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
Autism spectrum disorders (ASDs) are characterized by repetitive behaviors and impairments of sociability and communication. About 1% of ASD cases are caused by mutations of SHANK3, a major scaffolding protein of the postsynaptic density. We studied the role of SHANK3 in plastic changes of excitatory synapses within the central nervous system by employing mild traumatic brain injury (mTBI) in WT and Shank3 knockout mice. In WT mice, mTBI triggered ipsi- and contralateral loss of hippocampal dendritic spines and excitatory synapses with a partial recovery over time. In contrast, no significant synaptic alterations were detected in Shank3∆11-/- mice, which showed fewer dendritic spines and excitatory synapses at baseline. In line, mTBI induced the upregulation of synaptic plasticity-related proteins Arc and p-cofilin only in WT mice. Interestingly, microglia proliferation was observed in WT mice after mTBI but not in Shank3∆11-/- mice. Finally, we detected TBI-induced increased fear memory at the behavioral level, whereas in Shank3∆11-/- animals, the already-enhanced fear memory levels increased only slightly after mTBI. Our data show the lack of structural synaptic plasticity in Shank3 knockout mice that might explain at least in part the rigidity of behaviors, problems in adjusting to new situations and cognitive deficits seen in ASDs.
Asunto(s)
Trastorno Autístico , Lesiones Traumáticas del Encéfalo , Animales , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Lesiones Traumáticas del Encéfalo/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/genética , Sinapsis/metabolismoRESUMEN
The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/ß-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/ß-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts.
Asunto(s)
Ojo/embriología , Proteínas Activadoras de GTPasa/fisiología , Cristalino/embriología , Cristalino/crecimiento & desarrollo , Receptor EphA4/fisiología , Vía de Señalización Wnt/fisiología , Animales , Animales Modificados Genéticamente , Catarata/genética , Diferenciación Celular/genética , Embrión no Mamífero , Ojo/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Cristalino/metabolismo , Organogénesis/genética , Unión Proteica , Receptor EphA4/metabolismo , Xenopus/embriología , Xenopus/genéticaRESUMEN
Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by deficits in social interaction and communication, and repetitive behaviors. In addition, co-morbidities such as gastro-intestinal problems have frequently been reported. Mutations and deletion of proteins of the SH3 and multiple ankyrin repeat domains (SHANK) gene-family were identified in patients with ASD, and Shank knock-out mouse models display autism-like phenotypes. SHANK3 proteins are not only expressed in the central nervous system (CNS). Here, we show expression in gastrointestinal (GI) epithelium and report a significantly different GI morphology in Shank3 knock-out (KO) mice. Further, we detected a significantly altered microbiota composition measured in feces of Shank3 KO mice that may contribute to inflammatory responses affecting brain development. In line with this, we found higher E. coli lipopolysaccharide levels in liver samples of Shank3 KO mice, and detected an increase in Interleukin-6 and activated astrocytes in Shank3 KO mice. We conclude that apart from its well-known role in the CNS, SHANK3 plays a specific role in the GI tract that may contribute to the ASD phenotype by extracerebral mechanisms.
Asunto(s)
Trastorno del Espectro Autista/microbiología , Microbioma Gastrointestinal , Proteínas del Tejido Nervioso/genética , Animales , Astrocitos/metabolismo , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Interleucina-6/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Lipopolisacáridos/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de MicrofilamentosRESUMEN
Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2(-/-) mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2(-/-) mutants with ProSAP2/Shank3αß(-/-) mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Trastorno Autístico/genética , Conducta Animal/fisiología , Proteínas del Tejido Nervioso/genética , Agitación Psicomotora/genética , Animales , Trastorno Autístico/patología , Espinas Dendríticas/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Agitación Psicomotora/patología , Receptores Ionotrópicos de Glutamato/metabolismo , Sinapsis/metabolismo , Regulación hacia Arriba , Vocalización Animal/fisiologíaRESUMEN
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
SHANK3 (also known as ProSAP2) regulates the structural organization of dendritic spines and is a binding partner of neuroligins; genes encoding neuroligins are mutated in autism and Asperger syndrome. Here, we report that a mutation of a single copy of SHANK3 on chromosome 22q13 can result in language and/or social communication disorders. These mutations concern only a small number of individuals, but they shed light on one gene dosage-sensitive synaptic pathway that is involved in autism spectrum disorders.
