RESUMEN
The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.
Asunto(s)
Cuerpo Celular , Conos de Crecimiento , Neuronas/metabolismo , Membrana Celular , Hipocampo , Células CultivadasRESUMEN
The RNA-binding protein Ataxin-2 binds to and stabilizes a number of mRNA sequences, including that of the transactive response DNA-binding protein of 43 kDa (TDP-43). Ataxin-2 is additionally involved in several processes requiring translation, such as germline formation, long-term habituation, and circadian rhythm formation. However, it has yet to be unambiguously demonstrated that Ataxin-2 is actually involved in activating the translation of its target mRNAs. Here we provide direct evidence from a polysome profile analysis showing that Ataxin-2 enhances translation of target mRNAs. Our recently established method for transcriptional pulse-chase analysis under conditions of suppressing deadenylation revealed that Ataxin-2 promotes post-transcriptional polyadenylation of the target mRNAs. Furthermore, Ataxin-2 binds to a poly(A)-binding protein PABPC1 and a noncanonical poly(A) polymerase PAPD4 via its intrinsically disordered region (amino acids 906-1095) to recruit PAPD4 to the targets. Post-transcriptional polyadenylation by Ataxin-2 explains not only how it activates translation but also how it stabilizes target mRNAs, including TDP-43 mRNA. Ataxin-2 is known to be a potent modifier of TDP-43 proteinopathies and to play a causative role in the neurodegenerative disease spinocerebellar ataxia type 2, so these findings suggest that Ataxin-2-induced cytoplasmic polyadenylation and activation of translation might impact neurodegeneration (i.e. TDP-43 proteinopathies), and this process could be a therapeutic target for Ataxin-2-related neurodegenerative disorders.
Asunto(s)
Ataxina-2/metabolismo , Citoplasma/metabolismo , Poliadenilación , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/metabolismo , Ataxina-2/genética , Citoplasma/genética , Células HEK293 , Células HeLa , Humanos , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Polinucleotido Adenililtransferasa/genética , Polinucleotido Adenililtransferasa/metabolismo , Unión Proteica , ARN Mensajero/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.
Asunto(s)
Proteínas ADAMTS/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas ADAMTS/genética , Animales , Cerebelo/crecimiento & desarrollo , Corteza Cerebral/crecimiento & desarrollo , Femenino , Células HEK293 , Hipocampo/crecimiento & desarrollo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteolisis , Proteína ReelinaRESUMEN
Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid ß peptide (Aß). Therefore, augmentation of Reelin activity may ameliorate Alzheimer's disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aß by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with "next-generation" AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aß significantly in AppNL-F mice, which produce human wild-type Aß. On the other hand, reducing ADAMTS-3 had no effect in AppNL-G-F mice, which produce the Arctic mutant Aß (E22G) that forms protofibrils more efficiently than does wild-type Aß. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aß.
Asunto(s)
Proteínas ADAMTS/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas ADAMTS/antagonistas & inhibidores , Proteínas ADAMTS/genética , Enfermedad de Alzheimer , Animales , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteína Reelina , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismoRESUMEN
The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.
Asunto(s)
Proteínas ADAMTS/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Unión Proteica , Proteína ReelinaRESUMEN
Reelin is a secreted protein essential for the development and function of the mammalian brain. The receptors for Reelin, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, belong to the low-density lipoprotein receptor family, but it is not known whether Reelin is involved in the brain lipid metabolism. In the present study, we performed lipidomic analysis of the cerebral cortex of wild-type and Reelin-deficient (reeler) mice, and found that reeler mice exhibited several compositional changes in phospholipids. First, the ratio of phospholipids containing one saturated fatty acid (FA) and one docosahexaenoic acid (DHA) or arachidonic acid (ARA) decreased. Secondly, the ratio of phospholipids containing one monounsaturated FA (MUFA) and one DHA or ARA increased. Thirdly, the ratio of phospholipids containing 5,8,11-eicosatrienoic acid, or Mead acid (MA), increased. Finally, the expression of stearoyl-CoA desaturase-1 (SCD-1) increased. As the increase of MA is seen as an index of polyunsaturated FA (PUFA) deficiency, and the expression of SCD-1 is suppressed by PUFA, these results strongly suggest that the loss of Reelin leads to PUFA deficiency. Hence, MUFA and MA are synthesized in response to this deficiency, in part by inducing SCD-1 expression. This is the first report of changes of FA composition in the reeler mouse brain and provides a basis for further investigating the new role of Reelin in the development and function of the brain.
