RESUMEN
Astrocytes are in constant communication with neurons during the establishment and maturation of functional networks in the developing brain. Astrocytes release extracellular vesicles (EVs) containing microRNA (miRNA) cargo that regulates transcript stability in recipient cells. Astrocyte released factors are thought to be involved in neurodevelopmental disorders. Healthy astrocytes partially rescue Rett Syndrome (RTT) neuron function. EVs isolated from stem cell progeny also correct aspects of RTT. EVs cross the blood-brain barrier (BBB) and their cargo is found in peripheral blood which may allow non-invasive detection of EV cargo as biomarkers produced by healthy astrocytes. Here we characterize miRNA cargo and sequence motifs in healthy human astrocyte derived EVs (ADEVs). First, human induced Pluripotent Stem Cells (iPSC) were differentiated into Neural Progenitor Cells (NPCs) and subsequently into astrocytes using a rapid differentiation protocol. iPSC derived astrocytes expressed specific markers, displayed intracellular calcium transients and secreted ADEVs. miRNAs were identified by RNA-Seq on astrocytes and ADEVs and target gene pathway analysis detected brain and immune related terms. The miRNA profile was consistent with astrocyte identity, and included approximately 80 miRNAs found in astrocytes that were relatively depleted in ADEVs suggestive of passive loading. About 120 miRNAs were relatively enriched in ADEVs and motif analysis discovered binding sites for RNA binding proteins FUS, SRSF7 and CELF5. miR-483-5p was the most significantly enriched in ADEVs. This miRNA regulates MECP2 expression in neurons and has been found differentially expressed in blood samples from RTT patients. Our results identify potential miRNA biomarkers selectively sorted into ADEVs and implicate RNA binding protein sequence dependent mechanisms for miRNA cargo loading.
Asunto(s)
Astrocitos , Vesículas Extracelulares , Células Madre Pluripotentes Inducidas , MicroARNs , Neuronas , Humanos , Vesículas Extracelulares/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Astrocitos/metabolismo , Neuronas/metabolismo , Diferenciación Celular , Células Cultivadas , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citologíaRESUMEN
The human UBE3A gene, which is essential for normal neurodevelopment, encodes three Ubiquitin E3 ligase A (UBE3A) protein isoforms. However, the subcellular localization and relative abundance of these human UBE3A isoforms are unknown. We found, as previously reported in mice, that UBE3A is predominantly nuclear in human neurons. However, this conserved subcellular distribution is achieved by strikingly distinct cis-acting mechanisms. A single amino-acid deletion in the N-terminus of human hUBE3A-Iso3, which is homologous to cytosolic mouse mUBE3A-Iso2, results in its translocation to the nucleus. This singe amino-acid deletion is shared with apes and Old World monkeys and was preceded by the appearance of the cytosolic hUBE3A-Iso2 isoform. This hUBE3A-Iso2 isoform arose after the lineage of New World monkeys and Old World monkeys separated from the Tarsiers (Tarsiidae). Due to the loss of a single nucleotide in a non-coding exon, this exon became in frame with the remainder of the UBE3A protein. RNA-seq analysis of human brain samples showed that the human UBE3A isoforms arise by alternative splicing. Consistent with the predominant nuclear enrichment of UBE3A in human neurons, the two nuclear-localized isoforms, hUBE3A-Iso1 and -Iso3, are the most abundantly expressed isoforms of UBE3A, while hUBE3A-Iso2 maintains a small pool of cytosolic UBE3A. Our findings provide new insight into UBE3A localization and evolution and may have important implications for gene therapy approaches in Angelman syndrome.