Asunto(s)
Trastorno Autístico/genética , Proteínas Portadoras/genética , Secuencia de Bases , Análisis Mutacional de ADN , Femenino , Pruebas Genéticas , Humanos , Hibridación Fluorescente in Situ , Masculino , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso , LinajeRESUMEN
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
BACKGROUND: Autism spectrum disorder (ASD) is mainly characterized by deficits in social interaction and communication and repetitive behaviors. Known causes of ASD are mutations of certain risk genes like the postsynaptic protein SHANK3 and environmental factors including prenatal infections. METHODS: To analyze the gene-environment interplay in ASD, we combined the Shank3Δ11-/- ASD mouse model with maternal immune activation (MIA) via an intraperitoneal injection of polyinosinic/polycytidylic acid (Poly I:C) on gestational day 12.5. The offspring of the injected dams was further analyzed for autistic-like behaviors and comorbidities followed by biochemical experiments with a focus on synaptic analysis. RESULTS: We show that the two-hit mice exhibit excessive grooming and deficits in social behavior more prominently than the Shank3Δ11-/- mice. Interestingly, these behavioral changes were accompanied by an unexpected upregulation of postsynaptic density (PSD) proteins at excitatory synapses in striatum, hippocampus and prefrontal cortex. LIMITATIONS: We found several PSD proteins to be increased in the two-hit mice; however, we can only speculate about possible pathways behind the worsening of the autistic phenotype in those mice. CONCLUSIONS: With this study, we demonstrate that there is an interplay between genetic susceptibility and environmental factors defining the severity of ASD symptoms. Moreover, we show that a general misbalance of PSD proteins at excitatory synapses is linked to ASD symptoms, making this two-hit model a promising tool for the investigation of the complex pathophysiology of neurodevelopmental disorders.
Asunto(s)
Trastorno del Espectro Autista , Trastorno Autístico , Femenino , Embarazo , Ratones , Animales , Trastorno del Espectro Autista/genética , Conducta Animal/fisiología , Trastorno Autístico/genética , Conducta Social , Sinapsis , Modelos Animales de Enfermedad , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/genéticaRESUMEN
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
Memory formation in the brain is thought to be depending upon long lasting plastic changes of synaptic contacts that require alterations on the transcriptional level. Here, we characterize LAPSER1, a putative cytokinetic tumor suppressor that binds directly to ProSAP2/Shank3 and the synaptic Rap-Gap protein SPAR1 as a novel postsynaptic density component. Postsynaptic LAPSER1 is in complex with all important members of the canonical Wnt pathway including beta-catenin. Upon N-methyl-D-aspartate receptor-dependent activation, LAPSER1 and beta-catenin comigrate from the postsynaptic density to the nucleus and induce the transcription and translation of known beta-catenin target genes, including Tcfe2a and c-Myc. The nuclear export and cytoplasmic redistribution of beta-catenin is tightly regulated by LAPSER1. We postulate a postsynaptic cross-talk between N-methyl-D-aspartate receptors and a LAPSER1-beta-catenin complex that results in a self-regulated, synaptic activity-dependent expression of beta-catenin target genes. This calls for a novel role of Tcfe2a and c-Myc in plastic changes of neural tissue.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Unión al ADN/química , Receptores de N-Metil-D-Aspartato/química , Proteínas Supresoras de Tumor/química , beta Catenina/química , Animales , Células COS , Calcio/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis , Técnicas del Sistema de Dos HíbridosRESUMEN
OBJECTIVE: Heterozygous SHANK3 mutations or partial deletions of the long arm of chromosome 22, also known as Phelan-McDermid syndrome, result in a syndromic form of the autism spectrum as well as in global developmental delay, intellectual disability, and several neuropsychiatric comorbidities. The exact pathophysiological mechanisms underlying the disease are still far from being deciphered but studies of SHANK3 models have contributed to the understanding of how the loss of the synaptic protein SHANK3 affects neuronal function. METHODS AND RESULTS: Diffusion tensor imaging-based and automatic volumetric brain mapping were performed in 12 SHANK3-deficient participants (mean age 19 ± 15 years) versus 14 age- and gender-matched controls (mean age 29 ± 5 years). Using whole brain-based spatial statistics, we observed a highly significant pattern of white matter alterations in participants with SHANK3 mutations with focus on the long association fiber tracts, particularly the uncinate tract and the inferior fronto-occipital fasciculus. In contrast, only subtle gray matter volumetric abnormalities were detectable. In a back-translational approach, we observed similar white matter alterations in heterozygous isoform-specific Shank3 knockout (KO) mice. Here, in the baseline data sets, the comparison of Shank3 heterozygous KO vs wildtype showed significant fractional anisotropy reduction of the long fiber tract systems in the KO model. The multiparametric Magnetic Resonance Imaging (MRI) analysis by DTI and volumetry demonstrated a pathology pattern with severe white matter alterations and only subtle gray matter changes in the animal model. INTERPRETATION: In summary, these translational data provide strong evidence that the SHANK3-deficiency-associated pathomechanism presents predominantly with a white matter disease. Further studies should concentrate on the role of SHANK3 during early axonal pathfinding/wiring and in myelin formation.
