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International audience; Rationale:The genetic heterogeneity of autism spectrum disorders (ASDs) is enormous, and the neurobiology of proteins encoded by genes associated with ASD is very diverse. Revealing the mechanisms on which different neurobiological pathways in ASD pathogenesis converge may lead to the identification of drug targets.; Objective:The main objective is firstly to outline the main molecular networks and neuronal mechanisms in which ASD gene products participate and secondly to answer the question how these converge. Finally, we aim to pinpoint drug targets within these mechanisms.; Method:Literature review of the neurobiological properties of ASD gene products with a special focus on the developmental consequences of genetic defects and the possibility to reverse these by genetic or pharmacological interventions.; Results:The regulation of activity-dependent protein synthesis appears central in the pathogenesis of ASD. Through sequential consequences for axodendritic function, neuronal disabilities arise expressed as behavioral abnormalities and autistic symptoms in ASD patients. Several known ASD gene products have their effect on this central process by affecting protein synthesis intrinsically, e.g., through enhancing the mammalian target of rapamycin (mTOR) signal transduction pathway or through impairing synaptic function in general. These are interrelated processes and can be targeted by compounds from various directions: inhibition of protein synthesis through Lovastatin, mTOR inhibition using rapamycin, or mGluR-related modulation of synaptic activity.; Conclusions:ASD gene products may all feed into a central process of translational control that is important for adequate glutamatergic regulation of dendritic properties. This process can be modulated by available compounds but may also be targeted by yet unexplored routes.

We have recently isolated a novel proline-rich synapse-associated protein-1 (ProSAP1) that is highly enriched in postsynaptic density (PSD). A closely related multidomain protein, ProSAP2, shares a highly conserved PDZ (PSD-95/discs-large/ZO-1) domain (80% identity), a ppI domain that mediates the interaction with cortactin, and a C-terminal SAM (sterile alpha-motif) domain. In addition, ProSAP2 codes for five ankyrin repeats and a SH3 (Src homology 3) domain. Transcripts for both proteins are coexpressed in many regions of rat brain, but show a distinct expression pattern in the cerebellum. Using the PDZ domains of ProSAP1 and 2 as bait in the yeast two-hybrid system, we isolated several clones of the SAPAP/GKAP (SAP90/PSD-95-associated protein/guanylate kinase-associated protein) family. The association of the proteins was verified by coimmunoprecipitation and cotransfection in HEK cells. Therefore, proteins of the ProSAP family represent a novel link between SAP90/PSD-95 bound membrane receptors and the cytoskeleton at glutamatergic synapses of the central nervous system.

Using antibodies against synaptic protein preparations, we cloned the cDNA of a new Ca2+-binding protein. Its C-terminal portion displays significant similarity with calmodulin and contains two EF-hand motifs. The corresponding mRNA is highly expressed in rat brain, primarily in cerebral cortex, hippocampus, and cerebellum; its expression appears to be restricted to neurons. Transcript levels increase during postnatal development. A recombinant C-terminal protein fragment binds Ca2+ as indicated by a Ca2+-induced mobility shift in SDS-polyacrylamide gel electrophoresis. Antisera generated against the bacterial fusion protein recognize a brain-specific protein doublet with apparent molecular masses of 33 and 36 kDa. These data are confirmed by in vitro translation, which generates a single 36-kDa polypeptide, and by the heterologous expression in 293 cells, which yields a 33/36-kDa doublet comparable to that found in brain. On two-dimensional gels, the 33-kDa band separates into a chain of spots plausibly due to differential phosphorylation. This view is supported by in situ phosphorylation studies in hippocampal slices. Most of the immunoreactivity is detectable in cytoskeletal preparations with a further enrichment in the synapse-associated cytomatrix. These biochemical data, together with the ultra-structural localization in dendrites and the postsynaptic density, strongly suggest an association with the somato-dendritic cytoskeleton. Therefore, this novel Ca2+-binding protein was named caldendrin.

International audience; 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.

Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.

Nanocarriers can be useful tools for delivering drugs to the central nervous system (CNS). Their distribution within the brain and their interaction with CNS cells must be assessed accurately before they can be proposed for therapeutic use. In this paper, we investigated these issues by employing poly-lactide-co-glycolide nanoparticles (NPs) specifically engineered with a glycopeptide (g7) conferring to NPs the ability to cross the blood brain barrier (BBB) at a concentration of up to 10% of the injected dose. g7-NPs display increased in vitro uptake in neurons and glial cells. Our results show that in vivo administration of g7-NPs leads to a region- and cell type-specific enrichment of NPs within the brain. We provide evidence that g7-NPs are endocytosed in a clathrin-dependent manner and transported into a specific subset of early endosomes positive for Rab5 in vitro and in vivo. The differential Rab5 expression level is strictly correlated with the amount of g7-NP accumulation. These findings show that g7-NPs can cross the BBB and target specific brain cell populations, suggesting that these NPs can be promising carriers for the treatment of neuropsychiatric and neurodegenerative diseases.