Asunto(s)
Síndrome de Angelman/genética , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Empalme Alternativo/genética , Síndrome de Angelman/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Impresión Genómica/genética , Humanos , Ratones , Neuronas/patología , Isoformas de Proteínas/genéticaRESUMEN
Developmental disorders (DD), characterized by malformations/dysmorphism and/or intellectual disability, affecting around 3% of worldwide population, are mostly linked to genetic anomalies. Despite clinical exome sequencing (cES) centered on genes involved in human genetic disorders, the majority of patients affected by DD remain undiagnosed after solo-cES. Trio-based strategy is expected to facilitate variant selection thanks to rapid parental segregation. We performed a second step trio-ES (not only focusing on genes involved in human disorders) analysis in 70 patients with negative results after solo-cES. All candidate variants were shared with a MatchMaking exchange system to identify additional patients carrying variants in the same genes and with similar phenotype. In 18/70 patients (26%), we confirmed causal implication of nine OMIM-morbid genes and identified nine new strong candidate genes (eight de novo and one compound heterozygous variants). These nine new candidate genes were validated through the identification of patients with similar phenotype and genotype thanks to data sharing. Moreover, 11 genes harbored variants of unknown significance in 10/70 patients (14%). In DD, a second step trio-based ES analysis appears an efficient strategy in diagnostic and translational research to identify highly candidate genes and improve diagnostic yield.
Asunto(s)
Discapacidades del Desarrollo/genética , Exoma/genética , Predisposición Genética a la Enfermedad/genética , Discapacidad Intelectual/genética , Femenino , Genómica/métodos , Humanos , Masculino , Fenotipo , Secuenciación del Exoma/métodosRESUMEN
Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Proteínas de la Membrana/genética , Células Precursoras de Oligodendrocitos/metabolismo , Esquizofrenia/genética , Adulto , Antígenos/genética , Diferenciación Celular/fisiología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Imagen de Difusión Tensora , Familia , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Mutación/genética , Células Precursoras de Oligodendrocitos/fisiología , Oligodendroglía/metabolismo , Linaje , Proteoglicanos/genética , Esquizofrenia/metabolismo , Sustancia Blanca/metabolismoRESUMEN
Noncoding RNAs have been widely recognized as essential mediators of gene regulation. However, in contrast to protein-coding genes, much less is known about the influence of noncoding RNAs on human diseases. Here we examined the association of genetic variants located in primary microRNA sequences and long noncoding RNAs (lncRNAs) with Alzheimer disease (AD) by leveraging data from the largest genome-wide association meta-analysis of late-onset AD. Variants annotated to 5 miRNAs and 10 lncRNAs (in seven distinct loci) exceeded the Bonferroni-corrected significance threshold (p < 1.02 × 10-6 ). Among these, a leading variant (rs2526377:A>G) at the 17q22 locus annotated to two noncoding RNAs (MIR142 and BZRAP1-AS) was significantly associated with a reduced risk of AD and fulfilled predefined criteria for being a functional variant. Our functional genomic analyses revealed that rs2526377 affects the promoter activity and decreases the expression of miR-142. Moreover, differential expression analysis by RNA-Seq in human iPSC-derived neural progenitor cells and the hippocampus of miR-142 knockout mice demonstrated multiple target genes of miR-142 in the brain that are likely to be involved in the inflammatory and neurodegenerative manifestations of AD. These include TGFBR1 and PICALM, of which their derepression in the brain due to reduced expression levels of miR-142-3p may reduce the risk of AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Variación Genética , MicroARNs/genética , Regiones Promotoras Genéticas , Alelos , Enfermedad de Alzheimer/metabolismo , Animales , Línea Celular , Mapeo Cromosómico , Biología Computacional/métodos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Estudios de Asociación Genética , Estudio de Asociación del Genoma Completo , Hipocampo/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Polimorfismo de Nucleótido Simple , Interferencia de ARN , ARN no TraducidoRESUMEN
Zinc homeostasis is a highly regulated process in mammalian cells that is critical for normal growth and development. Movement of zinc across cell compartments is controlled by two classes of transporters: Slc39a family members transport zinc into the cytosol from either the extracellular space or intracellular stores such as the endoplasmic reticulum (ER), whereas the SLC30A family mediates zinc efflux from the cytosol. In this study, we report that genetic ablation of SLC39A7 (ZIP7) results in decreased cytosolic zinc levels, increased ER zinc levels, impaired cell proliferation, and induction of ER stress. Confirmatory of impaired zinc transport as the causal mechanism, both the increased ER stress and impaired cell proliferation were rescued by increasing cytosolic zinc. Furthermore, using these robust cellular phenotypes, we implemented a small-molecule library screen with 2800 compounds and identified one small molecule capable of rescuing ER stress and cell proliferation in ZIP7-deficient cells in the low micromolar range.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Zinc/metabolismo , Proteínas de Transporte de Catión/genética , Línea Celular , Proliferación Celular/fisiología , Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/fisiología , HumanosRESUMEN
