RESUMEN

The Shank3 gene encodes the major postsynaptic scaffolding protein SHANK3. Its mutation causes a syndromic form of autism spectrum disorder (ASD): Phelan-McDermid Syndrome (PMDS). It is characterized by global developmental delay, intellectual disorders (ID), ASD behavior, affective symptoms, as well as extra-cerebral symptoms. Although Shank3 deficiency causes a variety of molecular alterations, they do not suffice to explain all clinical aspects of this heterogenic syndrome. Since global gene expression alterations in Shank3 deficiency remain inadequately studied, we explored the transcriptome in vitro in primary hippocampal cells from Shank3∆11(-/-) mice, under control and lithium (Li) treatment conditions, and confirmed the findings in vivo. The Shank3∆11(-/-) genotype affected the overall transcriptome. Remarkably, extracellular matrix (ECM) and cell cycle transcriptional programs were disrupted. Accordingly, in the hippocampi of adolescent Shank3∆11(-/-) mice we found proteins of the collagen family and core cell cycle proteins downregulated. In vitro Li treatment of Shank3∆11(-/-) cells had a rescue-like effect on the ECM and cell cycle gene sets. Reversed ECM gene sets were part of a network, regulated by common transcription factors (TF) such as cAMP responsive element binding protein 1 (CREB1) and ß-Catenin (CTNNB1), which are known downstream effectors of synaptic activity and targets of Li. These TFs were less abundant and/or hypo-phosphorylated in hippocampi of Shank3∆11(-/-) mice and could be rescued with Li in vitro and in vivo. Our investigations suggest the ECM compartment and cell cycle genes as new players in the pathophysiology of Shank3 deficiency, and imply involvement of transcriptional regulators, which can be modulated by Li. This work supports Li as potential drug in the management of PMDS symptoms, where a Phase III study is ongoing.

Asunto(s)

Matriz Extracelular , Hipocampo , Ratones Noqueados , Proteínas del Tejido Nervioso , beta Catenina , Animales , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Hipocampo/metabolismo , Matriz Extracelular/metabolismo , Ratones , beta Catenina/metabolismo , beta Catenina/genética , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/metabolismo , Deleción Cromosómica , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Trastorno Autístico/genética , Trastorno Autístico/metabolismo , Cromosomas Humanos Par 22/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Masculino , Transcriptoma/genética , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/tratamiento farmacológico , Ratones Endogámicos C57BL , Litio/farmacología , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Células Cultivadas

RESUMEN

Phelan-McDermid syndrome (PMS) is a syndromic form of Autism Spectrum Disorders (ASD) classified as a rare genetic neurodevelopmental disorder featuring global developmental delay, absent or delayed speech, ASD-like behaviour and neonatal skeletal muscle hypotonia. PMS is caused by a heterozygous deletion of the distal end of chromosome 22q13.3 or SHANK3 mutations. We analyzed striated muscles of newborn Shank3Δ11(-/-) animals and found a significant enlargement of the sarcoplasmic reticulum as previously seen in adult Shank3Δ11(-/-) mice, indicative of a Shank3-dependent and not compensatory mechanism for this structural alteration. We analyzed transcriptional differences by RNA-sequencing of muscle tissue of neonatal Shank3Δ11(-/-) mice and compared those to Shank3(+/+) controls. We found significant differences in gene expression of ion channels crucial for muscle contraction and for molecules involved in calcium ion regulation. In addition, calcium storage- [i.e., Calsequestrin (CSQ)], calcium secretion- and calcium-related signaling-proteins were found to be affected. By immunostainings and Western blot analyses we could confirm these findings both in Shank3Δ11(-/-) mice and PMS patient muscle tissue. Moreover, alterations could be induced in vitro by the selective downregulation of Shank3 in C2C12 myotubes. Our results emphasize that SHANK3 levels directly or indirectly regulate calcium homeostasis in a cell autonomous manner that might contribute to muscular hypotonia especially seen in the newborn.

