RESUMEN
Members of the Shank protein family are master scaffolds of the postsynaptic architecture and mutations within the SHANK genes are causally associated with autism spectrum disorders (ASDs). We generated a Shank2-Shank3 double knockout mouse that is showing severe autism related core symptoms, as well as a broad spectrum of comorbidities. We exploited this animal model to identify cortical brain areas linked to specific autistic traits by locally deleting Shank2 and Shank3 simultaneously. Our screening of 10 cortical subregions revealed that a Shank2/3 deletion within the retrosplenial area severely impairs social memory, a core symptom of ASD. Notably, DREADD-mediated neuronal activation could rescue the social impairment triggered by Shank2/3 depletion. Data indicate that the retrosplenial area has to be added to the list of defined brain regions that contribute to the spectrum of behavioural alterations seen in ASDs.
Asunto(s)
Trastorno del Espectro Autista , Giro del Cíngulo , Interacción Social , Animales , Ratones , Trastorno del Espectro Autista/genética , Proteínas de Microfilamentos/genética , Mutación , Proteínas del Tejido Nervioso/genética , Neuronas/fisiología , Giro del Cíngulo/metabolismo , Giro del Cíngulo/patologíaRESUMEN
Cytosolic accumulation of TAR DNA binding protein 43 (TDP-43) is a major neuropathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the mechanisms involved in TDP-43 accumulation remain largely unknown. Previously, we reported that inhibitors of cyclin-dependent kinases (CDKs) prevented cytosolic stress granule accumulation of TDP-43, correlating with depletion of heterogeneous ribonucleoprotein (hnRNP) K from stress granules. In the present study, we further investigated the relationship between TDP-43 and hnRNP K and their control by CDKs. Inhibition of CDK2 abrogated the accumulation of TDP-43 into stress granules. Phosphorylated CDK2 co-localized with accumulated TDP-43 and phosphorylated hnRNP K in stress granules. Inhibition of CDK2 phosphorylation blocked phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevented accumulation of TDP-43. Mutation of Ser216 and Ser284 phosphorylation sites on hnRNP K inhibited hnRNP K- and TDP-43-positive stress granule formation in transfected cells. The interaction between hnRNP K and TDP-43 was further confirmed by the loss of TDP-43 accumulation following siRNA-mediated inhibition of hnRNP K expression. A substantial decrease of CDK2 and hnRNP K expression in spinal cord motor neurons in ALS patients demonstrates a potential key role for these proteins in ALS and TDP-43 accumulation, indicating that further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Citosol/metabolismo , Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Humanos , Ratones , Mutación Missense , FosforilaciónRESUMEN
Rap GTPase-activating proteins (RapGAPs) are essential for synaptic function as they tightly regulate synaptic Rap signaling. Among the most abundant synaptic RapGAPs in brain are the Spine-associated RapGAPs (SPARs) Sipa1l1/SPAR and Sipa1l2/SPAR2, whereas nothing has been reported on Sipa1l3/SPAR3. In this study, we show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in the developing rat brain and is localized at excitatory postsynapses. We further demonstrate that the Sipa1l3/SPAR3 C-terminus is required for postsynaptic targeting and represents an interaction module for Fezzins such as ProSAPiP1/Lzts3, a binding partner of the postsynaptic scaffold protein Shank3. Taken together, our data imply that Sipa1l3/SPAR3 is a hitherto unknown synaptic RapGAP, which is targeted to postsynaptic specializations and interacts with Fezzins. Spine-associated RapGAPs (SPARs) are essential modulators of synaptic signaling. Our study is the first to characterize the SPAR family member Sipa1l3/SPAR3 in neuronal tissue. We show that Sipa1l3/SPAR3 is conserved across species, has a distinct expression pattern in brain and is localized to excitatory postsynapses via its C-terminus, which represents an interaction module for other postsynaptic proteins including the Fezzin ProSAPiP1/Lzts3.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas de la Membrana/biosíntesis , Sinapsis/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Animales , Encéfalo/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Perros , Femenino , Humanos , Masculino , Ratones , Pan troglodytes , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity-dependent remodelling of excitatory synapses. Here, we report that Zn(2+) ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member-specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA-mediated knockdown, we could demonstrate that only the overexpression of zinc-sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn(2+) along with the knockdown of zinc-insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD-95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn(2+) is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hipocampo/citología , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Densidad Postsináptica/metabolismo , Sinapsis/fisiología , Zinc/metabolismo , Animales , Western Blotting , Quelantes , Cromatografía en Gel , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Modelos Biológicos , Oligonucleótidos/genética , Quinolonas , Interferencia de ARN , Ratas , Compuestos de Tosilo , TransfecciónRESUMEN
Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (nâ=â396 patients and nâ=â659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (Pâ=â0.004, ORâ=â2.37, 95% CIâ=â1.23-4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (Pâ=â0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11-q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the "multiple hit model" for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.