Hepatitis E virus (HEV), as a hepatotropic virus, is supposed to exclusively infect the liver and only cause hepatitis. However, a broad range of extrahepatic manifestations (in particular, idiopathic neurological disorders) have been recently reported in association with its infection. In this study, we have demonstrated that various human neural cell lines (embryonic stem cell-derived neural lineage cells) induced pluripotent stem cell-derived human neurons and primary mouse neurons are highly susceptible to HEV infection. Treatment with interferon-α or ribavirin, the off-label antiviral drugs for chronic hepatitis E, exerted potent antiviral activities against HEV infection in neural cells. More importantly, in mice and monkey peripherally inoculated with HEV particles, viral RNA and protein were detected in brain tissues. Finally, patients with HEV-associated neurological disorders shed the virus into cerebrospinal fluid, indicating a direct infection of their nervous system. Thus, HEV is neurotropic in vitro, and in mice, monkeys, and possibly humans. These results challenge the dogma of HEV as a pure hepatotropic virus and suggest that HEV infection should be considered in the differential diagnosis of idiopathic neurological disorders.
Asunto(s)
Encéfalo/virología , Virus de la Hepatitis E/patogenicidad , Hepatitis E/patología , Neuronas/virología , Adulto , Anciano , Animales , Antivirales/farmacología , Encéfalo/patología , Línea Celular Tumoral , Líquido Cefalorraquídeo/virología , Femenino , Síndrome de Guillain-Barré/virología , Hepatitis E/tratamiento farmacológico , Humanos , Interferón-alfa/farmacología , Hígado/patología , Hígado/virología , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neuronas/patología , ARN Viral/análisis , Ribavirina/farmacología , Replicación Viral/efectos de los fármacos , Esparcimiento de VirusRESUMEN
Microphysiological systems (MPSs) are cellular models that replicate aspects of organ and tissue functions in vitro. In contrast with conventional cell cultures, MPSs often provide physiological mechanical cues to cells, include fluid flow and can be interlinked (hence, they are often referred to as microfluidic tissue chips or organs-on-chips). Here, by means of examples of MPSs of the vascular system, intestine, brain and heart, we advocate for the development of standards that allow for comparisons of quantitative physiological features in MPSs and humans. Such standards should ensure that the in vivo relevance and predictive value of MPSs can be properly assessed as fit-for-purpose in specific applications, such as the assessment of drug toxicity, the identification of therapeutics or the understanding of human physiology or disease. Specifically, we distinguish designed features, which can be controlled via the design of the MPS, from emergent features, which describe cellular function, and propose methods for improving MPSs with readouts and sensors for the quantitative monitoring of complex physiology towards enabling wider end-user adoption and regulatory acceptance.
RESUMEN
Human brain organoid technology has the potential to generate unprecedented insight into normal and aberrant brain development. It opens up a developmental time window in which the effects of gene or environmental perturbations can be experimentally tested. However, detection sensitivity and correct interpretation of phenotypes are hampered by notable batch-to-batch variability and low reproducibility of cell and regional identities. Here, we describe a detailed, simplified protocol for the robust and reproducible generation of brain organoids with cortical identity from feeder-independent induced pluripotent stem cells (iPSCs). This self-patterning approach minimizes media supplements and handling steps, resulting in cortical brain organoids that can be maintained over prolonged periods and that contain radial glial and intermediate progenitors, deep and upper layer neurons, and astrocytes.
RESUMEN
Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (PolÏ´) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Sistemas CRISPR-Cas/genética , Roturas del ADN de Doble Cadena , Mutación/genética , Reparación del ADN por Unión de ExtremidadesRESUMEN
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection is associated with a diverse spectrum of neurological complications during the acute and postacute stages. The pathogenesis of these complications is complex and dependent on many factors. For accurate and consistent interpretation of experimental data in this fast-growing field of research, it is essential to use terminology consistently. In this article, we outline the distinctions between neuroinvasiveness, neurotropism, and neurovirulence. Additionally, we discuss current knowledge of these distinct features underlying the pathogenesis of SARS-CoV-2-associated neurological complications. Lastly, we briefly discuss the advantages and limitations of different experimental models, and how these approaches can further be leveraged to advance the field.