Asunto(s)
Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/deficiencia , Proteínas de la Matriz Extracelular/deficiencia , Lípidos/química , Proteínas del Tejido Nervioso/deficiencia , Serina Endopeptidasas/deficiencia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Ácido Araquidónico/metabolismo , Encéfalo/embriología , Moléculas de Adhesión Celular Neuronal/genética , Ácidos Docosahexaenoicos/metabolismo , Proteínas de la Matriz Extracelular/genética , Ácidos Grasos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Metabolismo de los Lípidos , Ratones Endogámicos ICR , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/genética , Fosfolípidos/metabolismo , Proteína Reelina , Serina Endopeptidasas/genética , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismoRESUMEN
Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)âCas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPRâCas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.
Asunto(s)
ARN Guía de Sistemas CRISPR-Cas , Ribosomas , Animales , Codón Iniciador/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Silenciador del Gen , Biosíntesis de Proteínas/genética , Iniciación de la Cadena Peptídica Traduccional , Mamíferos/genéticaRESUMEN
Gomafu (also referred to as RNCR2/MIAT) was originally identified as a noncoding RNA expressed in a particular set of neurons. Unlike protein-coding mRNAs, the Gomafu RNA escapes nuclear export and stably accumulates in the nucleus, making a unique nuclear compartment. Although recent studies have revealed the functional relevance of Gomafu in a series of physiological processes, the underlying molecular mechanism remains largely uncharacterized. In this report, we identified a chicken homologue of Gomafu using a comparative genomic approach to search for functionally important and conserved sequence motifs among evolutionarily distant species. Unexpectedly, we found that all Gomafu RNA examined shared a distinctive feature: tandem repeats of UACUAAC, a sequence that has been identified as a conserved intron branch point in the yeast Saccharomyces cerevisiae. The tandem UACUAAC Gomafu RNA repeats bind to the SF1 splicing factor with a higher affinity than the divergent branch point sequence in mammals, which affects the kinetics of the splicing reaction in vitro. We propose that the Gomafu RNA regulates splicing efficiency by changing the local concentration of splicing factors within the nucleus.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ARN no Traducido/metabolismo , Secuencias Repetidas en Tándem , Factores de Transcripción/metabolismo , Empalme Alternativo , Animales , Secuencia de Bases , Unión Competitiva , Western Blotting , Línea Celular Tumoral , Núcleo Celular/metabolismo , Pollos , Proteínas de Unión al ADN/genética , Exones/genética , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Intrones/genética , Cinética , Unión Proteica , Interferencia de ARN , Factores de Empalme de ARN , ARN Largo no Codificante , ARN no Traducido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Factores de Transcripción/genéticaRESUMEN
Reelin is a secreted protein that plays versatile roles in neuronal development and function. The strength of Reelin signaling is regulated by proteolytic processing, but its importance in vivo is not yet fully understood. Here, we generated Reelin knock-in (PA-DV KI) mice in which the key cleavage site of Reelin was abolished by mutation. As expected, the cleavage of Reelin was severely abrogated in the cerebral cortex and hippocampus of PA-DV KI mice. The amount of Dab1, whose degradation is induced by Reelin signaling, decreased in these tissues, indicating that the signaling strength of Reelin was augmented. The brains of PA-DV KI mice were largely structurally normal, but unexpectedly, the hippocampal layer was disturbed. This phenotype was ameliorated in hemizygote PA-DV KI mice, indicating that excess Reelin signaling is detrimental to hippocampal layer formation. The neuronal dendrites of PA-DV KI mice had more branches and were elongated compared to wild-type mice. These results present the first direct evidence of the physiological importance of Reelin cleavage.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Animales , Encéfalo/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Técnicas de Sustitución del Gen , Inmunohistoquímica , Ratones , Ratones Transgénicos , Proteolisis , Proteína ReelinaRESUMEN
Intracellular mislocalization of TAR DNA-binding protein 43 (TDP-43), a nuclear DNA/RNA-binding protein involved in RNA metabolism, is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although the aggregation-prone, TDP-43 C-terminal domain is widely considered as a key component of TDP-43 pathology in ALS, recent studies including ours suggest that TDP-43 N-terminal fragments (TDP-∆C) may also contribute to the motor dysfunction in ALS. However, the specific pathological functions of TDP-43 N-terminal fragments in mice have not been elucidated. Here, we established TDP-∆C knock-in mice missing a part of exon 6 of murine Tardbp gene, which encodes the C-terminal region of TDP-43. Homozygous TDP-∆C mice showed embryonic lethality, indicating that the N-terminal domain of TDP-43 alone is not sufficient for normal development. In contrast, heterozygous TDP-∆C mice developed normally but exhibited age-dependent mild motor dysfunction with a loss of C-boutons, large cholinergic synaptic terminals on spinal α-motor neurons. TDP-∆C protein broadly perturbed gene expression in the spinal cords of aged heterozygous TDP-∆C mice, including downregulation of Notch1 mRNA. Moreover, the level of Notch1 mRNA was suppressed both by TDP-43 depletion and TDP-∆C expression in Neuro2a cells. Decreased Notch1 mRNA expression in aged TDP-∆C mice was associated with the age-dependent motor dysfunction and loss of Akt surviving signal. Our findings indicate that the N-terminal region of TDP-43 derived from TDP-∆C induces the age-dependent motor dysfunction associated with impaired Notch1-Akt axis in mice.