Asunto(s)
Trastornos Generalizados del Desarrollo Infantil/genética , Proteínas del Tejido Nervioso/genética , Eliminación de Secuencia/genética , Sinapsis/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Empalme Alternativo/genética , Línea Celular , Niño , Preescolar , Femenino , Dosificación de Gen/genética , Regulación de la Expresión Génica , Humanos , Masculino , Neuronas/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sitios de Empalme de ARN/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Sinapsis/patología , Distribución Tisular , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The dynactin p150glued subunit, encoded by the gene DCTN1 is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport. This subunit is a candidate modifier for neurodegenerative diseases, in particular motoneuron and extrapyramidal diseases. Based on an extensive screening effort of all 32 exons in more than 2,500 ALS/MND patients, patients suffering from Parkinsonian Syndromes and controls, we investigated 24 sequence variants of p150 in cell-based studies. We used both non-neuronal cell lines and primary rodent spinal motoneurons and report on cell biological abnormalities in five of these sequence alterations and also briefly report on the clinical features. Our results suggest the presence of biological changes caused by some p150 mutants pointing to a potential pathogenetic significance as modifier of the phenotype of the human disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas Asociadas a Microtúbulos/genética , Neuronas Motoras/metabolismo , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/patología , Proteínas Adaptadoras Transductoras de Señales , Esclerosis Amiotrófica Lateral/patología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Complejo Dinactina , Embrión de Mamíferos , Femenino , Proteínas Fluorescentes Verdes/genética , Humanos , Masculino , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuronas Motoras/patología , Neuronas Motoras/ultraestructura , Mutación/genética , Embarazo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Estudios Retrospectivos , Médula Espinal/citología , Factores de TiempoRESUMEN
The dynactin p150(Glued) subunit, encoded by the gene DCTN1, is part of the dynein-dynactin motor protein complex responsible for retrograde axonal transport in motor neurons. The p150 subunit is a candidate gene for neurodegenerative diseases, in particular motor neuron and extrapyramidal diseases. Tubulin-binding cofactors are believed to be involved in tubulin biogenesis and degradation and therefore to contribute to microtubule functional diversity and regulation. A yeast-two-hybrid screen for putative interacting proteins of dynactin p150(Glued) has revealed tubulin-folding cofactor B (TBCB). We analyzed the interaction of these proteins and investigated the impact of this complex on the microtubule network in cell lines and primary hippocampal neurons in vitro. We especially concentrated on neuronal morphology and synaptogenesis. Overexpression of both proteins or depletion of TBCB alone does not alter the microtubule network and/or neuronal morphology. The demonstration of the interaction of the transport molecule dynactin and the tubulin-regulating factor TBCB is thought to have an impact on several cellular mechanisms. TBCB expression levels have been found to have only a subtle influence on the microtubule network and neuronal morphology. However, overexpression of TBCB leads to the decreased localization of p150 to the microtubule network that might result in a functional modulation of this protein complex.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Células COS , Chlorocebus aethiops , Regulación hacia Abajo/genética , Complejo Dinactina , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Espacio Intracelular/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Unión Proteica/genética , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Sinapsis/metabolismoRESUMEN
BACKGROUND: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. METHODS AND RESULTS: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects. CONCLUSIONS: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.