Asunto(s)
COVID-19 , Enfermedades del Sistema Nervioso , Humanos , SARS-CoV-2RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with various neurological complications. Although the mechanism is not fully understood, several studies have shown that neuroinflammation occurs in the acute and post-acute phase. As these studies have predominantly been performed with isolates from 2020, it is unknown if there are differences among SARS-CoV-2 variants in their ability to cause neuroinflammation. Here, we compared the neuroinvasiveness, neurotropism and neurovirulence of the SARS-CoV-2 ancestral strain D614G, the Delta (B.1.617.2) and Omicron BA.1 (B.1.1.529) variants using in vitro and in vivo models. The Omicron BA.1 variant showed reduced neurotropism and neurovirulence compared to Delta and D614G in human induced pluripotent stem cell (hiPSC)-derived cortical neurons co-cultured with astrocytes. Similar differences were obtained in Syrian hamsters inoculated with D614G, Delta and the Omicron BA.1 variant 5 days post infection. Replication in the olfactory mucosa was observed in all hamsters, but most prominently in D614G inoculated hamsters. Furthermore, neuroinvasion into the CNS via the olfactory nerve was observed in D614G, but not Delta or Omicron BA.1 inoculated hamsters. Furthermore, neuroinvasion was associated with neuroinflammation in the olfactory bulb of hamsters inoculated with D614G. Altogether, our findings suggest differences in the neuroinvasive, neurotropic and neurovirulent potential between SARS-CoV-2 variants using in vitro hiPSC-derived neural cultures and in vivo in hamsters during the acute phase of the infection.
Asunto(s)
COVID-19 , Células Madre Pluripotentes Inducidas , Animales , Cricetinae , Humanos , Mesocricetus , SARS-CoV-2RESUMEN
Fragile X syndrome, the most common form of inherited intellectual disability, is caused by a lack of FMRP, which is the product of the Fmr1 gene. FMRP is an RNA-binding protein and a component of RNA-granules found in the dendrites of neurons. At the synapse, FMRP is involved in regulation of translation of specific target mRNAs upon stimulation of mGluR5 receptors. In this study, we test the effects of a new mGluR5 antagonist (AFQ056) on the prepulse inhibition of startle response in mice. We show that Fmr1 KO mice have a deficit in inhibition of the startle response after a prepulse and that AFQ056 can rescue this phenotype. We also studied the effect of AFQ056 on cultured Fmr1 KO hippocampal neurons; untreated neurons showed elongated spines and treatment resulted in shortened spines. These results suggest that AFQ056 might be a potent mGluR5 antagonist to rescue various aspects of the fragile X phenotype.
Asunto(s)
Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Reflejo de Sobresalto/efectos de los fármacos , Filtrado Sensorial/efectos de los fármacos , Animales , Células Cultivadas , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Hipocampo/metabolismo , Ratones , Ratones Noqueados , Neuronas/metabolismo , Receptor del Glutamato Metabotropico 5RESUMEN
Fragile X syndrome (FXS) is the most common inherited form of mental retardation and is caused by the lack of fragile X mental retardation protein (FMRP). In the brain, spine abnormalities have been reported in both patients with FXS and Fmr1 knockout mice. This altered spine morphology has been linked to disturbed synaptic transmission related to altered signaling in the excitatory metabotropic glutamate receptor 5 (mGluR5) pathway. We investigated hippocampal protrusion morphology in adult Fmr1 knockout mice. Our results show a hippocampal CA1-specific altered protrusion phenotype, which was absent in the CA3 region of the hippocampus. This suggests a subregion-specific function of FMRP in synaptic plasticity in the brain.