RESUMEN

Mutations of the postsynaptic scaffold protein Shank2 lead to autism spectrum disorders (ASD). These patients frequently suffer from higher fracture risk. Here, we investigated whether Shank2 directly regulates bone mass. We show that Shank2 is expressed in bone and that Shank2 levels are increased during osteoblastogenesis. Knockdown of Shank2 by siRNA targeting the encoding regions for PDZ and SAM domain inhibits osteoblastogenesis of primary murine calvarial osteoblasts. Shank2 knockout mice (Shank2 -/-) have a decreased bone mass due to reduced osteoblastogenesis and bone formation, whereas bone resorption remains unaffected. Induced pluripotent stem cells (iPSCs)-derived osteoblasts from a loss-of-function Shank2 mutation in a patient showed a significantly reduced osteoblast differentiation potential. Moreover, silencing of known Shank2 interacting proteins revealed that a majority of them promote osteoblast differentiation. From this we conclude that Shank2 and interacting proteins known from the central nervous system are decisive regulators in osteoblast differentiation. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

RESUMEN

Mutations or deletions of the SHANK3 gene are causative for Phelan-McDermid syndrome (PMDS), a syndromic form of autism spectrum disorders (ASDs). We analyzed Shank3Δ11(-/-) mice and organoids from PMDS individuals to study effects on myelin. SHANK3 was found to be expressed in oligodendrocytes and Schwann cells, and MRI analysis of Shank3Δ11(-/-) mice revealed a reduced volume of the corpus callosum as seen in PMDS patients. Myelin proteins including myelin basic protein showed significant temporal and regional differences with lower levels in the CNS but increased amounts in the PNS of Shank3Δ11(-/-) animals. Node, as well as paranode, lengths were increased and ultrastructural analysis revealed region-specific alterations of the myelin sheaths. In PMDS hiPSC-derived cerebral organoids we observed an altered number and delayed maturation of myelinating cells. These findings provide evidence that, in addition to a synaptic deregulation, impairment of myelin might profoundly contribute to the clinical manifestation of SHANK3 deficiency.

Asunto(s)

Trastorno del Espectro Autista , Trastornos de los Cromosomas , Proteínas de Microfilamentos , Vaina de Mielina , Proteínas del Tejido Nervioso , Animales , Trastorno del Espectro Autista/genética , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/metabolismo , Cromosomas Humanos Par 22 , Humanos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Vaina de Mielina/patología , Proteínas del Tejido Nervioso/genética , Sistema Nervioso Periférico/metabolismo

RESUMEN

Individuals with a SHANK3-related neurodevelopmental disorder, also termed Phelan-McDermid syndrome or abbreviated as PMS, exhibit significant global developmental delay, language impairment, and muscular hypotonia. Also common are repetitive behaviors and altered social interactions, in line with a diagnosis of autism spectrum disorders. This study investigated the developmental aspect of autism-related behaviors and other phenotypes in a Shank3-transgenic mouse model. The animals underwent two sets of identical behavioral experiments, spanning motor skills, social and repetitive behavior, and cognition: baseline began at 5 weeks of age, corresponding to human adolescence, and the follow-up was initiated when aged 13 weeks, resembling early adulthood in humans. Interestingly, the animals displayed relatively stable phenotypes. Moreover, motor coordination and endurance were impaired, while muscle strength was unchanged. Surprisingly, the animals displayed only minor impairments in social behavior, but pronounced stereotypic and repetitive behaviors. Some behavioral tests indicated increased avoidance and anxiety. While spatial learning and memory were unchanged, knockout animals displayed slightly impaired cognitive flexibility. Female animals had similar abnormalities as males in the paradigms testing avoidance, anxiety, and cognition, but were less pathological in motor function and repetitive behavior. In all test paradigms, heterozygous Shank3 knockout animals had either no abnormal or a milder phenotype. Accurate characterization of animal models for genetic diseases is a prerequisite for understanding the pathophysiology. This is subsequently the basis for finding suitable and, ideally, translational biomarkers for therapeutic approaches and, thereby reducing the number of animals needed for preclinical trials.