Shank/ProSAP proteins are major scaffold proteins of the postsynaptic density; mutations in the human SHANK3 gene are associated with intellectual disability or autism spectrum disorders. We have analyzed the functional relevance of several SHANK3 missense mutations affecting the N-terminal portion of the protein by expression of wild-type and mutant Shank3 in cultured neurons and by binding assays in heterologous cells. Postsynaptic targeting of recombinant Shank3 was unaltered. In electrophysiological experiments, both wild-type and L68P mutant forms of Shank3 were equally effective in restoring synaptic function after knockdown of endogenous Shank3. We observed that several mutations affected binding to interaction partners of the Shank3 ankyrin repeat region. One of these mutations, L68P, improved binding to both ligands. Leu-68 is located N-terminal to the ankyrin repeats, in a highly conserved region that we identify here as a novel domain termed the Shank/ProSAP N-terminal (SPN) domain. We show that the SPN domain interacts with the ankyrin repeats in an intramolecular manner, thereby restricting access of either Sharpin or α-fodrin. The L68P mutation disrupts this blockade, thus exposing the Shank3 ankyrin repeat region to its ligands. Our data identify a new type of regulation of Shank proteins and suggest that mutations in the SHANK3 gene do not necessarily induce a loss of function, but may represent a gain of function with respect to specific interaction partners. Background: Missense mutations in the SHANK3 gene have been detected in autism patients. Results: A mutation in the conserved SPN region of Shank3 improves ligand binding to the ankyrin repeats. Conclusion: The SPN domain regulates accessibility of the ankyrin repeats through an intramolecular interaction. Significance: Autism-associated mutations of Shank3 result in gain-of-function with respect to specific interaction partners.

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.

Mutations in several postsynaptic proteins have recently been implicated in the molecular pathogenesis of autism and autism spectrum disorders (ASDs), including Neuroligins, Neurexins, and members of the ProSAP/Shank family, thereby suggesting that these genetic forms of autism may share common synaptic mechanisms. Initial studies of ASD-associated mutations in ProSAP2/Shank3 support a role for this protein in glutamate receptor function and spine morphology, but these synaptic phenotypes are not universally penetrant, indicating that other core facets of ProSAP2/Shank3 function must underlie synaptic deficits in patients with ASDs. In the present study, we have examined whether the ability of ProSAP2/Shank3 to interact with the cytoplasmic tail of Neuroligins functions to coordinate pre/postsynaptic signaling through the Neurexin–Neuroligin signaling complex in hippocampal neurons ofRattus norvegicus. Indeed, we find that synaptic levels of ProSAP2/Shank3 regulate AMPA and NMDA receptor-mediated synaptic transmission and induce widespread changes in the levels of presynaptic and postsynaptic proteins via Neurexin–Neuroligin transsynaptic signaling. ASD-associated mutations in ProSAP2/Shank3 disrupt not only postsynaptic AMPA and NMDA receptor signaling but also interfere with the ability of ProSAP2/Shank3 to signal across the synapse to alter presynaptic structure and function. These data indicate that ASD-associated mutations in a subset of synaptic proteins may target core cellular pathways that coordinate the functional matching and maturation of excitatory synapses in the CNS.

Summary The family of calcium activated potassium channels of low and intermediate conductance, known as SK channels, consists of four members (SK1–4). These channels are widely expressed throughout the organism and involved in various cellular processes, such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date, the role of SK channels in developmental processes has been merely a marginal focus of investigation, although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently, several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein, we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells, neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly, we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro . The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis.

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.

International audience; 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/Shank3αβ(-/-) 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.

Abelson interactor protein 1 (Abi-1) localizes to postsynaptic densities (PSDs) of excitatory synapses and was shown to be transported from the PSD to the nucleus and back depending upon synaptic activation. We employed a yeast-two-hybrid screen to search for putative transport molecules. We found Kif26B a member of the Kif family of transport proteins that has not been characterized in the central nervous system as a direct interaction partner of Abi-1. We delineated a proline-rich motif within the cargo-binding domain of Kif26B to be responsible for this protein–protein interaction. Kif26B was able to recruit Abi-1 to the microtubule network and we found that the expression of Kif26B is responsible for the localization of Abi-1 to PSDs in maturing neurons. Taken together we report that Abi-1 is a cargo of Kif26B in primary hippocampal neurons, pointing to a role of this transport molecule in the movement of Abi-1 between different cell compartments. Additionally, we provide the first detailed investigation of Kif26B and its cargo molecules in neuronal cells.

Scaffold proteins are abundant and essential components of the postsynaptic density (PSD). They play a major role in many synaptic functions including the trafficking, anchoring, and clustering of glutamate receptors and adhesion molecules. Moreover, they link postsynaptic receptors with their downstream signaling proteins and regulate the dynamics of cytoskeletal structures. By definition, PSD scaffold proteins do not have intrinsic enzymatic activities but are formed by modular and specific domains deputed to form large protein networks. Here, we will discuss the latest findings regarding the structure and functions of major PSD scaffold proteins. Given that scaffold proteins are central components of PSD architecture, it is not surprising that deletion or mutations in their human genes cause severe neuropsychiatric disorders including autism, mental retardation, and schizophrenia. Thus, their dynamic organization and regulation are directly correlated with the essential structure of the PSD and the normal physiology of neuronal synapses.

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.

Scaffold proteins are abundant and essential components of the postsynaptic density (PSD). They play a major role in many synaptic functions including the trafficking, anchoring, and clustering of glutamate receptors and adhesion molecules. Moreover, they link postsynaptic receptors with their downstream signaling proteins and regulate the dynamics of cytoskeletal structures. By definition, PSD scaffold proteins do not have intrinsic enzymatic activities but are formed by modular and specific domains deputed to form large protein networks. Here, we will discuss the latest findings regarding the structure and functions of major PSD scaffold proteins.