Asunto(s)
Región CA1 Hipocampal/citología , Espinas Dendríticas/clasificación , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Células Piramidales/crecimiento & desarrollo , Animales , Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/metabolismo , Espinas Dendríticas/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Piramidales/citología , Células Piramidales/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a wide variety of neurological complications. Even though SARS-CoV-2 is rarely detected in the central nervous system (CNS) or cerebrospinal fluid, evidence is accumulating that SARS-CoV-2 might enter the CNS via the olfactory nerve. However, what happens after SARS-CoV-2 enters the CNS is poorly understood. Therefore, we investigated the replication kinetics, cell tropism, and associated immune responses of SARS-CoV-2 infection in different types of neural cultures derived from human induced pluripotent stem cells (hiPSCs). SARS-CoV-2 was compared to the neurotropic and highly pathogenic H5N1 influenza A virus. SARS-CoV-2 infected a minority of individual mature neurons, without subsequent virus replication and spread, despite angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and neuropilin-1 (NPR1) expression in all cultures. However, this sparse infection did result in the production of type III interferons and interleukin-8 (IL-8). In contrast, H5N1 virus replicated and spread very efficiently in all cell types in all cultures. Taken together, our findings support the hypothesis that neurological complications might result from local immune responses triggered by virus invasion, rather than abundant SARS-CoV-2 replication in the CNS. IMPORTANCE Infections with the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are often associated with neurological complications. Evidence suggests that SARS-CoV-2 enters the brain via the olfactory nerve; however, SARS-CoV-2 is only rarely detected in the central nervous system of COVID-19 patients. Here, we show that SARS-CoV-2 is able to infect neurons of human iPSC neural cultures but that this infection is abortive and does not result in virus spread to other cells. However, infection of neural cultures did result in the production of type III interferon and IL-8. This study suggests that SARS-CoV-2 might enter the CNS and infect individual neurons, triggering local immune responses that could contribute to the pathogenesis of SARS-CoV-2-associated CNS disease.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Subtipo H5N1 del Virus de la Influenza A/fisiología , Neuronas/virología , SARS-CoV-2/fisiología , Tropismo Viral , Replicación Viral , Animales , Encefalopatías/etiología , COVID-19/complicaciones , Chlorocebus aethiops , Perros , Humanos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Cinética , Células de Riñón Canino Madin Darby , SARS-CoV-2/inmunología , Células VeroRESUMEN
Fragile X syndrome is caused by lack of the protein FMRP. FMRP mediates mRNA binding, dendritic mRNA transport and translational control at spines. We examined the role of functional domains of FMRP in neuronal RNA-granule formation and dendritic transport using different FMRP variants, including the mutant FMRP_I304N and the splice-variant FMRP_Iso12. Both variants are absent from dendritic RNA-granules in Fmr1 knockout neurons. Co-transfection experiments showed that wild-type FMRP recruits both FMRP variants into dendritic RNA-granules. Co-transfection of FXR2, an FMRP homologue, also resulted in redistribution of both variants into dendritic RNA-granules. Furthermore, the capacity of the variants to transport their mRNAs and the mRNA localization of an FMR1 construct containing silent point-mutations affecting only the G-quartet-structure were investigated. In conclusion, we show that wild-type FMRP and FXR2P are able to recruit FMRP variants into RNA-granules and that the G-quartet-structure in FMR1 mRNA is not essential for its incorporation in RNA-granules.
