Simiate and the focal adhesion kinase FAK1 cooperate in the regulation of dendritogenesis.

Despite the crucial importance of dendritogenesis for the correct functioning of neurons, the molecular mechanisms underlying neuronal arborisation are still not well understood. Current models suggest that distinct parts and phases of dendritic development are regulated by the expression of distinct transcription factors, that are able to target the cytoskeleton. Two proteins recently implicated in dendritogenesis are the Focal Adhesion Kinase FAK1 and the Actin-binding protein Simiate. Using heterologous expression systems as well as mouse brain extracts in combination with coprecipitation assays, we show that Simiate is able to associate with FAK1. Differential centrifugation experiments further revealed the interaction to be present in cytosolic as well as nuclear fractions. Inside the nucleus though, Simiate preferentially binds to a FAK1 isoform of 80 kDa, which has previously been shown to regulate transcription factor activity. Investigating the function of both proteins in primary hippocampal cultures, we further found that FAK1 and Simiate have distinct roles in dendritogenesis: While FAK1 increases dendrite length and number, Simiate preferentially enhances growth and branching. However, if being confined to the nucleus, Simiate selectively triggers primary dendrite formation, enhancing transcription activity at the same time. Since the effect on primary dendrites is specifically re-normalized by a co-expression of FAK1 and Simiate in the nucleus, the data implies that the two proteins interact to counterbalance each other in order to control dendrite formation. Looking at the role of the cytosolic interaction of FAK1 and Simiate, we found that neurotrophin induced dendritogenesis causes a striking colocalisation of FAK1 and Simiate in dendritic growth cones, which is not present otherwise, thus suggesting that the cytosolic interaction stimulates growth cone mediated dendritogenesis in response to certain external signals. Taken together, the data show that FAK1 and Simiate exert several and distinct actions during the different phases of dendritogenesis and that these actions are related to their subcellular localisation and their interaction.

Of Men and Mice: Modeling the Fragile X Syndrome.

The Fragile X Syndrome (FXS) is one of the most common forms of inherited intellectual disability in all human societies. Caused by the transcriptional silencing of a single gene, the fragile x mental retardation gene FMR1, FXS is characterized by a variety of symptoms, which range from mental disabilities to autism and epilepsy. More than 20 years ago, a first animal model was described, the Fmr1 knock-out mouse. Several other models have been developed since then, including conditional knock-out mice, knock-out rats, a zebrafish and a drosophila model. Using these model systems, various targets for potential pharmaceutical treatments have been identified and many treatments have been shown to be efficient in preclinical studies. However, all attempts to turn these findings into a therapy for patients have failed thus far. In this review, I will discuss underlying difficulties and address potential alternatives for our future research.

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions.

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides  mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Special characteristics of the transcription and splicing machinery in photoreceptor cells of the mammalian retina.

Chromatin organization and the management of transcription and splicing are fundamental to the correct functioning of every cell but, in particular, for highly active cells such as photoreceptors, the sensory neurons of the retina. Rod photoreceptor cells of nocturnal animals have recently been shown to have an inverted chromatin architecture compared with rod photoreceptor cells of diurnal animals. The heterochromatin is concentrated in the center of the nucleus, whereas the genetically active euchromatin is positioned close to the nuclear membrane. This unique chromatin architecture suggests that the transcription and splicing machinery is also subject to specific adaptations in these cells. Recently, we described the protein Simiate, which is enriched in nuclear speckles and seems to be involved in transcription and splicing processes. Here, we examine the distribution of Simiate and nuclear speckles in neurons of mouse retinae. In retinal neurons of the inner nuclear and ganglion cell layer, Simiate is concentrated in a clustered pattern in the nuclear interior, whereas in rod and cone photoreceptor cells, Simiate is present at the nuclear periphery. Further staining with markers for the transcription and splicing machinery has confirmed the localization of nuclear speckle components at the periphery. Comparing the distribution of nuclear speckles in retinae of the nocturnal mouse with the diurnal degu, we found no differences in the arrangement of the transcription and splicing machinery in their photoreceptor cells, thus suggesting that the organization of these machineries is not related to the animal's lifestyle but rather represents a general characteristic of photoreceptor organization and function.

Simiate is an Actin binding protein involved in filopodia dynamics and arborization of neurons.

The Actin cytoskeleton constitutes the functional base for a multitude of cellular processes extending from motility and migration to cell mechanics and morphogenesis. The latter is particularly important to neuronal cells since the accurate functioning of the brain crucially depends on the correct arborization of neurons, a process that requires the formation of several dozens to hundreds of dendritic branches. Recently, a model was proposed where different transcription factors are detailed to distinct facets and phases of dendritogenesis and exert their function by acting on the Actin cytoskeleton, however, the proteins involved as well as the underlying molecular mechanisms are largely unknown. Here, we demonstrate that Simiate, a protein previously indicated to activate transcription, directly associates with both, G- and F-Actin and in doing so, affects Actin polymerization and Actin turnover in living cells. Imaging studies illustrate that Simiate particularly influences filopodia dynamics and specifically increases the branching of proximal, but not distal dendrites of developing neurons. The data suggests that Simiate functions as a direct molecular link between transcription regulation on one side, and dendritogenesis on the other, wherein Simiate serves to coordinate the development of proximal and distal dendrites by acting on the Actin cytoskeleton of filopodia and on transcription regulation, hence supporting the novel model.

Identification and characterisation of Simiate, a novel protein linked to the fragile X syndrome.