RESUMEN

Impairments in social relationships and awareness are features observed in autism spectrum disorders (ASDs). However, the underlying mechanisms remain poorly understood. Shank2 is a high-confidence ASD candidate gene and localizes primarily to postsynaptic densities (PSDs) of excitatory synapses in the central nervous system (CNS). We show here that loss of Shank2 in mice leads to a lack of social attachment and bonding behavior towards pubs independent of hormonal, cognitive, or sensitive deficits. Shank2-/- mice display functional changes in nuclei of the social attachment circuit that were most prominent in the medial preoptic area (MPOA) of the hypothalamus. Selective enhancement of MPOA activity by DREADD technology re-established social bonding behavior in Shank2-/- mice, providing evidence that the identified circuit might be crucial for explaining how social deficits in ASD can arise.

Asunto(s)

Trastorno Autístico/tratamiento farmacológico , Modelos Animales de Enfermedad , Relaciones Interpersonales , Conducta Materna/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Piperazinas/farmacología , Área Preóptica/efectos de los fármacos , Animales , Trastorno Autístico/etiología , Trastorno Autístico/metabolismo , Trastorno Autístico/patología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Área Preóptica/metabolismo , Área Preóptica/patología , Sinapsis

RESUMEN

Older patients with severe physical trauma are at high risk of developing neuropsychiatric syndromes with global impairment of cognition, attention, and consciousness. We employed a thoracic trauma (TxT) mouse model and thoroughly analyzed age-dependent spatial and temporal posttraumatic alterations in the central nervous system. Up to 5 days after trauma, we observed a transient 50% decrease in the number of excitatory synapses specifically in hippocampal pyramidal neurons accompanied by alterations in attention and motor activity and disruption of contextual memory consolidation. In parallel, hippocampal corticotropin-releasing hormone (CRH) expression was highly upregulated, and brain-derived neurotrophic factor (BDNF) levels were significantly reduced. In vitro experiments revealed that CRH application induced neuronal autophagy with rapid lysosomal degradation of BDNF via the NF-κB pathway. The subsequent synaptic loss was rescued by BDNF as well as by specific NF-κB and CRH receptor 1 (CRHR1) antagonists. In vivo, the chronic application of a CRHR1 antagonist after TxT resulted in reversal of the observed histological, molecular, and behavioral alterations. The data suggest that neuropsychiatric syndromes (i.e., delirium) after peripheral trauma might be at least in part due to the activation of the hippocampal CRH/NF-κB/BDNF pathway, which results in a dramatic loss of synaptic contacts. The successful rescue by stress hormone receptor antagonists should encourage clinical trials focusing on trauma-induced delirium and/or other posttraumatic syndromes.

Asunto(s)

Delirio , Neuronas , Animales , Hormona Liberadora de Corticotropina , Humanos , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/etiología , Ratones , Receptores de Hormona Liberadora de Corticotropina , Síndrome

RESUMEN

Local protein synthesis in dendrites enables neurons to selectively change the protein complement of individual postsynaptic sites. Though it is generally assumed that this mechanism requires tight translational control of dendritically transported mRNAs, it is unclear how translation of dendritic mRNAs is regulated. We have analyzed here translational control elements of the dendritically localized mRNA coding for the postsynaptic scaffold protein Shank1. In its 5' region, the human Shank1 mRNA exhibits two alternative translation initiation sites (AUG⁺¹ and AUG⁺²¹4), three canonical upstream open reading frames (uORFs1-3) and a high GC content. In reporter assays, fragments of the 5'UTR with high GC content inhibit translation, suggesting a contribution of secondary structures. uORF3 is most relevant to translation control as it overlaps with the first in frame start codon (AUG⁺¹), directing translation initiation to the second in frame start codon (AUG⁺²¹4). Surprisingly, our analysis points to an additional uORF initiated at a non-canonical ACG start codon. Mutation of this start site leads to an almost complete loss of translation initiation at AUG⁺¹, demonstrating that this unconventional uORF is required for Shank1 synthesis. Our data identify a novel mechanism whereby initiation at a non-canonical site allows for translation of the main Shank1 ORF despite a highly structured 5'UTR.