Over the last decade, mutations of genes coding for synaptic proteins including postsynaptic ProSAP/Shank scaffolds, were found to play a central role in autism pathogenesis. Strikingly, alterations within the human genes of all three ProSAP/Shank family members called SHANK1, PROSAP1/SHANK2 and PROSAP2/SHANK3 have been detected in patients with Autism Spectrum Disorders (ASDs). Due to the fact, that the patho-mechanisms caused by those genetic alterations are still far from being understood and that the development of therapeutic options crucially relies on the latter understanding, the generation and thorough analysis of animal models is an essential step. Here, we review existing mouse models of Shank1 and ProSAP1/Shank2 disruption with respect to neurobiological, neurophysiological and neurobehavioral phenotypes and give some future directions towards the conception of therapeutic strategies.

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 (n = 396 patients and n = 659 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 (P = 0.004, OR = 2.37, 95% CI = 1.23–4.70). 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 (P = 0.0013). 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., Author Summary Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While mutations in several genes have been identified in patients with ASD, little is known about their effects on neuronal function and their interaction with other genetic variations. Using a combination of genetic and functional approaches, we identified novel SHANK2 mutations including a de novo loss of one copy of the SHANK2 gene in a patient with autism and several mutations observed in patients that reduced neuronal cell contacts in vitro. Further genomic analysis of three patients carrying de novo SHANK2 deletions identified additional rare genomic imbalances previously associated with neuropsychiatric disorders. Taken together, 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.

One of the major functional properties of the mature motoneuron is its ability to generate and conduct signals from the central nervous system (CNS) to the peripheral muscle cell in order to induce and control muscle contraction [1]. The molecular composition of the neuromuscular junction (NMj) is crucial for its function and maintenance whereas dysregulation of endplate physiology is considered to be involved in denervation of the muscle cells and subsequent motoneuron degeneration [2, 3]. At early developmental stages of the neuron-to-muscle synaptogenesis, a large number of spinal motoneurons die, presumably because they fail to form adequate connections with the target muscle. In fact, if the limb bud (the precursor of limb muscles) is removed before the formation of neuromuscular connections all the corresponding motoneurons eventually degenerate [4]. In vitro, various cell systems are utilized to search for developmental and functional characteristics of the motoneuron system. Both, primary cultures and stem cellderived motoneurons are used for various questions. Pluripotent embryonic stem cells [5] from mouse and human origin [6] had been shown to be able to generate motoneurons in vitro. Since the first discovery and invention of the induced pluripotent stem cell (iPSC) technology by Takahashi and Yamanaka [7], it is now possible to analyze and study cell development and differentiation on the basis of a gene defect in patient specific settings [8, 9]. For comparison all these studies are crucially dependent upon the analysis of human cell differentiation, morphology and protein expression under

Intact synaptic homeostasis is a fundamental prerequisite for a healthy brain. Thus, it is not surprising that altered synaptic morphology and function are involved in the molecular pathogenesis of so-called synaptopathies including autism, schizophrenia (SCZ) and Alzheimer's disease (AD). Intriguingly, various recent studies revealed a crucial role of postsynaptic ProSAP/Shank scaffold proteins in all of the aforementioned disorders. Considering these findings, we follow the hypothesis that ProSAP/Shank proteins are key regulators of synaptic development and plasticity with clear-cut isoform-specific roles. We thus propose a model where ProSAP/Shank proteins are in the center of a postsynaptic signaling pathway that is disrupted in several neuropsychiatric disorders.

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.

BackgroundThe 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 findingsWe 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.ConclusionsOur 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.

Intact synaptic function and plasticity are fundamental prerequisites to a healthy brain. Therefore, synaptic proteins are one of the major targets for drugs used as neuro-chemical therapeutics. Unfortunately, the majority of drugs is not able to cross the blood-brain barrier (BBB) and is therefore distributed within the CNS parenchyma. Here, we report the development of novel biodegradable Nanoparticles (NPs), made of poly-lactide-co-glycolide (PLGA) conjugated with glycopeptides that are able to cross the BBB and deliver for example Zn(2+) ions. We also provide a thorough characterization of loaded and unloaded NPs for their stability, cellular uptake, release properties, toxicity, and impact on cell trafficking. Our data reveal that these NPs are biocompatible, and can be used to elevate intracellular levels of Zn(2+). Importantly, by engineering the surface of NPs with antibodies against NCAM1 and CD44, we were able to selectively target neurons or glial cells, respectively. Our results indicate that these biodegradable NPs provide a potential new venue for the delivery Zn(2+) to the CNS and thus a means to explore the influence of altered zinc levels linked to neuropsychological disorders such as depression.

Members of the ProSAP/Shank family are important scaffolding proteins of the postsynaptic density (PSD). We investigated for the first time the expression of the three family members named Shank1, ProSAP1/Shank2, and ProSAP2/Shank3 during Xenopus laevis development. Shank1 is expressed in the neural tube, the retina, and the cranial ganglions. In contrast, ProSAP1/Shank2 transcripts could be visualized in the otic vesicle, the pronephros, the liver, the neural tube, and the retina. ProSAP2/Shank3 could be detected in the cardiovascular network, the neural tube, the pronephros, and the retina. Furthermore, we showed that LAPSER1 interacts with all three ProSAP/Shank family members in Xenopus embryos and co-localizes with ProSAP/Shank in a cell-based assay. In Xenopus, LAPSER1 is expressed in somites, brain, proctodeum, pronephros, and in some cranial ganglions. Thus, we suggest that members of the ProSAP/Shank family and LAPSER1 not only play a role in PSD formation and plasticity, but also during embryonic development. Developmental Dynamics 240:1528–1536, 2011. © 2011 Wiley-Liss, Inc.

