RESUMEN
The ionotropic glutamate delta receptor GluD1, encoded by the GRID1 gene, is involved in synapse formation, function, and plasticity. GluD1 does not bind glutamate, but instead cerebellin and D-serine, which allow the formation of trans-synaptic bridges, and trigger transmembrane signaling. Despite wide expression in the nervous system, pathogenic GRID1 variants have not been characterized in humans so far. We report homozygous missense GRID1 variants in five individuals from two unrelated consanguineous families presenting with intellectual disability and spastic paraplegia, without (p.Thr752Met) or with (p.Arg161His) diagnosis of glaucoma, a threefold phenotypic association whose genetic bases had not been elucidated previously. Molecular modeling and electrophysiological recordings indicated that Arg161His and Thr752Met mutations alter the hinge between GluD1 cerebellin and D-serine binding domains and the function of this latter domain, respectively. Expression, trafficking, physical interaction with metabotropic glutamate receptor mGlu1, and cerebellin binding of GluD1 mutants were not conspicuously altered. Conversely, upon expression in neurons of dissociated or organotypic slice cultures, we found that both GluD1 mutants hampered metabotropic glutamate receptor mGlu1/5 signaling via Ca2+ and the ERK pathway and impaired dendrite morphology and excitatory synapse density. These results show that the clinical phenotypes are distinct entities segregating in the families as an autosomal recessive trait, and caused by pathophysiological effects of GluD1 mutants involving metabotropic glutamate receptor signaling and neuronal connectivity. Our findings unravel the importance of GluD1 receptor signaling in sensory, cognitive and motor functions of the human nervous system.
Asunto(s)
Discapacidad Intelectual , Receptores de Glutamato Metabotrópico , Transducción de Señal , Sinapsis , Humanos , Discapacidad Intelectual/genética , Masculino , Sinapsis/metabolismo , Sinapsis/genética , Femenino , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/genética , Homocigoto , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/genética , Linaje , Adulto , Paraplejía/genética , Paraplejía/metabolismo , Animales , Niño , Neuronas/metabolismo , Adolescente , Células HEK293 , Mutación/genéticaRESUMEN
The ubiquitin pathway, one of the main actors regulating cell signaling processes and cellular protein homeostasis, is directly involved in the pathophysiology of amyotrophic lateral sclerosis (ALS). We first analyzed, by a next-generation sequencing (NGS) strategy, a series of genes of the ubiquitin pathway in two cohorts of familial and sporadic ALS patients comprising 176 ALS patients. We identified several pathogenic variants in different genes of this ubiquitin pathway already described in ALS, such as FUS, CCNF and UBQLN2. Other variants of interest were discovered in new genes studied in this disease, in particular in the HECW1 gene. We have shown that the HECT E3 ligase called NEDL1, encoded by the HECW1 gene, is expressed in neurons, mainly in their somas. Its overexpression is associated with increased cell death in vitro and, very interestingly, with the cytoplasmic mislocalization of TDP-43, a major protein involved in ALS. These results give new support for the role of the ubiquitin pathway in ALS, and suggest further studies of the HECW1 gene and its protein NEDL1 in the pathophysiology of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Neuronas/metabolismo , Transducción de Señal/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
The X-linked PTCHD1 gene, encoding a synaptic membrane protein, has been involved in neurodevelopmental disorders with the description of deleterious genomic microdeletions or truncating coding mutations. Missense variants were also identified, however, without any functional evidence supporting their pathogenicity level. We investigated 13 missense variants of PTCHD1, including eight previously described (c.152G>A,p.(Ser51Asn); c.217C>T,p.(Leu73Phe); c.517A>G,p.(Ile173Val); c.542A>C,p.(Lys181Thr); c.583G>A,p.(Val195Ile); c.1076A>G,p.(His359Arg); c.1409C>A,p.(Ala470Asp); c.1436A>G,p.(Glu479Gly)), and five novel ones (c.95C>T,p.(Pro32Leu); c.95C>G,p.(Pro32Arg); c.638A>G,p.(Tyr213Cys); c.898G>C,p.(Gly300Arg); c.928G>C,p.(Ala310Pro)) identified in male patients with intellectual disability (ID) and/or autism spectrum disorder (ASD). Interestingly, several of these variants involve amino acids localized in structural domains such as transmembrane segments. To evaluate their potentially deleterious impact on PTCHD1 protein function, we performed in vitro overexpression experiments of the wild-type and mutated forms of PTCHD1-GFP in HEK 293T and in Neuro-2a cell lines as well as in mouse hippocampal primary neuronal cultures. We found that six variants impaired the expression level of the PTCHD1 protein, and were retained in the endoplasmic reticulum suggesting abnormal protein folding. Our functional analyses thus provided evidence of the pathogenic impact of missense variants in PTCHD1, which reinforces the involvement of the PTCHD1 gene in ID and in ASD.
Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Proteínas de la Membrana , Animales , Trastorno del Espectro Autista/genética , Membrana Celular/metabolismo , Humanos , Discapacidad Intelectual/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Mutación MissenseRESUMEN
The 22q11.2 deletion syndrome (22q11DS) is associated with a wide spectrum of cognitive and psychiatric symptoms. Despite the considerable work performed over the past 20 years, the genetic etiology of the neurodevelopmental phenotype remains speculative. Here, we report de novo heterozygous truncating variants in the HIRA (Histone cell cycle regulation defective, S. Cerevisiae, homolog of, A) gene associated with a neurodevelopmental disorder in two unrelated patients. HIRA is located within the commonly deleted region of the 22q11DS and encodes a histone chaperone that regulates neural progenitor proliferation and neurogenesis, and that belongs to the WD40 Repeat (WDR) protein family involved in brain development and neuronal connectivity. To address the specific impact of HIRA haploinsufficiency in the neurodevelopmental phenotype of 22q11DS, we combined Hira knock-down strategies in developing mouse primary hippocampal neurons, and the direct study of brains from heterozygous Hira+/- mice. Our in vitro analyses revealed that Hira gene is mostly expressed during neuritogenesis and early dendritogenesis stages in mouse total brain and in developing primary hippocampal neurons. Moreover, shRNA knock-down experiments showed that a twofold decrease of endogenous Hira expression level resulted in an impaired dendritic growth and branching in primary developing hippocampal neuronal cultures. In parallel, in vivo analyses demonstrated that Hira+/- mice displayed subtle neuroanatomical defects including a reduced size of the hippocampus, the fornix and the corpus callosum. Our results suggest that HIRA haploinsufficiency would likely contribute to the complex pathophysiology of the neurodevelopmental phenotype of 22q11DS by impairing key processes in neurogenesis and by causing neuroanatomical defects during cerebral development.
Asunto(s)
Proteínas de Ciclo Celular/genética , Síndrome de DiGeorge/genética , Haploinsuficiencia , Chaperonas de Histonas/genética , Trastornos del Neurodesarrollo/genética , Plasticidad Neuronal/genética , Neuronas/metabolismo , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Niño , Preescolar , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Síndrome de DiGeorge/metabolismo , Síndrome de DiGeorge/patología , Femenino , Fórnix/metabolismo , Fórnix/patología , Expresión Génica , Heterocigoto , Hipocampo/metabolismo , Hipocampo/patología , Chaperonas de Histonas/antagonistas & inhibidores , Chaperonas de Histonas/deficiencia , Chaperonas de Histonas/metabolismo , Humanos , Ratones , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Neurogénesis/genética , Neuronas/patología , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: Pax6 is a key regulatory gene for eye, brain, and pancreas development. It acts as a transcriptional activator and repressor. Loss-of-function of Pax6 results in down- and upregulation of a comparable number of genes, although many are secondary targets. Recently, we found a prototype of a Pax6-binding site that acts as a transcriptional repressor. We also identified the Trpm3 gene as a Pax6-direct target containing the miR-204 gene located in intron 6. Thus, there are multiple Pax6-dependent mechanisms of transcriptional repression in the cell. More than 50 Pax6 missense mutations have been identified in humans and mice. Two of these mutations, N50K (Leca4) and R128C (Leca2), were analyzed in depth resulting in different numbers of regulated genes and different ratios of down- and upregulated targets. Thus, additional studies of these mutants are warranted to better understand the molecular mechanisms of the mutants' action. METHODS: Mutations in PAX6 and PAX6(5a), including G18W, R26G, N50K, G64V, R128C, and R242T, were generated with site-directed mutagenesis. A panel of ten luciferase reporters driven by six copies of Pax6-binding sites representing a spectrum of sites that act as repressors, moderate activators, and strong activators were used. Two additional reporters, including the Pax6-regulated enhancer from mouse Trpm3 and six copies of its individual Pax6-binding site, were also tested in P19 cells. RESULTS: PAX6 (N50K) acted either as a loss-of-function or neutral mutation. In contrast, PAX6 (R128C) and (R242T) acted as loss-, neutral, and gain-of-function mutations. With three distinct reporters, the PAX6 (N50K) mutation broke the pattern of effects produced by substitutions in the surrounding helices of the N-terminal region of the paired domain. All six mutations tested acted as loss-of-function using the Trpm3 Pax6-binding site. CONCLUSIONS: These studies highlight the complexity of Pax6-dependent transcriptional activation and repression mechanisms, and identify the N50K and R128C substitutions as valuable tools for testing interactions between Pax6, Pax6 (N50K), and Pax6 (R128C) with other regulatory proteins, including chromatin remodelers.
Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , MicroARNs/genética , Mutación Missense/genética , Factores de Transcripción Paired Box/química , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Canales Catiónicos TRPM/genética , Transcripción Genética , Animales , Secuencia de Bases , Proteínas del Ojo/genética , Genes Reporteros , Proteínas de Homeodominio/genética , Humanos , Luciferasas , Ratones , MicroARNs/metabolismo , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Homología Estructural de Proteína , Relación Estructura-Actividad , Canales Catiónicos TRPM/metabolismo , Activación Transcripcional/genéticaRESUMEN
The dihydropyrimidinase-like (DPYSL) proteins, also designated as the collapsin response mediators (CRMP) proteins, constitute a family of five cytosolic phosphoproteins abundantly expressed in the developing nervous system but down-regulated in the adult mouse brain. The DPYSL proteins were initially identified as effectors of semaphorin 3A (Sema3A) signaling and consequently involved in regulation of growth cone collapse in young developing neurons. To date, it has been established that DPYSL proteins mediate signals for numerous intracellular/extracellular pathways and play major roles in variety of cellular process including cell migration, neurite extension, axonal guidance, dendritic spine development and synaptic plasticity through their phosphorylation status. The roles of DPYSL proteins at early stages of brain development have been described in the past years, particularly for DPYSL2 and DPYSL5 proteins. The recent characterization of pathogenic genetic variants in DPYSL2 and in DPYSL5 human genes associated with intellectual disability and brain malformations, such as agenesis of the corpus callosum and cerebellar dysplasia, highlighted the pivotal role of these actors in the fundamental processes of brain formation and organization. In this review, we sought to establish a detailed update on the knowledge regarding the functions of DPYSL genes and proteins in brain and to highlight their involvement in synaptic processing in later stages of neurodevelopment, as well as their particular contribution in human neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD) and intellectual disability (ID).
RESUMEN
BACKGROUND: Mutations in the X-linked gene DDX3X account for approximately 2% of intellectual disability in females, often comorbid with behavioral problems, motor deficits, and brain malformations. DDX3X encodes an RNA helicase with emerging functions in corticogenesis and synaptogenesis. METHODS: We generated a Ddx3x haploinsufficient mouse (Ddx3x+/- females) with construct validity for DDX3X loss-of-function mutations. We used standardized batteries to assess developmental milestones and adult behaviors, as well as magnetic resonance imaging and immunostaining of cortical projection neurons to capture early postnatal changes in brain development. RESULTS: Ddx3x+/- females showed physical, sensory, and motor delays that evolved into behavioral anomalies in adulthood, including hyperactivity, anxiety-like behaviors, cognitive impairments in specific tasks (e.g., contextual fear memory but not novel object recognition memory), and motor deficits. Motor function declined with age but not if mice were previously exposed to behavioral training. Developmental and behavioral changes were associated with a reduction in brain volume, with some regions (e.g., cortex and amygdala) disproportionally affected. Cortical thinning was accompanied by defective cortical lamination, indicating that Ddx3x regulates the balance of glutamatergic neurons in the developing cortex. CONCLUSIONS: These data shed new light on the developmental mechanisms driving DDX3X syndrome and support construct and face validity of this novel preclinical mouse model.