Asunto(s)
Proteínas del Tejido Nervioso/deficiencia , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Trastornos del Neurodesarrollo/fisiopatología , Sustancia Blanca/patología , Adolescente , Adulto , Animales , Trastorno del Espectro Autista/diagnóstico por imagen , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Trastorno del Espectro Autista/fisiopatología , Niño , Preescolar , Deleción Cromosómica , Trastornos de los Cromosomas/diagnóstico por imagen , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/patología , Trastornos de los Cromosomas/fisiopatología , Cromosomas Humanos Par 22/genética , Imagen de Difusión Tensora , Modelos Animales de Enfermedad , Femenino , Sustancia Gris/diagnóstico por imagen , Sustancia Gris/patología , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Persona de Mediana Edad , Trastornos del Neurodesarrollo/diagnóstico por imagen , Investigación Biomédica Traslacional , Sustancia Blanca/diagnóstico por imagen , Adulto JovenRESUMEN
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
Research efforts over the past decades have unraveled both genetic and environmental factors, which contribute to the development of autism spectrum disorders (ASD). It is, to date, largely unknown how different underlying causes result in a common phenotype. However, the individual course of development and the different comorbidities might reflect the heterogeneous genetic and non-genetic contributions. Therefore, it is reasonable to identify commonalities and differences in models of these disorders at the different hierarchical levels of brain function, including genetics/environment, cellular/synaptic functions, brain regions, connectivity, and behavior. To that end, we investigated Shank3 transgenic mouse lines and compared them with a prenatal zinc-deficient (PZD) mouse model of ASD at the level of brain structural alterations in an 11,7 T small animal magnetic resonance imaging (MRI). Animals were measured at 4 and 9 weeks of age. We identified a decreased total brain volume (TBV) and hippocampal size of Shank3-/- mice but a convergent increase of basal ganglia (striatum and globus pallidus) in most mouse lines. Moreover, Shank3 transgenic mice had smaller thalami, whereas PZD mice had this region enlarged. Intriguingly, Shank3 heterozygous knockout mice mostly showed minor abnormalities to full knockouts, which might reflect the importance of proper Shank3 dosage in neuronal cells. Most reported volume changes seemed to be more pronounced at younger age. Our results indicate both convergent and divergent brain region abnormalities in genetic and non-genetic models of ASD. These alterations of brain structures might be mirrored in the reported behavior of both models, which have not been assessed in this study.