A strict regulation of protein expression during developmental stages and in response to environmental signals is essential to every cell and organism. Recent research has shown that the mammalian brain is particularly sensitive to alterations in expression patterns of specific proteins and cognitive deficits as well as autistic behaviours have been linked to dysregulated protein expression. An intellectual disability characterised by changes in the expression of a variety of proteins is the fragile X syndrome. Due to the loss of a single mRNA binding protein, the Fragile X Mental Retardation Protein FMRP, vast misregulation of the mRNA metabolism is taking place in the disease. Here, we present the identification and characterisation of a novel protein named Simiate, whose mRNA contains several FMRP recognition motifs and associates with FMRP upon co-precipitation. Sequence analysis revealed that the protein evolved app. 1.7 billion years ago when eukaryotes developed. Applying antibodies generated against Simiate, the protein is detected in a variety of tissues, including the mammalian brain. On the subcellular level, Simiate localises to somata and nuclear speckles. We show that Simiate and nuclear speckles experience specific alterations in FMR1(-/-) mice. An antibody-based block of endogenous Simiate revealed that the protein is essential for cell survival. These findings suggest not only an important role for Simiate in gene transcription and/or RNA splicing, but also provide evidence for a function of nuclear speckles in the fragile X syndrome. Indeed, transcription and splicing are two fundamental mechanisms to control protein expression, that underlie not only synaptic plasticity and memory formation, but are also affected in several diseases associated with mental disabilities.

Altered neuroligin expression is involved in social deficits in a mouse model of the fragile X syndrome.

The fragile X syndrome (FXS) is the most common form of inherited mental retardation. Caused by a transcriptional silencing of the fragile X mental retardation protein (FMRP), a mRNA binding protein itself, misregulated translation is thought to be the leading cause of the fragile X syndrome. Interestingly, recent results indicated several neuroligin interacting proteins to be affected by this misregulation, including neurexin1 and PSD95, which have also been implicated in autism spectrum disorders. Using co-immunoprecipitation assays and RT-PCR, FMRP is shown to interact with neuroligin1- and 2-mRNA, while no interaction with neuroligin3-mRNA is observed. In line with FMRP's role in translation regulation, Western blot as well as immunohistochemistry analysis reveal changes in protein expression levels suggesting impaired synaptic function. As increasing evidence indicates neuroligin expression to be critical for synapse maturation and function, consequences of impaired neuroligin1 expression in FXS are assessed by overexpressing HA-neuroligin1 in FMR1-/- mice, a model for FXS. Behavioural assessments demonstrate that enhanced neuroligin1 expression improves social behaviour in FMR1-/- mice, whereas no positive effect on learning and memory is seen. These results provide for the first time evidence for an involvement of a neuroligin-neurexin protein network in core symptoms of FXS.

Overexpression of the cell adhesion protein neuroligin-1 induces learning deficits and impairs synaptic plasticity by altering the ratio of excitation to inhibition in the hippocampus.

Trans-synaptic cell-adhesion molecules have been implicated in regulating CNS synaptogenesis. Among these, the Neuroligin (NL) family (NLs 1-4) of postsynaptic adhesion proteins has been shown to promote the development and specification of excitatory versus inhibitory synapses. NLs form a heterophilic complex with the presynaptic transmembrane protein Neurexin (NRX). A differential association of NLs with postsynaptic scaffolding proteins and NRX isoforms has been suggested to regulate the ratio of excitatory to inhibitory synapses (E/I ratio). Using transgenic mice, we have tested this hypothesis by overexpressing NL1 in vivo to determine whether the relative levels of these cell adhesion molecules may influence synapse maturation, long-term potentiation (LTP), and/or learning. We found that NL1-overexpressing mice show significant deficits in memory acquisition, but not in memory retrieval. Golgi and electron microscopy analysis revealed changes in synapse morphology indicative of increased maturation of excitatory synapses. In parallel, electrophysiological examination indicated a shift in the synaptic activity toward increased excitation as well as impairment in LTP induction. Our results demonstrate that altered balance in the expression of molecules necessary for synapse specification and development (such as NL1) can lead to defects in memory formation and synaptic plasticity and outline the importance of rigidly controlled synaptic maturation processes.

Synaptic imbalance, stereotypies, and impaired social interactions in mice with altered neuroligin 2 expression.

The level of excitation in the brain is kept under control through inhibitory signals mainly exerted by GABA neurons. However, the molecular machinery that regulates the balance between excitation and inhibition (E/I) remains unclear. Candidate molecules implicated in this process are neuroligin (NL) adhesion molecules, which are differentially enriched at either excitatory or inhibitory contacts. In this study, we use transgenic mouse models expressing NL1 or NL2 to examine whether enhanced expression of specific NLs results in synaptic imbalance and altered neuronal excitability and animal behavior. Our analysis reveals several abnormalities selectively manifested in transgenic mice with enhanced expression of NL2 but not NL1. A small change in NL2 expression results in enlarged synaptic contact size and vesicle reserve pool in frontal cortex synapses and an overall reduction in the E/I ratio. The frequency of miniature inhibitory synaptic currents was also found to be increased in the frontal cortex of transgenic NL2 mice. These animals also manifested stereotyped jumping behavior, anxiety, impaired social interactions, and enhanced incidence of spike-wave discharges, as depicted by EEG analysis in freely moving animals. These findings may provide the neural basis for E/I imbalance and altered behavior associated with neurodevelopmental disorders.

EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling.

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin-EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.