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells.Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation.Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus.Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.

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.

Recent studies indicate that the selective serotonin reuptake inhibitor (SSRI) fluoxetine is not solely effective by the instant inhibition of the serotonin transporter (SERT) but also by its influence on mitotic and/or apoptotic processes.To investigate the effects of the compound in vitro, we treated neurons from different brain areas with increasing concentrations of fluoxetine. Additionally, human embryonic kidney (HEK-293) cells and HEK-293 cells stably expressing the SERT were used. Cell viability was quantified by MTT-assay and apoptosis via fluorescence-activated cell-sorting analyses. Fluoxetine's effect on the γ-aminobutyric acid (GABA) receptor was electrophysiologically investigated to test the hypothesis if a GABA-mimetic effect exists that might lead - additionally to the well-known N-methyl-D-aspartate (NMDA)-antagonism - to increased apoptosis in immature neurons.In hippocampal, cortical, and both types of HEK-293 cells, viability decreased and apoptosis increased in a dose-dependent manner (0.5-75 μM). In contrast, in mesencephalic and striatal cells the viability was unchanged or even slightly stimulated up to 20 μM fluoxetine. An anti-apoptotic effect of concentrations below 10 μM was observed in these cells. The GABA(A) receptor was directly activated by fluoxetine.We conclude that fluoxetine affects apoptotic processes independently from SERT expression. Since especially the combined GABA-mimetic and NMDA-antagonistic effects increase apoptosis in developing neuronal cells, whereas both effects are neuroprotective in adult neurons we hypothesise that these mechanisms explain the discrepancy of in vitro and in vivo studies.

Proline-rich synapse-associated protein-1 and 2 (ProSAP1/Shank2 and ProSAP2/Shank3) were originally found as synapse-associated protein 90/postsynaptic density protein-95-associated protein (SAPAP)/guanylate-kinase-associated protein (GKAP) interaction partners and also isolated from synaptic junctional protein preparations of rat brain. They are essential components of the postsynaptic density (PSD) and are specifically targeted to excitatory asymmetric type 1 synapses. Functionally, the members of the ProSAP/Shank family are one of the postsynaptic key elements since they link and attach the postsynaptic signaling apparatus, for example N-methyl-d-aspartic acid (NMDA)-receptors via direct and indirect protein interactions to the actin-based cytoskeleton. The functional significance of ProSAP1/2 for synaptic transmission and the paucity of data with respect to the molecular composition of PSDs of the peripheral nervous system (PNS) stimulated us to investigate neuromuscular junctions (NMJs), synapses of the superior cervical ganglion (SCG), and synapses in myenteric ganglia as representative synaptic junctions of the PNS. Confocal imaging revealed ProSAP1/2-immunoreactivity (-iry) in NMJs of rat and mouse sternomastoid and tibialis anterior muscles. In contrast, ProSAP1/2-iry was only negligibly found in motor endplates of striated esophageal muscle probably caused by antigen masking or a different postsynaptic molecular anatomy at these synapses. ProSAP1/2-iry was furthermore detected in cell bodies and dendrites of superior cervical ganglion neurons and myenteric neurons in esophagus and stomach. Ultrastructural analysis of ProSAP1/2 expression in myenteric ganglia demonstrated that ProSAP1 and ProSAP2 antibodies specifically labelled PSDs of myenteric neurons. Thus, scaffolding proteins ProSAP1/2 were found within the postsynaptic specializations of synapses within the PNS, indicating a similar molecular assembly of central and peripheral postsynapses.

There is increasing evidence that not only the monoaminergic but also the glutamatergic system is involved in the pathophysiology of attention-deficit hyperactivity disorder (ADHD). Hyperactivity of glutamate metabolism might be causally related to a hypoactive state in the dopaminergic system. Atomoxetine, a selective noradrenaline reuptake inhibitor, is the first non-stimulant approved for the treatment of this disorder. Here we have evaluated the effects of atomoxetine on glutamate receptors in vitro.The whole-cell configuration of the patch-clamp technique was used to analyse the effect of atomoxetine on N-methyl-d-aspartate (NMDA) receptors in cultured rodent cortical and hippocampal neurons as well as on NMDA receptors heterologously expressed in human TsA cells.Atomoxetine blocked NMDA-induced membrane currents. Half-maximal inhibition emerged at about 3 microM which is in the range of clinically relevant concentrations found in plasma of patients treated with this drug. The inhibition was voltage-dependent, indicating an open-channel blocking mechanism. Furthermore, the inhibitory potency of atomoxetine did not vary when measured on NMDA receptors from different brain regions or with different subunit compositions.The effective NMDA receptor antagonism by atomoxetine at low micromolar concentrations may be relevant to its clinical effects in the treatment of ADHD. Our data provide further evidence that altered glutamatergic transmission might play a role in ADHD pathophysiology.