Asunto(s)

Dendritas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Codón Iniciador/metabolismo , Análisis Mutacional de ADN , Eliminación de Gen , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Sitio de Iniciación de la Transcripción

RESUMEN

Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.

Asunto(s)

Hipocampo/citología , Neuronas/citología , Sinapsis/metabolismo , Animales , Células Cultivadas , Feto/citología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Sinapsis/ultraestructura

RESUMEN

Pre- and post-synaptic targeting of synaptic molecules is depending upon specific targeting signals that are encoded within defined regions of the respective protein. For the post-synaptic scaffolding proteins of excitatory synapses, ProSAP1/Shank2 and ProSAP2/Shank3 this targeting information is located within about 460aa of the C-terminus. We found the C-terminal targeting signal to be bipartite composed of a 135aa stretch and the SAM (sterile alpha motif) domain embedding a relatively large variable spacer region. Based on this we developed a new GFP vector system called pSDTarget to easily clone proteins of interest as GFP fusion proteins flanked by a bipartite targeting signal for post-synaptic densities (PSDs) of excitatory synapses. The targeting signal has been derived from the PSD scaffolding protein ProSAP1/Shank2. In hippocampal neuron culture we could effectively localize and attach i.e. Glutathion-S-transferase (GST) at PSDs of excitatory synapses already during early synaptogenesis. Moreover, Gephyrin, an important scaffold molecule of inhibitory post-synapses was succesfully targeted to excitatory synapses followed by the subsequent recruitment of GABAergic receptors leading to hybrid synaptic contacts. In light of the role of specific protein domains for plastic changes of the post-synaptic compartment or investigations focusing on synaptogenesis, signalling and/or transsynaptic crosstalk this vector system provides a powerful and innovative tool for the functional analysis of molecular mechanisms and structural changes in a small but well defined neuronal compartment.

Asunto(s)

Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Animales , Células Cultivadas , Vectores Genéticos , Hipocampo/citología , Neuronas/metabolismo , Plásmidos/genética , Transporte de Proteínas , Ratas , Transfección

RESUMEN

The PDZ domain-containing protein Shank is a master scaffolding protein of the neuronal postsynaptic density and directly or indirectly links neurotransmitter receptors and cell adhesion molecules to the actin-based cytoskeleton. ProSAP/Shank proteins have recently also been detected in several non-neuronal cells in which they are mostly concentrated in the apical subplasmalemmal cytoplasm. In contrast, we have previously reported a more widespread cytoplasmic immunostaining pattern for the ProSAP1/Shank2 protein in endocrine cells at the light-microscopic level. Therefore, in the present study, we have determined the ultrastructural localization of ProSAP1/Shank2 and the ProSAP/Shank-interacting proteins ProSAPiP1 and IRSp53 in pancreatic islet and adenohypophyseal cells by using immunogold staining techniques. Dense immunolabeling of secretory granules including the granule core in cells such as hypophyseal somatotrophs and pancreatic B-cells indicates the unexpected presence of ProSAP/Shank and ProSAP/Shank-interacting proteins in the hormone-storing compartment of endocrine cells. Thus, ProSAP/Shank and certain ProSAP/Shank-interacting proteins exhibit distinct subcellular localizations in the different cell types, raising the possibility that the function of ProSAP/Shank proteins is more diverse than has been envisaged to date.