Asunto(s)
Diferenciación Celular/fisiología , Sistema de Conducción Cardíaco/citología , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Canales de Potasio Calcio-Activados/fisiología , Animales , Bencimidazoles/farmacología , Agonistas de los Canales de Calcio/farmacología , Línea Celular , Proliferación Celular , Citoesqueleto/fisiología , Sistema de Conducción Cardíaco/fisiología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Miocitos Cardíacos/fisiología , Células Madre Pluripotentes/fisiología , Canales de Potasio Calcio-Activados/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The postsynaptic density (PSD) is a highly specialized structure that is located juxtaposed to the presynaptic active zone of excitatory synapses. It is composed of a variety of proteins that include receptors, signaling molecules, cytoskeletal components and scaffolding proteins. ProSAP/Shank proteins are large multidomain proteins that facilitate multiple functions within the PSD. They build large scaffolds that are the structural basis for the direct and/or indirect connection between receptor proteins and the actin based cytoskeleton. Here, we characterize a novel interaction partner of ProSAP2/Shank3, named ProSAP interacting protein 2 (ProSAPiP2) that does not show any close homology to other known proteins. It binds to the PDZ domain of ProSAP2/Shank3 and is highly expressed in the neuronal system. ProSAPiP2 is located in dendrites and spines, is enriched in the PSD and interacts with actin. Therefore ProSAPiP2 could be involved in the linkage between molecules of the PSD and the cytoskeleton.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
One major pathophysiological hallmark of Alzheimer's disease (AD) is senile plaques composed of amyloid ß (Aß). In the amyloidogenic pathway, cleavage of the amyloid precursor protein (APP) is shifted towards Aß production and soluble APPß (sAPPß) levels. Aß is known to impair synaptic function; however, much less is known about the physiological functions of sAPPß. The neurotrophic properties of sAPPα, derived from the non-amyloidogenic pathway of APP cleavage, are well-established, whereas only a few, conflicting studies on sAPPß exist. The intracellular pathways of sAPPß are largely unknown. Since sAPPß is generated alongside Aß by ß-secretase (BACE1) cleavage, we tested the hypothesis that sAPPß effects differ from sAPPα effects as a neurotrophic factor. We therefore performed a head-to-head comparison of both mammalian recombinant peptides in developing primary hippocampal neurons (PHN). We found that sAPPα significantly increases axon length (pâ¯=â¯0.0002) and that both sAPPα and sAPPß increase neurite number (pâ¯<â¯0.0001) of PHN at 7â¯days in culture (DIV7) but not at DIV4. Moreover, both sAPPα- and sAPPß-treated neurons showed a higher neuritic complexity in Sholl analysis. The number of glutamatergic synapses (pâ¯<â¯0.0001), as well as layer thickness of postsynaptic densities (PSDs), were significantly increased, and GABAergic synapses decreased upon sAPP overexpression in PHN. Furthermore, we showed that sAPPα enhances ERK and CREB1 phosphorylation upon glutamate stimulation at DIV7, but not DIV4 or DIV14. These neurotrophic effects are further associated with increased glutamate sensitivity and CREB1-signaling. Finally, we found that sAPPα levels are significantly reduced in brain homogenates of AD patients compared to control subjects. Taken together, our data indicate critical stage-dependent roles of sAPPs in the developing glutamatergic system in vitro, which might help to understand deleterious consequences of altered APP shedding in AD patients, beyond Aß pathophysiology.
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Precursor de Proteína beta-Amiloide/metabolismo , Calcio/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Hipocampo/patología , Homeostasis/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Neuronas/patología , Transducción de Señal/fisiologíaRESUMEN
Fused in Sarcoma (FUS) is a multifunctional RNA-/DNA-binding protein, which is involved in the pathogenesis of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A common hallmark of these disorders is the abnormal accumulation of mutated FUS protein in the cytoplasm. Under normal conditions FUS is confined to the nuclear compartment, in neurons, however, additional somatodendritic localization can be observed. In this study, we carefully analyzed the subcellular localization of endogenous FUS at synaptic sites of hippocampal neurons which are among the most affected cell types in FTD with FUS pathology. We could confirm a strong nuclear localization of FUS as well as its prominent and widespread neuronal expression throughout the adult and developing rat brain, particularly in the hippocampus, the cerebellum and the outer layers of the cortex. Intriguingly, FUS was also consistently observed at synaptic sites as detected by neuronal subcellular fractionation as well as by immunolabeling. To define a pre- and/or postsynaptic localization of FUS, we employed super-resolution fluorescence localization microscopy. FUS was found to be localized within the axon terminal in close proximity to the presynaptic vesicle protein Synaptophysin1 and adjacent to the active zone protein Bassoon, but well separated from the postsynaptic protein PSD-95. Having shown the presynaptic localization of FUS in the nervous system, a novel extranuclear role of FUS at neuronal contact sites has to be considered. Since there is growing evidence that local presynaptic translation might also be an important mechanism for plasticity, FUS - like the fragile X mental retardation protein FMRP - might act as one of the presynaptic RNA-binding proteins regulating this machinery. Our observation of presynaptic FUS should foster further investigations to determine its role in neurodegenerative diseases such as ALS and FTD.