The dissection course (DC) is an essential part of the preclinical medical curriculum that mediates professionalism. The process of dissecting, however, has an inherent additional stress potential. Our study determined student mental stress, their need of psychological support and factors influencing this need. A quantitative longitudinal query before, during and after the DC was performed including the Brief Symptom Inventory (BSI) as well as self-formulated questions used a 5-point Likert scale. Half of the students who anticipated dissection to be a stress factor reported that this declined significantly over time. Instead, student fear of not being able to cope with the work load increased significantly. As many as 64% of the students favored psychological support on the first course day, while 75% rejected this during the period of dissection and 39% appreciated this after the course. Moreover, 42% emphasized the importance of the funeral ceremony. Additionally, 75% documented their need for support in coping with stress and learning strategies. Gender, previous medical training, and BSI levels were identified as psychosocial influence factors. A majority of students named friends, members of their family or workmates as partners with whom they could talk about mental stress. Our results document the need to develop an optimum support during the DC taking into account the ascertained indicators. Exemplarily the Institute of Anatomy and Cell Biology at Ulm University suggests several options like a step by step approach for optimization. These measures reduce mental stress and help students to cope with it by the development of "detached concern" towards their "first patient" as this will decisively influence their future professional behavior.

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.

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.

IRSp53 is an essential intermediate between the activation of Rac and Cdc42 GTPases and the formation of cellular protrusions; it affects cell shape by coupling membrane-deforming activity with the actin cytoskeleton. IRSp53 is highly expressed in neurons where it is also an abundant component of the postsynaptic density (PSD). Here we analyze the physiological function of this protein in the mouse brain by generating IRSp53-deficient mice. Neurons in the hippocampus of young and adult knock-out (KO) mice do not exhibit morphological abnormalities in vivo. Conversely, primary cultured neurons derived from IRSp53 KO mice display retarded dendritic development in vitro. On a molecular level, Eps8 cooperates with IRSp53 to enhance actin bundling and interacts with IRSp53 in developing neurons. However, postsynaptic Shank proteins which are expressed at high levels in mature neurons compete with Eps8 to block actin bundling. In electrophysiological experiments the removal of IRSp53 increases synaptic plasticity as measured by augmented long term potentiation and paired-pulse facilitation. A primarily postsynaptic role of IRSp53 is underscored by the decreased size of the PSDs, which display increased levels of N-methyl-d-aspartate receptor subunits in IRSp53 KO animals. Our data suggest that the incorporation of IRSp53 into the PSD enables the protein to limit the number of postsynaptic glutamate receptors and thereby affect synaptic plasticity rather than dendritic morphology. Consistent with altered synaptic plasticity, IRSp53-deficient mice exhibit cognitive deficits in the contextual fear-conditioning paradigm.

Correction for: Dieterich DC, Karpova A, Mikhaylova M, Zdobnova I, Konig I, et al. (2008) Caldendrin–Jacob: A protein liaison that couples NMDA receptor signalling to the nucleus. PLoS Biol 6(2): e34. doi:10.1371/journal.pbio.0060034 The version of Figure 3 originally published in this article was not the one intended for publication, and therefore does not correspond to the accompanying legend. The correct version of Figure 3 is published below. Figure 3 Jacob's Association with Subcellular Structures Additionally, the authors would like to clarify the legend that accompanies Figure 3. The original final sentence is ambiguous, and should be replaced by “Jacob is only present in fraction P, which contains RNA Polymerase II and represents the euchromatin.”

The ability of the mammalian brain to store and recall information is based on synaptic plasticity due to constant remodeling of synaptic contacts. Although various classes of proteins such as neurotransmitter receptors, cytoskeletal components and protein kinases were already identified as modulators of memory formation, their specific interactions and crosstalks are still poorly understood. Genetic variants of the scaffolding protein KIBRA (kidney brain) a substrate of the memory-related protein kinase C zeta and component of the neuronal cytoskeleton, were recently shown to be associated with human memory performance. However, the function of KIBRA on the cellular and physiological level is still unclear. To gain more insights into the temporal and spatial expression of KIBRA, we performed in situ hybridization assays and immunohistological staining of human and rodent (rat) brain. Our data demonstrate that KIBRA is mainly expressed in memory-related regions of the brain (hippocampus, cortex) but is also found in the cerebellum and the hypothalamus. In primary hippocampal neurons, KIBRA displays a somatodendritic distribution and an enrichment at the postsynaptic density. Binding studies further show that KIBRA is able to form head-to-tail homodimers and that dimerization is mediated by the internal C2-like domain. Our data indicate that KIBRA is involved in brain development and memory formation as a postsynaptic scaffold protein connecting cytoskeletal and signaling molecules.

Summary Because of a new legislation concerning fees for study in Germany, novel financial opportunities for improvement of medical education have been provided. Some of these additional funds were invested at Ulm University in the Institute of Anatomy and Cell Biology to realize a teaching operating theatre similar to the ancient anatomical theatre in an amazingly short period of time. The anatomical theatre is a unique teaching facility in Germany, a direct implementation of joint preclinical and clinical education in the medical curriculum as required by the new Federal Medical Licensing Regulations (Approbationsordnung). It was set up in January of 2008. In this teaching operating theatre students and doctors will meet to study anatomical structures. In a simulated operating setting, surgeons will demonstrate invasive procedures and operative techniques on donated bodies. About 50 students can be positioned on a grandstand at a short distance to the operating table to follow the “operation” or anatomical demonstrations. Students can also learn how to behave in a sterile environment before they are actually confronted with this new situation during their first clinical clerkship. The anatomical theatre is supposed to be integrated in preclinical and clinical medical education as well as in advanced professional education for suitable sub-specialties. We are confident that the anatomical theatre will further improve students’ motivation and their efforts in gaining knowledge in the field of gross anatomy because of its clinical relevance.