Asunto(s)
Trastorno Autístico , Encéfalo/diagnóstico por imagen , Desnutrición/complicaciones , Proteínas del Tejido Nervioso/genética , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Zinc/deficiencia , Factores de Edad , Animales , Trastorno Autístico/inducido químicamente , Trastorno Autístico/diagnóstico por imagen , Trastorno Autístico/genética , Trastorno Autístico/patología , Modelos Animales de Enfermedad , Femenino , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/metabolismo , EmbarazoRESUMEN
Phelan McDermid Syndrome (PMDS) is a genetic disorder characterized by features of Autism spectrum disorders. Similar to reports of Zn deficiency in autistic children, we have previously reported high incidence of Zn deficiency in PMDS. However, the underlying mechanisms are currently not well understood. Here, using inductively coupled plasma mass-spectrometry to measure the concentration of Zinc (Zn) and Copper (Cu) in hair samples from individuals with PMDS with 22q13.3 deletion including SHANK3 (SH3 and multiple ankyrin repeat domains 3), we report a high rate of abnormally low Zn/Cu ratios. To investigate possible underlying mechanisms, we generated enterocytes from PMDS patient-derived induced pluripotent stem cells and used Caco-2 cells with knockdown of SHANK3. We detected decreased expression of Zn uptake transporters ZIP2 and ZIP4 on mRNA and protein level correlating with SHANK3 expression levels, and found reduced levels of ZIP4 protein co-localizing with SHANK3 at the plasma membrane. We demonstrated that especially ZIP4 exists in a complex with SHANK3. Furthermore, we performed immunohistochemistry on gut sections from Shank3αß knockout mice and confirmed a link between enterocytic SHANK3, ZIP2 and ZIP4. We conclude that apart from its well-known role in the CNS, SHANK3 might play a specific role in the GI tract.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Trastornos de los Cromosomas/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Zinc/deficiencia , Adolescente , Células CACO-2 , Proteínas de Transporte de Catión/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Niño , Preescolar , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 22/metabolismo , Cobre , Enterocitos/citología , Enterocitos/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Cabello/química , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Adulto JovenRESUMEN
Disruption of the human SHANK3 gene can cause several neuropsychiatric disease entities including Phelan-McDermid syndrome, autism spectrum disorder (ASD), and intellectual disability. Although, a wide array of neurobiological studies strongly supports a major role for SHANK3 in organizing the post-synaptic protein scaffold, the molecular processes at synapses of individuals harboring SHANK3 mutations are still far from being understood. In this study, we biochemically isolated the post-synaptic density (PSD) fraction from striatum and hippocampus of adult Shank3Δ11-/- mutant mice and performed ion-mobility enhanced data-independent label-free LC-MS/MS to obtain the corresponding PSD proteomes (Data are available via ProteomeXchange with identifier PXD005192). This unbiased approach to identify molecular disturbances at Shank3 mutant PSDs revealed hitherto unknown brain region specific alterations including a striatal decrease of several molecules encoded by ASD susceptibility genes such as the serine/threonine kinase Cdkl5 and the potassium channel KCa1.1. Being the first comprehensive analysis of brain region specific PSD proteomes from a Shank3 mutant line, our study provides crucial information on molecular alterations that could foster translational treatment studies for SHANK3 mutation-associated synaptopathies and possibly also ASD in general.
RESUMEN
BACKGROUND: The majority of neurons within the central nervous system receive their excitatory inputs via small, actin-rich protrusions called dendritic spines. Spines can undergo rapid morphological alterations according to synaptic activity. This mechanism is implicated in learning and memory formation as it is ultimately altering the number and distribution of receptors and proteins at the post-synaptic membrane, thereby regulating synaptic input. The Rho-family GTPases play an important role in regulating this spine plasticity by the interaction with cytoskeletal components and several signaling pathways within the spine compartment. Rho-GAP interacting CIP4 homologue2/RICH2 is a Rho-GAP protein regulating small GTPases and was identified as an interaction partner of the scaffolding protein SHANK3 at post-synaptic densities. RESULTS: Here, we characterize the loss of RICH2 in a novel mouse model. Our results show that RICH2 KO animals display a selective and highly significant fear of novel objects and increased stereotypic behavior as well as impairment of motor learning. We found an increase in multiple spine synapses in the hippocampus and cerebellum along with alterations in receptor composition and actin polymerization. Furthermore, we observed that the loss of RICH2 leads to a disinhibition of synaptic RAC1 in vivo. CONCLUSIONS: The results are in line with the reported role of RAC1 activity being essential for activity-dependent spine enlargement. Since SHANK3 mutations are known to be causative for neuropsychiatric diseases of the Autism Spectrum (ASD), a disintegrated SHANK3/RICH2 complex at synaptic sites might at least in part be responsible for abnormal spine formation and plasticity in ASDs.
Asunto(s)
Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Proteínas Activadoras de GTPasa/deficiencia , Trastornos Fóbicos/metabolismo , Animales , Conducta Animal , Glutamatos/metabolismo , Ratones Mutantes , Actividad Motora , Trastornos Fóbicos/fisiopatología , Transducción de Señal , Sinapsis/metabolismoRESUMEN
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.
Asunto(s)
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
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.