RESUMEN
Proteolytic processing of amyloid-ß precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aß), which accumulates in senile plaques in Alzheimer's disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aß generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPß, and Aß upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Encéfalo/metabolismo , Línea Celular , Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Familia de Multigenes , Unión Proteica , Proteolisis , Eliminación de SecuenciaRESUMEN
Recently, mutations in ProSAP2/Shank3 have been discovered as one of the genetic factors for schizophrenia (SCZ). Here, we show that the postsynaptic density protein ProSAP2/Shank3 undergoes activity dependent synapse-to-nucleus shuttling in hippocampal neurons. Our study shows that the de novo mutation (R1117X) in ProSAP2/Shank3 that was identified in a patient with SCZ leads to an accumulation of mutated ProSAP2/Shank3 within the nucleus independent of synaptic activity. Furthermore, we identified novel nuclear ProSAP2/Shank3 interaction partners. Nuclear localization of mutated ProSAP2/Shank3 alters transcription of several genes, among them already identified genetic risk factors for SCZ such as Synaptotagmin 1 and LRRTM1. Comparing the SCZ mutation of ProSAP2/Shank3 to the knockdown of ProSAP2/Shank3 we found some shared features such as reduced synaptic density in neuronal cultures. However, hippocampal neurons expressing the ProSAP2/Shank3 SCZ mutation furthermore show altered E/I ratio and reduced dendritic branching. Thus, we conclude that the uncoupling of ProSAP2/Shank3 nuclear shuttling from synaptic activity may represent a molecular mechanism that contributes to the pathology of SCZ in patients with mutations in ProSAP2/Shank3.
Asunto(s)
Núcleo Celular/metabolismo , Regulación de la Expresión Génica/genética , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Animales , Arginina/genética , Células COS , Células Cultivadas , Chlorocebus aethiops , Embrión de Mamíferos , Hipocampo/citología , Humanos , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/ultraestructura , Ratas , Factores de TiempoRESUMEN
Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs) and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.
RESUMEN
BACKGROUND: Abelson-interacting protein 1 (Abi-1) plays an important role for dendritic branching and synapse formation in the central nervous system. It is localized at the postsynaptic density (PSD) and rapidly translocates to the nucleus upon synaptic stimulation. At PSDs Abi-1 is in a complex with several other proteins including WASP/WAVE or cortactin thereby regulating the actin cytoskeleton via the Arp 2/3 complex. PRINCIPAL FINDINGS: We identified heterogeneous nuclear ribonucleoprotein K (hnRNPK), a 65 kDa ssDNA/RNA-binding-protein that is involved in multiple intracellular signaling cascades, as a binding partner of Abi-1 at postsynaptic sites. The interaction with the Abi-1 SH3 domain is mediated by the hnRNPK-interaction (KI) domain. We further show that during brain development, hnRNPK expression becomes more and more restricted to granule cells of the cerebellum and hippocampal neurons where it localizes in the cell nucleus as well as in the spine/dendritic compartment. The downregulation of hnRNPK in cultured hippocampal neurons by RNAi results in an enlarged dendritic tree and a significant increase in filopodia formation. This is accompanied by a decrease in the number of mature synapses. Both effects therefore mimic the neuronal morphology after downregulation of Abi-1 mRNA in neurons. CONCLUSIONS: Our findings demonstrate a novel interplay between hnRNPK and Abi-1 in the nucleus and at synaptic sites and show obvious similarities regarding both protein knockdown phenotypes. This indicates that hnRNPK and Abi-1 act synergistic in a multiprotein complex that regulates the crucial balance between filopodia formation and synaptic maturation in neurons.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Espinas Dendríticas/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Sinapsis/fisiología , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Western Blotting , Células COS , Núcleo Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Ribonucleoproteína Heterogénea-Nuclear Grupo K/antagonistas & inhibidores , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Hipocampo/citología , Técnicas para Inmunoenzimas , Inmunoprecipitación , Hibridación in Situ , Ratones , Células 3T3 NIH , Neuronas/citología , Fenotipo , Seudópodos/metabolismo , ARN Interferente Pequeño/genética , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell. PRINCIPAL FINDINGS: We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners. CONCLUSIONS: Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos Multiproteicos/metabolismo , Neurogénesis , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Secuencia de Aminoácidos , Animales , Apamina/farmacología , Bencimidazoles/farmacología , Carbazoles/farmacología , Forma de la Célula/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Activación del Canal Iónico/efectos de los fármacos , Datos de Secuencia Molecular , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Propanolaminas/farmacología , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Ratas , Ratas Sprague-Dawley , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/químicaRESUMEN