In recent years, magnetic resonance imaging (MRI) has emerged as a preferred tool for the diagnosis of amyotrophic lateral sclerosis (ALS) in humans. A widely used animal model for human ALS is the G93A-superoxide dismutase 1 (G93A-SOD1) transgenic mouse model. However, the mechanisms for the selective degeneration of motor neurons in the brainstem and spinal cord are still uncertain. In our study, we applied MRI at 4.7 Tesla to non-invasively evaluate pathological alterations in the brainstem of this animal model and to follow the progression of the disease. Extending previous investigation, we used the relaxation parameter T(2) as a suitable measure for the progression of ALS, and evaluated the potential agreement with histological evaluation and behavioural data of open-field tests. In the brainstem of G93A-SOD1 mice, T(2) values were significantly increased in the motor nuclei Nc. V, Nc. VII and Nc. XII, as early as Day 80, i.e. before the average disease onset at about Day 90. Moreover, this increase is associated with a progressive development of vacuoles in the brainstem motor nuclei and a significantly decreased performance in behavioural tests. Overall, MRI is a very sensitive tool to obtain correlates for neuronal degeneration in vivo. Furthermore, MRI enables us to investigate a follow up at different time points of the disease. These advantages are especially useful for therapeutic studies with respect to survival rates of motor neurons using mouse models. Finally, our data suggest that MRI does not only resemble the findings of behavioural tests, but is potentially superior to behavioural studies.

NSCs are found in the developing brain, as well as in the adult brain. They are self-renewing cells that maintain the capacity to differentiate into all major brain-specific cell types, such as glial cells and neurons. However, it is still unclear whether these cells are capable of gaining full functionality, which is one of the major prerequisites for NSC-based cell replacement strategies of neurological diseases. The ability to establish and maintain polarized excitatory synaptic contacts would be one of the basic requirements for intercellular communication and functional integration into existing neuronal networks. In primary cultures of hippocampal neurons, it has already been shown that synaptogenesis is characterized by a well-ordered, time-dependent targeting and recruitment of pre- and postsynaptic proteins. In this study, we investigated the expression and localization of important pre- and postsynaptic proteins, including Bassoon and synaptophysin, as well as proteins of the ProSAP/Shank family, in differentiating rat fetal mesencephalic NSCs. Moreover, we analyzed the ultrastructural features of neuronal cell-cell contacts during synaptogenesis. We show that NSCs express and localize cytoskeletal and scaffolding molecules of the pre- and postsynaptic specializations in a well-defined temporal order, leading to mature synaptic contacts after 14 days of differentiation. The temporal and spatial pattern of synaptic maturation is comparable to synaptogenesis of hippocampal neurons grown in primary culture. Therefore, with respect to the general ability to create mature synaptic contacts, NSCs seem to be well equipped to potentially compensate for lost or injured brain tissue. Disclosure of potential conflicts of interest is found at the end of this article.

Glutamatergic synapses in the central nervous system are characterized by an electron-dense web underneath the postsynaptic membrane; this web is called the postsynaptic density (PSD). PSDs are composed of a dense network of several hundred proteins, creating a macromolecular complex that serves a wide range of functions. Prominent PSD proteins such as members of the MaGuk or ProSAP/Shank family build up a dense scaffold that creates an interface between clustered membrane-bound receptors, cell adhesion molecules and the actin-based cytoskeleton. Moreover, kinases, phosphatases and several proteins of different signalling pathways are specifically localized within the spine/PSD compartment. Small GTPases and regulating proteins are also enriched in PSDs being the molecular basis for regulated structural changes of cytoskeletal components within the synapse in response to external or internal stimuli, e.g. synaptic activation. This synaptic rearrangement (structural plasticity) is a rapid process and is believed to underlie learning and memory formation. The characterization of synapse/PSD proteins is especially important in the light of recent data suggesting that several mental disorders have their molecular defect at the synapse/PSD level.

The postsynaptic density (PSD) is a complex assembly of proteins associated with the postsynaptic membrane that organizes neurotransmitter receptors, signaling pathways, and regulatory elements within a cytoskeletal matrix. Here we show that the sterile alpha motif domain of rat Shank3/ProSAP2, a master scaffolding protein located deep within the PSD, can form large sheets composed of helical fibers stacked side by side. Zn 2+ , which is found in high concentrations in the PSD, binds tightly to Shank3 and may regulate assembly. Sheets of the Shank protein could form a platform for the construction of the PSD complex.