Asunto(s)

Proteínas Portadoras/metabolismo , Islotes Pancreáticos/citología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Hipófisis/citología , Vesículas Secretoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Femenino , Masculino , Neuronas , Transporte de Proteínas , Ratas , Ratas Wistar , Vesículas Secretoras/ultraestructura

RESUMEN

PSD95-DLG-ZO1 domain-containing proteins of the ProSAP/Shank family are major scaffolding proteins of the neuronal postsynaptic density which play a pivotal role in the linkage of membrane receptors to downstream signal effectors and the actin-based cytoskeleton. Recently, ProSAP1/Shank2 has also been localized in various non-neuronal cells where it may fulfill similar functions as in neurons. We now complement these data by the study of ProSAP/Shank expression at the mRNA and protein level in a primary lymphoid organ, i.e., the thymus. Transcripts for ProSAP1/Shank2, the spliceoform Shank2E, and ProSAP2/Shank3 could be clearly detected in the thymus. Western blot and immunocytochemical analyses verified the presence of ProSAP1/Shank2 and ProSAP2/Shank3 proteins in thymic tissue. Immunoreactivity was concentrated in the whole peripheral cytoplasm of thymocytes underneath the plasma membrane. Discrete subplasmalemmal areas of pronounced ProSAP/Shank immunoreactivity could be demonstrated inside several thymocytes by confocal laser scanning microscopy. Our results establish ProSAP/Shank as a constituent of the cell cortex of thymocytes and thus lead to the hypothesis that ProSAP/Shank proteins serve as a platform for the coordination of membrane receptor-dependent signal transduction in immune cells.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/análisis , Timo/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Animales , Western Blotting , Células Cultivadas , Citoesqueleto/química , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/aislamiento & purificación , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/citología , Timo/inmunología

RESUMEN

Synapses are specialized contact sites mediating communication between neurons. Synaptogenesis requires the specific assembly of protein clusters at both sides of the synaptic contact by mechanisms that are barely understood. We studied the synaptic targeting of multi-domain proteins of the ProSAP/Shank family thought to serve as master scaffolding molecules of the postsynaptic density. In contrast to Shank1, expression of green-fluorescent protein (GFP)-tagged ProSAP1/Shank2 and ProSAP2/Shank3 deletion constructs in hippocampal neurons revealed that their postsynaptic localization relies on the integrity of the C-termini. The shortest construct that was perfectly targeted to synaptic sites included the last 417 amino acids of ProSAP1/Shank2 and included the C-terminal sterile alpha motif (SAM) domain. Removal of 54 residues from the N-terminus of this construct resulted in a diffuse distribution in the cytoplasm. Altogether, our data delineate a hitherto unknown targeting signal in both ProSAP1/Shank2 and ProSAP2/Shank3 and provide evidence for an implication of these proteins and their close homologue, Shank1, in distinct molecular pathways.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Proteínas Recombinantes de Fusión/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transfección

RESUMEN

Shank proteins are scaffolding proteins in the postsynaptic density of excitatory synapses in the mammalian brain. In situ hybridization revealed that Shank1/SSTRIP and Shank2/ProSAP1 mRNAs are widely expressed early in postnatal brain development whereas Shank3/ProSAP2 expression increases during postnatal development especially in the cerebellum and thalamus. Shank1 and Shank3 (but not Shank2) mRNAs are present in the molecular layers of the hippocampus, consistent with a dendritic transcript localization. Shank1 and Shank2 transcripts are detectable in the dendritic fields of Purkinje cells, whereas Shank3 mRNA is restricted to cerebellar granule cells. The appearance of dendritic Shank mRNAs in cerebellar Purkinje cells coincides with the onset of dendrite formation. Expression of reporter transcripts in hippocampal neurons identifies a 200-nucleotide dendritic targeting element (DTE) in the Shank1 mRNA. The widespread presence of Shank mRNAs in dendrites suggests a role for local synthesis of Shanks in response to stimuli that induce alterations in synaptic morphology.