Because evasion of apoptosis can cause radioresistance of glioblastoma, there is a need to design rational strategies that counter apoptosis resistance. In the present study, we investigated the potential of targeting the antiapoptotic protein XIAP for the radiosensitization of glioblastoma. Here, we report that small-molecule XIAP inhibitors significantly enhance gamma-irradiation-induced loss of viability and apoptosis and cooperate with gamma-irradiation to suppress clonogenic survival of glioblastoma cells. Analysis of molecular mechanisms reveals that XIAP inhibitors act in concert with gamma-irradiation to cause mitochondrial outer membrane permeabilization, caspase activation, and caspase-dependent apoptosis. Importantly, XIAP inhibitors also sensitize primary cultured glioblastoma cells derived from surgical specimens as well as glioblastoma-initiating stemlike cancer stem cells for gamma-irradiation. In contrast, they do not increase the toxicity of gamma-irradiation on some nonmalignant cells of the central nervous system, including rat neurons or glial cells, pointing to some tumor selectivity. In conclusion, by demonstrating for the first time that small-molecule XIAP inhibitors increase the radiosensitivity of glioblastoma cells while sparing normal cells of the central nervous system, our findings build the rationale for further (pre)clinical development of XIAP inhibitors in combination with gamma-irradiation in glioblastoma.
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Apoptosis/efectos de los fármacos , Rayos gamma/uso terapéutico , Glioblastoma/terapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Animales , Apoptosis/efectos de la radiación , Western Blotting , Caspasas/efectos de los fármacos , Terapia Combinada , Fragmentación del ADN/efectos de los fármacos , Fragmentación del ADN/efectos de la radiación , Citometría de Flujo , Humanos , Inmunohistoquímica , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de la radiación , Tolerancia a Radiación , Ratas , Proteína Inhibidora de la Apoptosis Ligada a X/efectos de los fármacosAsunto(s)
Células Madre Pluripotentes Inducidas/citología , Neuronas Motoras/citología , Animales , Diferenciación Celular , Forma de la Célula , Células Cultivadas , Técnicas de Cocultivo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/ultraestructura , Canales Iónicos/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Sinapsis/metabolismo , Factores de TiempoRESUMEN
Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1-ethyl-2-benzimidazolinone (1-EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50=2+/- 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+-channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non-differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1-EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre-incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels.
Asunto(s)
Neuronas/fisiología , Canales de Potasio Calcio-Activados/fisiología , Seudópodos/fisiología , Células Madre/citología , Animales , Bencimidazoles/farmacología , Agonistas de los Canales de Calcio/farmacología , Células Cultivadas , Interacciones Farmacológicas , Embrión de Mamíferos , Femenino , Inmunohistoquímica/métodos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Mesencéfalo/citología , Microscopía Inmunoelectrónica/métodos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Embarazo , Seudópodos/efectos de los fármacos , Seudópodos/ultraestructura , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Venenos de Escorpión/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Células Madre/efectos de los fármacosRESUMEN
Synaptogenesis and synaptic plasticity depend crucially on the dynamic and locally specific regulation of the actin cytoskeleton. We identified an important component for controlled actin assembly, abelson interacting protein-1 (Abi-1), as a binding partner for the postsynaptic density (PSD) protein ProSAP2/Shank3. During early neuronal development, Abi-1 is localized in neurites and growth cones; at later stages, the protein is enriched in dendritic spines and PSDs, as are components of a trimeric complex consisting of Abi-1, Eps8 and Sos-1. Abi-1 translocates upon NMDA application from PSDs to nuclei. Nuclear entry depends on abelson kinase activity. Abi-1 co-immunoprecipitates with the transcription factor complex of Myc/Max proteins and enhances E-box-regulated gene transcription. Downregulation of Abi-1 by small interfering RNA results in excessive dendrite branching, immature spine and synapse morphology and a reduction of synapses, whereas overexpression of Abi-1 has the opposite effect. Data show that Abi-1 can act as a specific synapto-nuclear messenger and is essentially involved in dendrite and synapse formation.