RESUMEN
Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.
Asunto(s)
Microscopía por Crioelectrón , Hidroliasas , Transferasas Intramoleculares , Seudouridina , ARN de Transferencia , Humanos , Dominio Catalítico , Células HEK293 , Hidroliasas/metabolismo , Hidroliasas/genética , Hidroliasas/química , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/enzimología , Modelos Moleculares , Mutación , Unión Proteica , Seudouridina/metabolismo , Seudouridina/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Especificidad por SustratoRESUMEN
Nonsense-mediated mRNA decay (NMD) is one of the best characterized and most evolutionarily conserved cellular quality control mechanisms. Although NMD was first found to target one-third of mutated, disease-causing mRNAs, it is now known to also target ~10% of unmutated mammalian mRNAs to facilitate appropriate cellular responses - adaptation, differentiation or death - to environmental changes. Mutations in NMD genes in humans are associated with intellectual disability and cancer. In this Review, we discuss how NMD serves multiple purposes in human cells by degrading both mutated mRNAs to protect the integrity of the transcriptome and normal mRNAs to control the quantities of unmutated transcripts.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Discapacidad Intelectual/metabolismo , Mutación , Neoplasias/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Transcriptoma , Animales , Humanos , Discapacidad Intelectual/genética , Neoplasias/genética , ARN Mensajero/genética , ARN Neoplásico/genéticaRESUMEN
The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.
Asunto(s)
Epilepsia , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Humanos , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Encéfalo/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Lípidos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.
Asunto(s)
ADN Helicasas/metabolismo , Elementos de Facilitación Genéticos , Heterocigoto , Células Madre Embrionarias Humanas/metabolismo , Mutación Missense , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , ADN Helicasas/genética , Facies , Deformidades Congénitas del Pie/genética , Deformidades Congénitas del Pie/metabolismo , Deformidades Congénitas del Pie/patología , Antígeno 2 Relacionado con Fos/biosíntesis , Antígeno 2 Relacionado con Fos/genética , Células HEK293 , Células Madre Embrionarias Humanas/patología , Humanos , Hipotricosis/genética , Hipotricosis/metabolismo , Hipotricosis/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Proteínas Nucleares/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genéticaRESUMEN
Genes encoding the KDM5 family of transcriptional regulators are disrupted in individuals with intellectual disability (ID). To understand the link between KDM5 and ID, we characterized five Drosophila strains harboring missense alleles analogous to those observed in patients. These alleles disrupted neuroanatomical development, cognition and other behaviors, and displayed a transcriptional signature characterized by the downregulation of many ribosomal protein genes. A similar transcriptional profile was observed in KDM5C knockout iPSC-induced human glutamatergic neurons, suggesting an evolutionarily conserved role for KDM5 proteins in regulating this class of gene. In Drosophila, reducing KDM5 changed neuronal ribosome composition, lowered the translation efficiency of mRNAs required for mitochondrial function, and altered mitochondrial metabolism. These data highlight the cellular consequences of altered KDM5-regulated transcriptional programs that could contribute to cognitive and behavioral phenotypes. Moreover, they suggest that KDM5 may be part of a broader network of proteins that influence cognition by regulating protein synthesis.
Asunto(s)
Proteínas de Drosophila , Neuronas , Proteínas Ribosómicas , Animales , Humanos , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Neuronas/metabolismo , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Ribosomas/genética , Activación TranscripcionalRESUMEN
TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here, we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein-protein interaction.
Asunto(s)
Trastornos del Neurodesarrollo , Proteínas de Dominio T Box , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas/genética , Proteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
SATB2 is a schizophrenia risk gene and is genetically associated with human intelligence. How it affects cognition at molecular level is currently unknown. Here, we show that interactions between SATB2, a chromosomal scaffolding protein, and the inner nuclear membrane protein LEMD2 orchestrate the response of pyramidal neurons to neuronal activation. Exposure to novel environment in vivo causes changes in nuclear shape of CA1 hippocampal neurons via a SATB2-dependent mechanism. The activity-driven plasticity of the nuclear envelope requires not only SATB2, but also its protein interactor LEMD2 and the ESCRT-III/VPS4 membrane-remodeling complex. Furthermore, LEMD2 depletion in cortical neurons, similar to SATB2 ablation, affects neuronal activity-dependent regulation of multiple rapid and delayed primary response genes. In human genetic data, LEMD2-regulated genes are enriched for de novo mutations reported in intellectual disability and schizophrenia and are, like SATB2-regulated genes, enriched for common variants associated with schizophrenia and cognitive function. Hence, interactions between SATB2 and the inner nuclear membrane protein LEMD2 influence gene expression programs in pyramidal neurons that are linked to cognitive ability and psychiatric disorder etiology.
