-2 and (iii) three immune system-related proteins (C3, HLA-DR-gamma and CD99), were found consistently downregulated or upregulated in the EC of MTLE patients as compared with non-epileptic autopsy controls. Quantitative western blot analyses confirmed decreased expression of NPY1R in all eight MTLE patients tested. Immunohistochemistry experiments revealed the existence of a perivascular infiltration of C3 positive leucocytes and/or detected membrane attack complexes on a subset of neurons, within the EC of nine out of eleven MTLE patients. To summarize, a large-scale microarray expression study on the EC of MTLE patients led to the identification of six candidate genes for human MTLE pathophysiology. Altered expression of NPY1R and C3 was also demonstrated at the protein level. Overall, our data indicate that local dysregulation of the neurotransmission and complement systems in the EC is a frequent event in human MTLE.
Asunto(s)
Complemento C3/metabolismo , Corteza Entorrinal/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Neurotransmisores/metabolismo , Adulto , Complemento C3/genética , Complejo de Ataque a Membrana del Sistema Complemento , Regulación hacia Abajo , Electroforesis en Gel de Poliacrilamida/métodos , Corteza Entorrinal/inmunología , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/inmunología , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Neurotransmisores/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores de Neuropéptido Y/genética , Receptores de Neuropéptido Y/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Regulación hacia ArribaRESUMEN
BACKGROUND: Congenital cytomegalovirus infections are a leading cause of neurodevelopmental disorders in human and represent a major health care and socio-economical burden. In contrast with this medical importance, the pathophysiological events remain poorly known. Murine models of brain cytomegalovirus infection, mostly neonatal, have brought recent insights into the possible pathogenesis, with convergent evidence for the alteration and possible involvement of brain immune cells. OBJECTIVES AND METHODS: In order to confirm and expand those findings, particularly concerning the early developmental stages following infection of the fetal brain, we have created a model of in utero cytomegalovirus infection in the developing rat brain. Rat cytomegalovirus was injected intraventricularly at embryonic day 15 (E15) and the brains analyzed at various stages until the first postnatal day, using a combination of gene expression analysis, immunohistochemistry and multicolor flow cytometry experiments. RESULTS: Rat cytomegalovirus infection was increasingly seen in various brain areas including the choroid plexi and the ventricular and subventricular areas and was prominently detected in CD45low/int, CD11b+ microglial cells, in CD45high, CD11b+ cells of the myeloid lineage including macrophages, and in CD45+, CD11b- lymphocytes and non-B non-T cells. In parallel, rat cytomegalovirus infection of the developing rat brain rapidly triggered a cascade of pathophysiological events comprising: chemokines upregulation, including CCL2-4, 7 and 12; infiltration by peripheral cells including B-cells and monocytes at E17 and P1, and T-cells at P1; and microglia activation at E17 and P1. CONCLUSION: In line with previous findings in neonatal murine models and in human specimen, our study further suggests that neuroimmune alterations might play critical roles in the early stages following cytomegalovirus infection of the brain in utero. Further studies are now needed to determine which role, whether favorable or detrimental, those putative double-edge swords events actually play.
Asunto(s)
Encéfalo/embriología , Infecciones por Citomegalovirus/patología , Microglía/patología , Muromegalovirus/patogenicidad , Animales , Linaje de la Célula , Infecciones por Citomegalovirus/inmunología , Citometría de Flujo , Activación de Macrófagos , Microglía/inmunología , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cotransporters represent a major class of proteins that make use of ion gradients to drive active transport of substrate into cells. A new human gene, KST1, encoding a member of the sodium/glucose cotransporter family, was identified onto human chromosome 16p12-p11. This genomic region contains a major gene responsible for a syndrome of infantile convulsions and paroxysmal dyskinesia (ICCA syndrome), inherited as an autosomal dominant trait, as well as for benign familial infantile convulsions (BFIC). The entire coding sequence of the human KST1 gene was determined using a combination of methods including in silico comparison of its rabbit orthologous DNA complementary to RNA (cDNA) to the corresponding human genomic sequences, reverse transcription-polymerase chain reaction on human brain RNA, 5' and 3' rapid amplification of cDNA ends. The gene is divided into 16 exons and the predicted protein of 675 amino acids contains 14 transmembrane domains. It shares significant homology to the sodium-glucose transporter 1 cotransporter proteins. An alternatively spliced transcript resulting from the skipping of exon 6 led to a predicted protein lacking the 4th transmembrane domain. As ion transporters are good candidates for a large variety of human diseases, including paroxysmal disorders, a mutation search was performed in four families with ICCA or BFIC syndromes. No pathogenic mutation was found, although several polymorphic variants with amino acids exchanges were identified. Due to its broad expression in human tissues, the human KST1 gene could be involved in several other diseases mapped to human chromosome 16p12-p11.