Glutamate receptor interacting protein (GRIP) homologues, initially characterized in synaptic glutamate receptor trafficking, consist of seven PDZ domains (PDZDs), whose conserved arrangement is of unknown significance. The Drosophila GRIP homologue (DGrip) is needed for proper guidance of embryonic somatic muscles towards epidermal attachment sites, with both excessive and reduced DGrip activity producing specific phenotypes in separate muscle groups. These phenotypes were utilized to analyze the molecular architecture underlying DGrip signaling function in vivo. Surprisingly, removing PDZDs 1-3 (DGripDelta1-3) or deleting ligand binding in PDZDs 1 or 2 convert DGrip to excessive in vivo activity mediated by ligand binding to PDZD 7. Yeast two-hybrid screening identifies the cell adhesion protein Echinoid's (Ed) type II PDZD-interaction motif as binding PDZDs 1, 2 and 7 of DGrip. ed loss-of-function alleles exhibit muscle defects, enhance defects caused by reduced DGrip activity and suppress the dominant DGripDelta1-3 effect during embryonic muscle formation. We propose that Ed and DGrip form a signaling complex, where competition between N-terminal and the C-terminal PDZDs of DGrip for Ed binding controls signaling function.

Recent studies suggest that central nervous system synapses can persist for weeks, months, perhaps lifetimes, yet little is known as to how synapses maintain their structural and functional characteristics for so long. As a step toward a better understanding of synaptic maintenance we examined the loss, redistribution, reincorporation, and replenishment dynamics of Synapsin I and ProSAP2/Shank3, prominent presynaptic and postsynaptic matrix molecules, respectively. Fluorescence recovery after photobleaching and photoactivation experiments revealed that both molecules are continuously lost from, redistributed among, and reincorporated into synaptic structures at time-scales of minutes to hours. Exchange rates were not affected by inhibiting protein synthesis or proteasome-mediated protein degradation, were accelerated by stimulation, and greatly exceeded rates of replenishment from somatic sources. These findings indicate that the dynamics of key synaptic matrix molecules may be dominated by local protein exchange and redistribution, whereas protein synthesis and degradation serve to maintain and regulate the sizes of local, shared pools of these proteins.

Proline-rich synapse-associated protein-1 (ProSAP1) is a neuronal PDZ domain-containing protein that has recently been identified as an essential element of the postsynaptic density. Via its interaction with the actin-binding protein cortactin and its integrative function in the organization of neurotransmitter receptors, ProSAP1 is believed to be involved in the linkage of the postsynaptic signaling machinery to the actin-based cytoskeleton, and may play a role in the cytoskeletal rearrangements that underlie synaptic plasticity. As a result of our ongoing studies on the distribution and function of this novel PDZ domain protein, we now report that the expression of ProSAP1 is restricted neither to neurons and interneuronal junctions nor to the nervous system. Using immunohistochemical techniques in conjunction with specific antibodies, we found that, in the CNS, ProSAP1 can be detected in certain glial cells, such as ependymal cells, tanycytes, subpial/radial astrocytes, and in the choroid plexus epithelium. Moreover, our immunohistochemical analyses revealed the presence of ProSAP1 in endocrine cells of the adenohypophysis and of the pancreas, as well as in non-neuronal cell types of other organs. In the pancreas, ProSAP1 immunoreactivity was also localized in the duct system of the exocrine parenchyma. Our findings demonstrate that, in addition to neurons, ProSAP1 is present in various non-neuronal cells, in which it may play a crucial role in the dynamics of the actin-based cytoskeleton. (J Histochem Cytochem 49:639–648, 2001)

The postsynaptic density is a highly dynamic structure, which is reorganized in an activity-dependent manner. An animal model for temporal lobe epilepsy, i.e. kainate-induced limbic seizures in rats, was used to study changes in postsynaptic density composition after extensive synaptic activity. Six hours after kainate injection, the protein content of the postsynaptic density fractions from rats that developed strong seizures was increased three-fold compared to saline-treated controls. Immunoblot analysis revealed that the relative amounts of metabotropic glutamate receptor 1alpha, N-ethylmaleimide-sensitive fusion protein, protein kinases C, Fyn and TrkB, as well as the neuronal nitric oxide synthase, were significantly higher in seizure-developing than in control rats. In contrast, the relative contents of the kainate receptor KA2 subunit, beta-actin, alpha-adducin and the membrane-associated guanylate kinase homolog SAP90/PSD-95 were decreased. The relative amounts of additional postsynaptic density proteins, including alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl-D-aspartate receptor subunits, calcium/calmodulin-dependent kinase type II, casein kinase 2, tubulin, microtubule-associated protein 2B, the membrane-associated guanylate kinase homolog SAP102, and proline-rich synapse-associated protein 1/cortactin binding protein 1/Shank2 remained essentially unchanged. To assess possible changes in postsynaptic performance, postsynaptic densities were isolated from control and epileptic rats, incorporated into giant liposomes and N-methyl-D-aspartate receptor currents were recorded. A significant reduction in the mean conductance was observed in patches containing postsynaptic densities from animals with high seizure activity. This was due to the presence of reduced conductance levels in each membrane patch compared to control postsynaptic density preparations. From these data, we suggest that intense synaptic activity associated with seizures modifies the composition of postsynaptic densities and has profound consequences on the function of the N-methyl-D-aspartate receptors present in them. This rearrangement may accompany impairment of synaptic plasticity.

Neurocan is a component of the extracellular matrix in brain. Due to its inhibition of neuronal adhesion and outgrowth in vitro and its expression pattern in vivo it was suggested to play an important role in axon guidance and neurite growth. To study the role of neurocan in brain development we generated neurocan-deficient mice by targeted disruption of the neurocan gene. These mice are viable and fertile and have no obvious deficits in reproduction and general performance. Brain anatomy, morphology, and ultrastructure are similar to those of wild-type mice. Perineuronal nets surrounding neurons appear largely normal. Mild deficits in synaptic plasticity may exist, as maintenance of late-phase hippocampal long-term potentiation is reduced. These data indicate that neurocan has either a redundant or a more subtle function in the development of the brain.