Asunto(s)
Dendritas/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Transporte de ARN , Animales , Células Cultivadas , Dendritas/ultraestructura , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Hipocampo/citología , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Microscopía Confocal , Mutación , Neuronas/citología , Conformación de Ácido Nucleico , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , TransfecciónRESUMEN
Loss of neurons in neurodegenerative diseases is usually preceded by the accumulation of protein deposits that contain components of the ubiquitin/proteasome system. Affected neurons in Alzheimer's disease often accumulate UBB(+1), a mutant ubiquitin carrying a 19-amino acid C-terminal extension generated by a transcriptional dinucleotide deletion. Here we show that UBB(+1) is a potent inhibitor of ubiquitin-dependent proteolysis in neuronal cells, and that this inhibitory activity correlates with induction of cell cycle arrest. Surprisingly, UBB(+1) is recognized as a ubiquitin fusion degradation (UFD) proteasome substrate and ubiquitinated at Lys29 and Lys48. Full blockade of proteolysis requires both ubiquitination sites. Moreover, the inhibitory effect was enhanced by the introduction of multiple UFD signals. Our findings suggest that the inhibitory activity of UBB(+1) may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Mutación , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Ubiquitina/genética , Ciclo Celular , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Lisina/metabolismo , Complejos Multienzimáticos/antagonistas & inhibidores , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/genética , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismo , Especificidad por Sustrato , Células Tumorales Cultivadas , Ubiquitina/antagonistas & inhibidores , Ubiquitina/metabolismoRESUMEN
Mutations affecting the gene encoding the ubiquitin ligase UBE3A cause Angelman syndrome. Although most studies focus on the synaptic function of UBE3A, we show that UBE3A is highly enriched in the nucleus of mouse and human neurons. We found that the two major isoforms of UBE3A exhibit highly distinct nuclear versus cytoplasmic subcellular localization. Both isoforms undergo nuclear import through direct binding to PSMD4 (also known as S5A or RPN10), but the amino terminus of the cytoplasmic isoform prevents nuclear retention. Mice lacking the nuclear UBE3A isoform recapitulate the behavioral and electrophysiological phenotypes of Ube3am-/p+ mice, whereas mice harboring a targeted deletion of the cytosolic isoform are unaffected. Finally, we identified Angelman syndrome-associated UBE3A missense mutations that interfere with either nuclear targeting or nuclear retention of UBE3A. Taken together, our findings elucidate the mechanisms underlying the subcellular localization of UBE3A, and indicate that the nuclear UBE3A isoform is the most critical for the pathophysiology of Angelman syndrome.
Asunto(s)
Síndrome de Angelman/genética , Síndrome de Angelman/psicología , Conducta Animal , Ubiquitina-Proteína Ligasas/genética , Animales , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/genética , Citosol/enzimología , Fenómenos Electrofisiológicos/genética , Femenino , Humanos , Isoenzimas/genética , Masculino , Ratones , Ratones Noqueados , Mutación Missense/genética , Comportamiento de Nidificación , Neuronas/enzimología , Desempeño Psicomotor , Proteínas de Unión al ARN , Natación/psicología , Dedos de ZincRESUMEN
Fragile X syndrome is a common inherited form of mental retardation and originates from the absence of expression of the FMR1 gene. This gene and its two homologues, FXR1 and FXR2, encode for a family of fragile X related (FXR) proteins with similar tissue distribution, together with sequence and functional homology. Based on these characteristics, it has been suggested that these proteins might partly complement one another. To unravel the function of Fxr2 protein, the expression pattern of 12,588 genes was studied in the brains of wild-type and Fxr2 knockout mice, an animal model which shows behavioral abnormalities partly similar to those observed in Fmr1-knockout mice. By genome expression profiling and stringent significance tests we identify genes and gene groups de-regulated in the brains of Fxr2 knockout mice. Differential expression of candidate genes was validated by real-time PCR, in situ hybridization, immunohistochemistry and western blot analysis. A number of differentially expressed genes associated with the Fxr2 phenotype have been previously involved in other memory or cognitive disorders.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Proteínas de Unión al ARN/fisiología , Animales , Western Blotting , Encéfalo/patología , Cerebelo/metabolismo , Cerebelo/patología , Regulación hacia Abajo , Perfilación de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Unión al ARN/genética , Receptores AMPA/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Lack of fragile X mental retardation protein (FMRP) causes Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA-binding protein and is a component of messenger ribonucleoprotein complexes, associated with brain polyribosomes, including dendritic polysomes. FMRP is therefore thought to be involved in translational control of specific mRNAs at synaptic sites. In mice lacking FMRP, protein synthesis-dependent synaptic plasticity is altered and structural malformations of dendritic protrusions occur. One hypothesized cause of the disease mechanism is based on exaggerated group I mGluR receptor activation. In this study, we examined the effect of the mGluR5 antagonist MPEP on Fragile X related behavior in Fmr1 KO mice. Our results demonstrate a clear defect in prepulse inhibition of startle in Fmr1 KO mice, that could be rescued by MPEP. Moreover, we show for the first time a structural rescue of Fragile X related protrusion morphology with two independent mGluR5 antagonists.