Asunto(s)
Proteínas de Transporte de Monosacáridos/genética , Simportadores , Secuencia de Aminoácidos , Atetosis/complicaciones , Atetosis/genética , Secuencia de Bases , Northern Blotting , Corea/complicaciones , Corea/genética , Clonación Molecular , Análisis Mutacional de ADN , ADN Complementario/química , ADN Complementario/genética , Epilepsia Benigna Neonatal/genética , Exones , Salud de la Familia , Expresión Génica , Genes/genética , Humanos , Lactante , Recién Nacido , Intrones , Datos de Secuencia Molecular , Mutación , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Convulsiones/complicaciones , Convulsiones/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteínas de Transporte de Sodio-GlucosaRESUMEN
OBJECTIVE: Whole genome sequencing and the screening of 103 families recently led us to identify PRRT2 (proline-rich-transmembrane protein) as the gene causing infantile convulsions (IC) with paroxysmal kinesigenic dyskinesia (PKD) (PKD/IC syndrome, formerly ICCA). There is interfamilial and intrafamilial variability and the patients may have IC or PKD. Association of IC with hemiplegic migraine (HM) has also been reported. In order to explore the mutational and clinical spectra, we analyzed 34 additional families with either typical PKD/IC or PKD/IC with migraine. METHODS: We performed Sanger sequencing of all PRRT2 coding exons and of exon-intron boundaries in the probands and in their relatives whenever appropriate. RESULTS: Two known and 2 novel PRRT2 mutations were detected in 18 families. The p.R217Pfs*8 recurrent mutation was found in ≈50% of typical PKD/IC, and the unreported p.R145Gfs*31 in one more typical family. PRRT2 mutations were also found in PKD/IC with migraine: p.R217Pfs*8 cosegregated with PKD associated with HM in one family, and was also detected in one IC patient having migraine with aura, in related PKD/IC familial patients having migraine without aura, and in one sporadic migraineur with abnormal MRI. Previously reported p.R240X was found in one patient with PKD with migraine without aura. The novel frameshift p.S248Afs*65 was identified in a PKD/IC family member with IC and migraine with aura. CONCLUSIONS: We extend the spectrum of PRRT2 mutations and phenotypes to HM and to other types of migraine in the context of PKD/IC, and emphasize the phenotypic pleiotropy seen in patients with PRRT2 mutations.
Asunto(s)
Discinesias/diagnóstico , Discinesias/genética , Epilepsia Benigna Neonatal/diagnóstico , Epilepsia Benigna Neonatal/genética , Ligamiento Genético/genética , Proteínas de la Membrana/genética , Trastornos Migrañosos/diagnóstico , Trastornos Migrañosos/genética , Proteínas del Tejido Nervioso/genética , Convulsiones/diagnóstico , Convulsiones/genética , Secuencia de Bases , Corea/diagnóstico , Corea/epidemiología , Corea/genética , Discinesias/epidemiología , Epilepsia Benigna Neonatal/epidemiología , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Trastornos Migrañosos/epidemiología , Datos de Secuencia Molecular , Mutación/genética , Linaje , Convulsiones/epidemiologíaRESUMEN
BACKGROUND: Benign infantile convulsions and paroxysmal dyskinesia are episodic cerebral disorders that can share common genetic bases. They can be co-inherited as one single autosomal dominant trait (ICCA syndrome); the disease ICCA gene maps at chromosome 16p12-q12. Despite intensive and conventional mutation screening, the ICCA gene remains unknown to date. The critical area displays highly complicated genomic architecture and is the site of deletions and duplications associated with various diseases. The possibility that the ICCA syndrome is related to the existence of large-scale genomic alterations was addressed in the present study. METHODOLOGY/PRINCIPAL FINDINGS: A combination of whole genome and dedicated oligonucleotide array comparative genomic hybridization coupled with quantitative polymerase chain reaction was used. Low copy number of a region corresponding to a genomic variant (Variation_7105) located at 16p11 nearby the centromere was detected with statistical significance at much higher frequency in patients from ICCA families than in ethnically matched controls. The genomic variant showed no apparent difference in size and copy number between patients and controls, making it very unlikely that the genomic alteration detected here is ICCA-specific. Furthermore, no other genomic alteration that would directly cause the ICCA syndrome in those nine families was detected in the ICCA critical area. CONCLUSIONS/SIGNIFICANCE: Our data excluded that inherited genomic deletion or duplication events directly cause the ICCA syndrome; rather, they help narrowing down the critical ICCA region dramatically and indicate that the disease ICCA genetic defect lies very close to or within Variation_7105 and hence should now be searched in the corresponding genomic area and its surrounding regions.
Asunto(s)
Corea/genética , Cromosomas Humanos Par 16 , Epilepsia Benigna Neonatal/genética , Dosificación de Gen , Estudios de Casos y Controles , Femenino , Humanos , Hibridación in Situ , Lactante , Masculino , Linaje , Reacción en Cadena de la Polimerasa , SíndromeRESUMEN
The rolandic and sylvian fissures divide the human cerebral hemispheres and the adjacent areas participate in speech processing. The relationship of rolandic (sylvian) seizure disorders with speech and cognitive impairments is well known, albeit poorly understood. We have identified the Xq22 gene SRPX2 as being responsible for rolandic seizures (RSs) associated with oral and speech dyspraxia and mental retardation (MR). SRPX2 is a secreted sushi-repeat containing protein expressed in neurons of the human adult brain, including the rolandic area. The disease-causing mutation (N327S) resulted in gain-of-glycosylation of the secreted mutant protein. A second mutation (Y72S) was identified within the first sushi domain of SRPX2 in a male with RSs and bilateral perisylvian polymicrogyria and his female relatives with mild MR or unaffected carrier status. In cultured cells, both mutations were associated with altered patterns of intracellular processing, suggesting protein misfolding. In the murine brain, Srpx2 protein expression appeared in neurons at birth. The involvement of SRPX2 in these disorders suggests an important role for SRPX2 in the perisylvian region critical for language and cognitive development.