The postsynaptic density (PSD) is crucially involved in the structural and functional organization of the postsynaptic neurotransmitter reception apparatus. Using antisera against rat brain synaptic junctional protein preparations, we isolated cDNAs coding for proline-rich synapse-associated protein-1 (ProSAP1), a PDZ-domain protein. This protein was found to be identical to the recently described cortactin-binding protein-1 (CortBP1). Homology screening identified a related protein, ProSAP2. Specific antisera raised against a C-terminal fusion construct and a central part of ProSAP1 detect a cluster of immunoreactive bands of 180 kDa in the particulate fraction of rat brain homogenates that copurify with the PSD fraction. Transcripts and immunoreactivity are widely distributed in the brain and are upregulated during the period of synapse formation in the brain. In addition, two short N-terminal insertions are detected; they are differentially regulated during brain development. Confocal microscopy of hippocampal neurons showed that ProSAP1 is predominantly localized in synapses, and immunoelectron microscopyin siturevealed a strong association with PSDs of hippocampal excitatory synapses. The accumulation of ProSAP1 at synaptic structures was analyzed in the developing cerebral cortex. During early postnatal development, strong immunoreactivity is detectable in neurites and somata, whereas from postnatal day 10 (P10) onward a punctate staining is observed. At the ultrastructural level, the immunoreactivity accumulates at developing PSDs starting from P8. Both interaction with the actin-binding protein cortactin and early appearance at postsynaptic sites suggest that ProSAP1/CortBP1 may be involved in the assembly of the PSD during neuronal differentiation.

Bassoon is a novel 420-kDa protein recently identified as a component of the cytoskeleton at presynaptic neurotransmitter release sites. Analysis of the rat and mouse sequences revealed a polyglutamine stretch in the C-terminal part of the protein. Since it is known for some proteins that abnormal amplification of such polyglutamine regions can cause late-onset neurodegeneration, we cloned and localized the human BASSOON gene (BSN). Phage clones spanning most of the open reading frame and the 3' untranslated region were isolated from a human genomic library and used for chromosomal localization of BSN to chromosome 3p21 by FISH. The localization was confirmed by PCR on rodent/human somatic cell hybrids; it is consistent with the localization of the murine Bsn gene at chromosome 9F. Sequencing revealed a polyglutamine stretch of only five residues in human, and PCR amplifications from 50 individuals showed no obvious length polymorphism in this region. Analysis of the primary structure of Bassoon and comparison to previous database entries provide evidence for a newly emerging protein family.

Patrick T Udvardi,1,2 Karl J Föhr,3 Carolin Henes,1,2 Stefan Liebau,2 Jens Dreyhaupt,4 Tobias M Boeckers,2 Andrea G Ludolph11Department of Child and Adolescent Psychiatry and Psychotherapy, 2Institute of Anatomy and Cell Biology, 3Department of Anaesthesiology, 4Institute of Epidemiology and Medical Biometry, University of Ulm, Ulm, GermanyAbstract: Attention-deficit/hyperactivity disorder (ADHD) is the most frequently diagnosed neurodevelopmental disorder. The norepinephrine transporter (NET) inhibitor atomoxetine, the first nonstimulant drug licensed for ADHD treatment, also acts as an N-methyl-D-aspartate receptor (NMDAR) antagonist. The compound's effects on gene expression and protein levels of NET and NMDAR subunits (1, 2A, and 2B) are unknown. Therefore, adolescent Sprague Dawley rats were treated with atomoxetine (3 mg/kg, intraperitoneal injection [ip]) or saline (0.9%, ip) for 21 consecutive days on postnatal days (PND) 21–41. In humans, atomoxetine's earliest clinical therapeutic effects emerge after 2–3 weeks. Material from prefrontal cortex, striatum (STR), mesencephalon (MES), and hippocampus (HC) was analyzed either directly after treatment (PND 42) or 2 months after termination of treatment (PND 101) to assess the compound's long-term effects. In rat brains analyzed immediately after treatment, protein analysis exhibited decreased levels of the NET in HC, and NMDAR subunit 2B in both STR and HC; the transcript levels were unaltered. In rat brains probed 2 months after final atomoxetine exposure, messenger RNA analysis also revealed significantly reduced levels of genes coding for NMDAR subunits in MES and STR. NMDAR protein levels were reduced in STR and HC. Furthermore, the levels of two SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, synaptophysin and synaptosomal-associated protein 25, were also significantly altered in both treatment groups. This in vivo study detected atomoxetine's effects beyond NET inhibition. Taken together, these data reveal that atomoxetine seems to decrease glutamatergic transmission in a brain region-specific manner. Long-term data show that the compound's impact is not due to an acute pharmacological effect but lasts or even amplifies after a drug-free period of 2 months, leading to altered development of synaptic composition. These alterations might contribute to atomoxetine's clinical effects in the treatment of ADHD, a neurodevelopmental disorder in which synaptic processes and especially a dysregulated glutamatergic metabolism seem to be involved.Keywords: attention-deficit hyperactivity disorder (ADHD), neurodevelopment, atomoxetine, in vivo study, altered gene expression, N-methyl-D-aspartate receptor