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Receptor Notch1/biosíntesis , Transducción de Señal/fisiología , Factores de Edad , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular Tumoral , Células Cultivadas , Proteínas de Unión al ADN/genética , Técnicas de Sustitución del Gen , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/genética , Receptor Notch1/genéticaRESUMEN
TDP-43 is an RNA-binding protein important for many aspects of RNA metabolism. Abnormal accumulation of TDP-43 in the cytoplasm of affected neurons is a pathological hallmark of the neurodegenerative diseases frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Several transgenic mouse models have been generated that recapitulate defects in TDP-43 accumulation, thus causing neurodegeneration and behavioural impairments. While aging is the key risk factor for neurodegenerative diseases, the specific effect of aging on phenotypes in TDP-43 transgenic mice has not been investigated. Here, we analyse age-dependent changes in TDP-43 transgenic mice that displayed impaired memory. We found the accumulation of abundant poly-ubiquitinated protein aggregates in the hippocampus of aged TDP-43 transgenic mice. Intriguingly, the aggregates contained some interneuron-specific proteins such as parvalbumin and calretinin, suggesting that GABAergic interneurons were degenerated in these mice. The abundance of aggregates significantly increased with age and with the overexpression of TDP-43. Gene array analyses in the hippocampus and other brain areas revealed dysregulation in genes linked to oxidative stress and neuronal function in TDP-43 transgenic mice. Our results indicate that the interneuron degeneration occurs upon aging, and TDP-43 accelerates age-dependent neuronal degeneration, which may be related to the impaired memory of TDP-43 transgenic mice.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interneuronas/patología , Trastornos de la Memoria/patología , Factores de Edad , Envejecimiento , Animales , Encéfalo/metabolismo , Encéfalo/patología , Muerte Celular , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Interneuronas/citología , Interneuronas/metabolismo , Masculino , Trastornos de la Memoria/genética , Trastornos de la Memoria/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Agregado de Proteínas , UbiquitinaciónRESUMEN
The discovery of C9orf72 mutations as the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has awakened a surge of interest in deciphering how mutations in this mysterious gene cause disease and what can be done to stop it. C9orf72 harbors a hexanucleotide repeat, GGGGCC, in a non-coding region of the gene and a massive expansion of this repeat causes ALS, FTD, or both (FTD/ALS). Many questions lie ahead. What does this gene normally do? What is the consequence of an enormous GGGGCC repeat expansion on that gene's function? Could that hexanucleotide repeat expansion have additional pathological actions unrelated to C9orf72 function? There has been tremendous progress on all fronts in the quest to define how C9orf72 mutations cause disease. Many new experimental models have been constructed and unleashed in powerful genetic screens. Studies in mouse and human patient samples, including iPS-derived neurons, have provided unprecedented insights into pathogenic mechanisms. Three major hypotheses have emerged and are still being hotly debated in the field. These include (1) loss of function owing to decrease in the abundance of C9orf72 protein and its ability to carryout its still unknown cellular role; (2) RNA toxicity from bidirectionally transcribed sense (GGGGCC) and antisense (GGCCCC) transcripts that accumulate in RNA foci and might sequester critical RNA-binding proteins; (3) proteotoxicity from dipeptide repeat proteins produced by an unconventional form of translation from the expanded nucleotide repeats. Here we review the evidence in favor and against each of these three hypotheses. We also suggest additional experiments and considerations that we propose will help clarify which mechanism(s) are most important for driving disease and therefore most critical for considering during the development of therapeutic interventions. This article is part of a Special Issue entitled SI:RNA Metabolism in Disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Mutación , Proteínas/genética , Animales , Proteína C9orf72 , Expansión de las Repeticiones de ADN , Dipéptidos/genética , Humanos , Ratones , ARN/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.