Asunto(s)

Regiones no Traducidas 3'/genética , Proteínas Adaptadoras Transductoras de Señales , Encéfalo/metabolismo , Proteínas Portadoras/genética , Dendritas/metabolismo , ARN Mensajero/genética , Membranas Sinápticas/metabolismo , Animales , Encéfalo/citología , Encéfalo/crecimiento & desarrollo , Corteza Cerebelosa/citología , Corteza Cerebelosa/crecimiento & desarrollo , Corteza Cerebelosa/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Genes Reguladores/genética , Genes Reporteros/genética , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/genética , ARN Mensajero/metabolismo , Ratas , Membranas Sinápticas/genética , Transmisión Sináptica/genética

RESUMEN

Proteins of the presynaptic exocytic machinery have been found associated with the acrosome of male germ cells, suggesting that the sperm acrosome reaction and neurotransmission at chemical synapses may share some common mechanisms. To substantiate this hypothesis, we studied the expression and ultrastructural localization of prominent pre- and postsynaptic protein components in rat testis. The presynaptic membrane trafficking proteins SV2 and complexin, the vesicular amino acid transporters VGLUT and VIAAT, the postsynaptic scaffolding protein ProSAP/Shank, and the postsynaptic calcium-sensor protein caldendrin, could be identified in germ line cells. Immunogold electron microscopy revealed an association of these proteins with the acrosome. In addition, evidence was obtained for the expression of the plasmalemmal glutamate transporters GLT1 and GLAST in rat sperm. The novel finding that not only presynaptic proteins, which are believed to be involved in membrane fusion processes, but also postsynaptic elements are present at the acrosome sheds new light on its structural organization. Moreover, our data point to a possible role for neuroactive amino acids in reproductive physiology.

Asunto(s)

Acrosoma/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Sinapsis/metabolismo , Testículo/metabolismo , Animales , Encéfalo/ultraestructura , Inmunohistoquímica , Masculino , Microscopía Inmunoelectrónica , Ratas , Ratas Wistar , Testículo/citología , Testículo/ultraestructura

RESUMEN

Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMP-CrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.

Asunto(s)

Adaptación Fisiológica/genética , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Sacarosa/metabolismo , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fructoquinasas/genética , Fructoquinasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADN , beta-Fructofuranosidasa

RESUMEN

The postsynaptic density (PSD) is a specialized electron-dense structure underneath the postsynaptic plasmamembrane of excitatory synapses. It is thought to anchor and cluster glutamate receptors exactly opposite to the presynaptic neurotransmitter release site. Various efforts to study the molecular structure of the PSD identified several new proteins including membrane receptors, cell adhesion molecules, components of signalling cascades, cytoskeletal elements and adaptor proteins with scaffolding functions to interconnect these PSD components. The characterization of a novel adaptor protein family, the ProSAPs or Shanks, sheds new light on the basic structural organization of the PSD. ProSAPs/Shanks are multidomain proteins that interact directly or indirectly with receptors of the postsynaptic membrane including NMDA-type and metabotropic glutamate receptors, and the actin-based cytoskeleton. These interactions suggest that ProSAP/Shanks may be important scaffolding molecules of the PSD with a crucial role in the assembly of the PSD during synaptogenesis, in synaptic plasticity and in the regulation of dendritic spine morphology. Moreover the analysis of a patient with 22q13.3 distal deletion syndrome revealed a balanced translocation with a breakpoint in the human ProSAP2/Shank3 gene. This ProSAP2/Shank3 haploinsufficiency may cause a syndrome that is characterized by severe expressive language delay, mild mental retardation and minor facial dysmorphisms.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Sinapsis/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genética , Humanos , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/genética , Receptores de Glutamato/metabolismo , Sinapsis/ultraestructura