Asunto(s)
Redes Reguladoras de Genes , Hipocampo/citología , Discapacidad Intelectual/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Esquizofrenia/genética , Factores de Transcripción/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Animales , Núcleo Celular/metabolismo , Plasticidad de la Célula , Células Cultivadas , Cognición , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células HeLa , Hipocampo/metabolismo , Humanos , Discapacidad Intelectual/metabolismo , Masculino , Proteínas de Unión a la Región de Fijación a la Matriz/química , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Neuronas/citología , Neuronas/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Esquizofrenia/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Covalent tRNA modifications play multi-faceted roles in tRNA stability, folding, and recognition, as well as the rate and fidelity of translation, and other cellular processes such as growth, development, and stress responses. Mutations in genes that are known to regulate tRNA modifications lead to a wide array of phenotypes and diseases including numerous cognitive and neurodevelopmental disorders, highlighting the critical role of tRNA modification in human disease. One such gene, THUMPD1, is involved in regulating tRNA N4-acetylcytidine modification (ac4C), and recently was proposed as a candidate gene for autosomal-recessive intellectual disability. Here, we present 13 individuals from 8 families who harbor rare loss-of-function variants in THUMPD1. Common phenotypic findings included global developmental delay, speech delay, moderate to severe intellectual deficiency, behavioral abnormalities such as angry outbursts, facial dysmorphism, and ophthalmological abnormalities. We demonstrate that the bi-allelic variants identified cause loss of function of THUMPD1 and that this defect results in a loss of ac4C modification in small RNAs, and of individually purified tRNA-Ser-CGA. We further corroborate this effect by showing a loss of tRNA acetylation in two CRISPR-Cas9-generated THUMPD1 KO cell lines. In addition, we also show the resultant amino acid substitution that occurs in a missense THUMPD1 allele identified in an individual with compound heterozygous variants results in a marked decrease in THUMPD1 stability and RNA-binding capacity. Taken together, these results suggest that the lack of tRNA acetylation due to THUMPD1 loss of function results in a syndromic form of intellectual disability associated with developmental delay, behavioral abnormalities, hearing loss, and facial dysmorphism.
Asunto(s)
Discapacidad Intelectual , Trastornos del Neurodesarrollo , Proteínas de Unión al ARN , Acetilación , Alelos , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Mutación/genética , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , ARN/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.
Asunto(s)
Quistes del Sistema Nervioso Central/genética , Trastornos Congénitos de Glicosilación/genética , Hamartoma/genética , Discapacidad Intelectual/genética , Oligosacáridos/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficiencia , Polimicrogiria/genética , alfa-Manosidasa/genética , Adolescente , Alelos , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Línea Celular Tumoral , Quistes del Sistema Nervioso Central/metabolismo , Quistes del Sistema Nervioso Central/patología , Vermis Cerebeloso/metabolismo , Vermis Cerebeloso/patología , Niño , Preescolar , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Femenino , Feto , Glicosilación , Hamartoma/metabolismo , Hamartoma/patología , Humanos , Hipotálamo/metabolismo , Hipotálamo/patología , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Leucocitos/metabolismo , Leucocitos/patología , Masculino , Manosa/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polimicrogiria/metabolismo , Polimicrogiria/patología , Lengua/metabolismo , Lengua/patología , alfa-Manosidasa/deficienciaRESUMEN
N-linked glycosylation is the most frequent modification of secreted and membrane-bound proteins in eukaryotic cells, disruption of which is the basis of the congenital disorders of glycosylation (CDGs). We describe a new type of CDG caused by mutations in the steroid 5alpha-reductase type 3 (SRD5A3) gene. Patients have mental retardation and ophthalmologic and cerebellar defects. We found that SRD5A3 is necessary for the reduction of the alpha-isoprene unit of polyprenols to form dolichols, required for synthesis of dolichol-linked monosaccharides, and the oligosaccharide precursor used for N-glycosylation. The presence of residual dolichol in cells depleted for this enzyme suggests the existence of an unexpected alternative pathway for dolichol de novo biosynthesis. Our results thus suggest that SRD5A3 is likely to be the long-sought polyprenol reductase and reveal the genetic basis of one of the earliest steps in protein N-linked glycosylation.
Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Anomalías Múltiples/metabolismo , Dolicoles/metabolismo , Discapacidad Intelectual/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Proteínas de Saccharomyces cerevisiae/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/genética , Animales , Butadienos/metabolismo , Consanguinidad , Embrión de Mamíferos/metabolismo , Estudio de Asociación del Genoma Completo , Glicosilación , Hemiterpenos/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Pentanos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Respuesta de Proteína DesplegadaRESUMEN
Rahman syndrome (RMNS) is a rare genetic disorder characterized by mild to severe intellectual disability, hypotonia, anxiety, autism spectrum disorder, vision problems, bone abnormalities and dysmorphic facies. RMNS is caused by de novo heterozygous mutations in the histone linker gene H1-4; however, mechanisms underlying impaired neurodevelopment in RMNS are not understood. All reported mutations associated with RMNS in H1-4 are small insertions or deletions that create a shared frameshift, resulting in a H1.4 protein that is both truncated and possessing an abnormal C-terminus frameshifted tail (H1.4 CFT). To expand understanding of mutations and phenotypes associated with mutant H1-4, we identified new variants at both the C- and N-terminus of H1.4. The clinical features of mutations identified at the C-terminus are consistent with other reports and strengthen the support of pathogenicity of H1.4 CFT. To understand how H1.4 CFT may disrupt brain function, we exogenously expressed wild-type or H1.4 CFT protein in rat hippocampal neurons and assessed neuronal structure and function. Genome-wide transcriptome analysis revealed ~ 400 genes altered in the presence of H1.4 CFT. Neuronal genes downregulated by H1.4 CFT were enriched for functional categories involved in synaptic communication and neuropeptide signaling. Neurons expressing H1.4 CFT also showed reduced neuronal activity on multielectrode arrays. These data are the first to characterize the transcriptional and functional consequence of H1.4 CFT in neurons. Our data provide insight into causes of neurodevelopmental impairments associated with frameshift mutations in the C-terminus of H1.4 and highlight the need for future studies on the function of histone H1.4 in neurons.
Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Animales , Trastorno del Espectro Autista/genética , Mutación del Sistema de Lectura/genética , Histonas/genética , Histonas/metabolismo , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Mutación , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Neuronas/metabolismo , RatasRESUMEN
ABHD16A (abhydrolase domain-containing protein 16A, phospholipase) encodes the major phosphatidylserine (PS) lipase in the brain. PS lipase synthesizes lysophosphatidylserine, an important signaling lipid that functions in the mammalian central nervous system. ABHD16A has not yet been associated with a human disease. In this report, we present a cohort of 11 affected individuals from six unrelated families with a complicated form of hereditary spastic paraplegia (HSP) who carry bi-allelic deleterious variants in ABHD16A. Affected individuals present with a similar phenotype consisting of global developmental delay/intellectual disability, progressive spasticity affecting the upper and lower limbs, and corpus callosum and white matter anomalies. Immunoblot analysis on extracts from fibroblasts from four affected individuals demonstrated little to no ABHD16A protein levels compared to controls. Our findings add ABHD16A to the growing list of lipid genes in which dysregulation can cause complicated forms of HSP and begin to describe the molecular etiology of this condition.
Asunto(s)
Parálisis Cerebral/patología , Discapacidad Intelectual/patología , Leucoencefalopatías/patología , Monoacilglicerol Lipasas/genética , Mutación , Paraplejía Espástica Hereditaria/patología , Adolescente , Adulto , Parálisis Cerebral/etiología , Parálisis Cerebral/metabolismo , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Discapacidad Intelectual/etiología , Discapacidad Intelectual/metabolismo , Leucoencefalopatías/etiología , Leucoencefalopatías/metabolismo , Masculino , Monoacilglicerol Lipasas/deficiencia , Linaje , Fenotipo , Paraplejía Espástica Hereditaria/etiología , Paraplejía Espástica Hereditaria/metabolismo , Adulto JovenRESUMEN
Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype.
Asunto(s)
Parálisis Cerebral/patología , Epilepsia/patología , Predisposición Genética a la Enfermedad , Variación Genética , Pérdida Auditiva/patología , Discapacidad Intelectual/patología , Espasticidad Muscular/patología , ATPasas Asociadas con Actividades Celulares Diversas/genética , Adolescente , Adulto , Alelos , Animales , Parálisis Cerebral/etiología , Parálisis Cerebral/metabolismo , Preescolar , Epilepsia/etiología , Epilepsia/metabolismo , Femenino , Pérdida Auditiva/etiología , Pérdida Auditiva/metabolismo , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/etiología , Discapacidad Intelectual/metabolismo , Masculino , Espasticidad Muscular/etiología , Espasticidad Muscular/metabolismo , Ratas , Adulto JovenRESUMEN
Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.
Asunto(s)
Encéfalo/metabolismo , Discapacidades del Desarrollo/genética , Epilepsia/genética , Discapacidad Intelectual/genética , Mutación , Receptores de Ácido Kaínico/genética , Adolescente , Adulto , Alelos , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Niño , Preescolar , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Epilepsia/diagnóstico por imagen , Epilepsia/metabolismo , Epilepsia/patología , Potenciales Evocados/fisiología , Regulación del Desarrollo de la Expresión Génica , Estudios de Asociación Genética , Heterocigoto , Homocigoto , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Activación del Canal Iónico , Masculino , Modelos Moleculares , Neuronas/metabolismo , Neuronas/patología , Conformación Proteica , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Receptor de Ácido Kaínico GluK2RESUMEN
Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.
Asunto(s)
Discapacidades del Desarrollo/genética , Proteínas de Drosophila/genética , Enfermedades Hereditarias del Ojo/genética , Discapacidad Intelectual/genética , Carioferinas/genética , Anomalías Musculoesqueléticas/genética , beta Carioferinas/genética , Proteína de Unión al GTP ran/genética , Alelos , Secuencia de Aminoácidos , Animales , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Hereditarias del Ojo/patología , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Genoma Humano , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Carioferinas/antagonistas & inhibidores , Carioferinas/metabolismo , Masculino , Anomalías Musculoesqueléticas/metabolismo , Anomalías Musculoesqueléticas/patología , Mutación , Neuronas/metabolismo , Neuronas/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Secuenciación Completa del Genoma , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.
Asunto(s)
Huesos/metabolismo , Proteína Coat de Complejo I/genética , Proteína Coatómero/genética , Discapacidades del Desarrollo/genética , Discapacidad Intelectual/genética , Osteoporosis/genética , Animales , Ácido Ascórbico/farmacología , Huesos/efectos de los fármacos , Huesos/patología , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Niño , Preescolar , Proteína Coat de Complejo I/deficiencia , Proteína Coatómero/química , Proteína Coatómero/deficiencia , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Discapacidades del Desarrollo/diagnóstico por imagen , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Embrión no Mamífero , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación del Desarrollo de la Expresión Génica , Aparato de Golgi , Haploinsuficiencia , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Masculino , Ratones , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoporosis/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Índice de Severidad de la Enfermedad , Pez CebraRESUMEN
Glypicans are a family of cell surface heparan sulfate proteoglycans that play critical roles in multiple cell signaling pathways. Glypicans consist of a globular core, an unstructured stalk modified with sulfated glycosaminoglycan chains, and a glycosylphosphatidylinositol anchor. Though these structural features are conserved, their individual contribution to glypican function remains obscure. Here, we investigate how glypican 3 (GPC3), which is mutated in Simpson-Golabi-Behmel tissue overgrowth syndrome, regulates Hedgehog signaling. We find that GPC3 is necessary for the Hedgehog response, surprisingly controlling a downstream signal transduction step. Purified GPC3 ectodomain rescues signaling when artificially recruited to the surface of GPC3-deficient cells but has dominant-negative activity when unattached. Strikingly, the purified stalk, modified with heparan sulfate but not chondroitin sulfate, is necessary and sufficient for activity. Our results demonstrate a novel function for GPC3-associated heparan sulfate and provide a framework for the functional dissection of glycosaminoglycans by in vivo biochemical complementation. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Anomalías Múltiples , Glipicanos , Proteínas Hedgehog , Heparitina Sulfato , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Arritmias Cardíacas , Enfermedades Genéticas Ligadas al Cromosoma X , Gigantismo , Glipicanos/genética , Glipicanos/metabolismo , Cardiopatías Congénitas , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteoglicanos de Heparán Sulfato , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Transducción de SeñalRESUMEN
Pitt Hopkins Syndrome (PTHS) is a rare syndromic form of autism spectrum disorder (ASD) caused by autosomal dominant mutations in the Transcription Factor 4 (TCF4) gene. TCF4 is a basic helix-loop-helix transcription factor that is critical for neurodevelopment and brain function through its binding to cis-regulatory elements of target genes. One potential therapeutic strategy for PTHS is to identify dysregulated target genes and normalize their dysfunction. Here, we propose that SCN10A is an important target gene of TCF4 that is an applicable therapeutic approach for PTHS. Scn10a encodes the voltage-gated sodium channel Nav1.8 and is consistently shown to be upregulated in PTHS mouse models. In this perspective, we review prior literature and present novel data that suggests inhibiting Nav1.8 in PTHS mouse models is effective at normalizing neuron function, brain circuit activity and behavioral abnormalities and posit this therapeutic approach as a treatment for PTHS.
Asunto(s)
Discapacidad Intelectual , Canal de Sodio Activado por Voltaje NAV1.8 , Animales , Ratones , Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Facies , Hiperventilación/genética , Discapacidad Intelectual/tratamiento farmacológico , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Factor de Transcripción 4/genética , Canal de Sodio Activado por Voltaje NAV1.8/química , Canal de Sodio Activado por Voltaje NAV1.8/metabolismoRESUMEN
Impairments of inhibitory circuits are at the basis of most, if not all, cognitive deficits. The impact of OPHN1, a gene associate with intellectual disability (ID), on inhibitory neurons remains elusive. We addressed this issue by analyzing the postnatal migration of inhibitory interneurons derived from the subventricular zone in a validated mouse model of ID (OPHN1-/y mice). We found that the speed and directionality of migrating neuroblasts were deeply perturbed in OPHN1-/y mice. The significant reduction in speed was due to altered chloride (Cl-) homeostasis, while the overactivation of the OPHN1 downstream signaling pathway, RhoA kinase (ROCK), caused abnormalities in the directionality of the neuroblast progression in mutants. Blocking the cation-Cl- cotransporter KCC2 almost completely rescued the migration speed while proper directionality was restored upon ROCK inhibition. Our data unveil a strong impact of OPHN1 on GABAergic inhibitory interneurons and identify putative targets for successful therapeutic approaches.
Asunto(s)
Proteínas del Citoesqueleto/genética , Neuronas GABAérgicas/metabolismo , Proteínas Activadoras de GTPasa/genética , Discapacidad Intelectual/metabolismo , Animales , Movimiento Celular/fisiología , Cloruros/metabolismo , Cloruros/fisiología , Proteínas del Citoesqueleto/metabolismo , Neuronas GABAérgicas/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Homeostasis , Discapacidad Intelectual/fisiopatología , Interneuronas/metabolismo , Interneuronas/fisiología , Masculino , Ratones , Modelos Animales , Células-Madre Neurales/metabolismo , Neurogénesis , Proteínas Nucleares/metabolismo , Prosencéfalo/metabolismo , Transducción de Señal , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Copy number variation (CNV) at the 16p11.2 locus is associated with neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia. CNVs of the 16p gene can manifest in opposing head sizes. Carriers of 16p11.2 deletion tend to have macrocephaly (or brain enlargement), while those with 16p11.2 duplication frequently have microcephaly. Increases in both gray and white matter volume have been observed in brain imaging studies in 16p11.2 deletion carriers with macrocephaly. Here, we use human induced pluripotent stem cells (hiPSCs) derived from controls and subjects with 16p11.2 deletion and 16p11.2 duplication to understand the underlying mechanisms regulating brain overgrowth. To model both gray and white matter, we differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). In both NPCs and OPCs, we show that CD47 (a "don't eat me" signal) is overexpressed in the 16p11.2 deletion carriers contributing to reduced phagocytosis both in vitro and in vivo. Furthermore, 16p11.2 deletion NPCs and OPCs up-regulate cell surface expression of calreticulin (a prophagocytic "eat me" signal) and its binding sites, indicating that these cells should be phagocytosed but fail to be eliminated due to elevations in CD47. Treatment of 16p11.2 deletion NPCs and OPCs with an anti-CD47 antibody to block CD47 restores phagocytosis to control levels. While the CD47 pathway is commonly implicated in cancer progression, we document a role for CD47 in psychiatric disorders associated with brain overgrowth.