Asunto(s)
Diferenciación Celular/genética , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Mutación Missense , Proteína FUS de Unión a ARN/genética , Adulto , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Secuencia de Bases , Células Cultivadas , Citosol/metabolismo , Salud de la Familia , Femenino , Edición Génica , Perfilación de la Expresión Génica/métodos , Heterocigoto , Homocigoto , Humanos , Células Madre Pluripotentes Inducidas/patología , Masculino , Microscopía Fluorescente , Modelos Genéticos , Neuronas Motoras/patología , Linaje , Proteína FUS de Unión a ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Two motor neuron diseases, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are caused by distinct genes involved in RNA metabolism, TDP-43 and FUS/TLS, and SMN, respectively. However, whether there is a shared defective mechanism in RNA metabolism common to these two diseases remains unclear. Here, we show that TDP-43 and FUS/TLS localize in nuclear Gems through an association with SMN, and that all three proteins function in spliceosome maintenance. We also show that in ALS, Gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology. This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome. These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Atrofia Muscular Espinal/metabolismo , Empalmosomas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Ratones Noqueados , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Unión Proteica , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas del Complejo SMN/genética , Proteínas del Complejo SMN/metabolismo , Empalmosomas/genéticaRESUMEN
To investigate conformations of C-cadherin associated with functional activity and physiological regulation, we generated monoclonal antibodies (mAbs) that bind differentially to monomeric or dimeric forms. These mAbs recognize conformational epitopes at multiple sites along the C-cadherin ectodomain aside from the well known Trp-2-mediated dimer interface in the N-terminal EC1 domain. Group 1 mAbs, which bind monomer better than dimer and the Trp-2-mutated protein (W2A) better than wild type, recognize epitopes in EC4 or EC5. Dimerization of the W2A mutant protein via a C-terminal immunoglobulin Fc domain restored the dimeric mAb-binding properties to EC4-5 and partial homophilic binding activity but did not restore full cell adhesion activity. Group 2 and Group 3 mAbs, which bind dimer better than monomer and wild type better than W2A, recognize epitopes in EC1 and the interface between EC1 and EC2, respectively. None of the mAbs could distinguish between different physiological states of C-cadherin at the cell surface of either Xenopus embryonic cells or Colo 205 cultured cells, demonstrating that changes in dimerization do not underlie regulation of adhesion activity. On the cell surface the EC3-EC5 domains are much less accessible to mAb binding than EC1-EC2, suggesting that they are masked by the state of cadherin organization or by other molecules. Thus, the EC2-EC5 domains either reflect, or are involved in, cadherin dimerization and organization at the cell surface.
Asunto(s)
Anticuerpos Monoclonales/química , Cadherinas/química , Epítopos/química , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Células CHO , Cadherinas/inmunología , Adhesión Celular/genética , Adhesión Celular/inmunología , Cricetinae , Cricetulus , Dimerización , Epítopos/genética , Epítopos/inmunología , Humanos , Mutación , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , XenopusRESUMEN
Recently, we identified dysadherin, a novel carcinoma-associated glycoprotein, and showed that overexpression of dysadherin in human hepatocarcinoma PLC/PRF/5 cells could suppress E-cadherin-mediated cell-cell adhesion and promote tumor metastasis. The present study shows evidence that dysadherin is actually O-glycosylated. This was based on a direct carbohydrate composition analysis of a chimera protein of an extracellular domain of dysadherin fused to an Fc fragment of immunoglobulin. To assess the importance of O-glycosylation in dysadherin function, dysadherin-transfected hepatocarcinoma cells were cultured in a medium containing benzyl-alpha-GalNAc, a modulator of O-glycosylation. This treatment facilitated homotypic cell adhesion among dysadherin transfectants accompanied with morphological changes, indicating that the anti-adhesive effect of dysadherin was weakened. Modification of O-glycan synthesis also resulted in down-regulation of dysadherin expression and up-regulation of E-cadherin expression in dysadherin transfectants but did not affect E-cadherin expression in mock transfectants. Structural analysis of O-glycans released from the dysadherin chimera proteins indicated that a series of O-glycans with core 1 and 2 structures are attached to dysadherin, and their sialylation is remarkably inhibited by benzyl-alpha-GalNAc treatment. However, sialidase treatment of the cells did not affect calcium-dependent cell aggregation, which excluded the possibility that sialic acid itself is directly involved in cell-cell adhesion. We suggest that aberrant O-glycosylation in carcinoma cells inhibits stable expression of dysadherin and leads to the up-regulation of E-cadherin expression by an unknown mechanism, resulting in increased cell-cell adhesion. The carbohydrate-directed approach to the regulation of dysadherin expression might be a new strategy for cancer therapy.
Asunto(s)
Acetilgalactosamina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Acetilgalactosamina/farmacología , Compuestos de Bencilo/farmacología , Western Blotting , Cadherinas/metabolismo , Calcio/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Glicosilación/efectos de los fármacos , Humanos , Inmunohistoquímica , Canales Iónicos , Glicoproteínas de Membrana/biosíntesis , Proteínas de Microfilamentos , Proteínas de Neoplasias/biosíntesis , Neuraminidasa/metabolismoRESUMEN
To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up-regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid-stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G-protein-coupled receptor. By real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, overexpression (>3-fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in beta-catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant beta-catenin into mouse hepatocytes in culture caused up-regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt-signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with beta-catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC.