Christian Proepper

Genetic and Functional Analyses of SHANK2 Mutations Suggest a Multiple Hit Model of Autism Spectrum Disorders

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.

Concerted action of zinc and ProSAP/Shank in synaptogenesis and synapse maturation

Neuronal morphology and number of synapses is not static, but can change in response to a variety of factors, a process called synaptic plasticity. These structural and molecular changes are believed to represent the basis for learning and memory, thereby underling both the developmental and activity‐dependent remodelling of excitatory synapses. Here, we report that Zn2+ ions, which are highly enriched within the postsynaptic density (PSD), are able to influence the recruitment of ProSAP/Shank proteins to PSDs in a family member‐specific manner during the course of synaptogenesis and synapse maturation. Through selectively overexpressing each family member at excitatory postsynapses and comparing this to shRNA‐mediated knockdown, we could demonstrate that only the overexpression of zinc‐sensitive ProSAP1/Shank2 or ProSAP2/Shank3 leads to increased synapse density, although all of them cause a decrease upon knockdown. Furthermore, depletion of synaptic Zn2+ along with the knockdown of zinc‐insensitive Shank1 causes the rapid disintegration of PSDs and the loss of several postsynaptic molecules including Homer1, PSD‐95 and NMDA receptors. These findings lead to the model that the concerted action of ProSAP/Shank and Zn2+ is essential for the structural integrity of PSDs and moreover that it is an important element of synapse formation, maturation and structural plasticity.

Abelson interacting protein 1 (Abi-1) is essential for dendrite morphogenesis and synapse formation

Synaptogenesis and synaptic plasticity depend crucially on the dynamic and locally specific regulation of the actin cytoskeleton. We identified an important component for controlled actin assembly, abelson interacting protein‐1 (Abi‐1), as a binding partner for the postsynaptic density (PSD) protein ProSAP2/Shank3. During early neuronal development, Abi‐1 is localized in neurites and growth cones; at later stages, the protein is enriched in dendritic spines and PSDs, as are components of a trimeric complex consisting of Abi‐1, Eps8 and Sos‐1. Abi‐1 translocates upon NMDA application from PSDs to nuclei. Nuclear entry depends on abelson kinase activity. Abi‐1 co‐immunoprecipitates with the transcription factor complex of Myc/Max proteins and enhances E‐box‐regulated gene transcription. Downregulation of Abi‐1 by small interfering RNA results in excessive dendrite branching, immature spine and synapse morphology and a reduction of synapses, whereas overexpression of Abi‐1 has the opposite effect. Data show that Abi‐1 can act as a specific synapto‐nuclear messenger and is essentially involved in dendrite and synapse formation.

Modulation of Calcium-Activated Potassium Channels Induces Cardiogenesis of Pluripotent Stem Cells and Enrichment of Pacemaker-Like Cells

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 Ca2+-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.

ProSAP-interacting Protein 1 (ProSAPiP1), a Novel Protein of the Postsynaptic Density That Links the Spine-associated Rap-Gap (SPAR) to the Scaffolding Protein ProSAP2/Shank3

ProSAPs/Shanks are a family of proteins that have a major scaffolding function for components of the postsynaptic density (PSD) of excitatory brain synapses. Members of the family harbor a variety of domains for protein-protein interactions, one of which is a unique PDZ domain that differs significantly from those of other proteins. We have identified a novel binding partner for this PDZ domain, termed ProSAPiP1, that is highly enriched in the PSD and shares significant sequence homology with the PSD protein PSD-Zip70. Both molecules code for a Fez1 domain that can be found in a total of four related proteins. ProSAPiP1 is widely expressed in rat brain and co-localizes with ProSAP2/Shank3 in excitatory spines and synapses. ProSAP2/Shank3 co-immunoprecipitates with ProSAPiP1 but not with PSD-Zip70. Both proteins, however, bind and recruit SPAR to synapses with a central coiled-coil region that harbors a leucine zipper motif. This region is also responsible for homo- and heteromultimerization of ProSAPiP1 and PSD-Zip70. Thus, ProSAPiP1 and PSD-Zip70 are founders of a novel family of scaffolding proteins, the “Fezzins,” which adds further complexity to the organization of the PSD protein network.

An SK3 Channel/nWASP/Abi-1 Complex Is Involved in Early Neurogenesis

Background The stabilization or regulated reorganization of the actin cytoskeleton is essential for cellular structure and function. Recently, we could show that the activation of the SK3-channel that represents the predominant SK-channel in neural stem cells, leads to a rapid local outgrowth of long filopodial processes. This observation indicates that the rearrangement of the actin based cytoskeleton via membrane bound SK3-channels might selectively be controlled in defined micro compartments of the cell. Principal Findings We found two important proteins for cytoskeletal rearrangement, the Abelson interacting protein 1, Abi-1 and the neural Wiskott Aldrich Syndrome Protein, nWASP, to be in complex with SK3- channels in neural stem cells (NSCs). Moreover, this interaction is also found in spines and postsynaptic compartments of developing primary hippocampal neurons and regulates neurite outgrowth during early phases of differentiation. Overexpression of the proteins or pharmacological activation of SK3 channels induces obvious structural changes in NSCs and hippocampal neurons. In both neuronal cell systems SK3 channels and nWASP act synergistic by strongly inducing filopodial outgrowth while Abi-1 behaves antagonistic to its interaction partners. Conclusions Our results give good evidence for a functional interplay of a trimeric complex that transforms incoming signals via SK3-channel activation into the local rearrangement of the cytoskeleton in early steps of neuronal differentiation involving nWASP and Abi-1 actin binding proteins.

Formation of cellular projections in neural progenitor cells depends on SK3 channel activity

Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1‐ethyl‐2‐benzimidazolinone (1‐EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50 = 2 ± 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+‐channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non‐differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1‐EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre‐incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels.

The PSD protein ProSAP2/Shank3 displays synapto-nuclear shuttling which is deregulated in a schizophrenia-associated mutation.

Recently, mutations in ProSAP2/Shank3 have been discovered as one of the genetic factors for schizophrenia (SCZ). Here, we show that the postsynaptic density protein ProSAP2/Shank3 undergoes activity dependent synapse-to-nucleus shuttling in hippocampal neurons. Our study shows that the de novo mutation (R1117X) in ProSAP2/Shank3 that was identified in a patient with SCZ leads to an accumulation of mutated ProSAP2/Shank3 within the nucleus independent of synaptic activity. Furthermore, we identified novel nuclear ProSAP2/Shank3 interaction partners. Nuclear localization of mutated ProSAP2/Shank3 alters transcription of several genes, among them already identified genetic risk factors for SCZ such as Synaptotagmin 1 and LRRTM1. Comparing the SCZ mutation of ProSAP2/Shank3 to the knockdown of ProSAP2/Shank3 we found some shared features such as reduced synaptic density in neuronal cultures. However, hippocampal neurons expressing the ProSAP2/Shank3 SCZ mutation furthermore show altered E/I ratio and reduced dendritic branching. Thus, we conclude that the uncoupling of ProSAP2/Shank3 nuclear shuttling from synaptic activity may represent a molecular mechanism that contributes to the pathology of SCZ in patients with mutations in ProSAP2/Shank3.

Heterogeneous nuclear ribonucleoprotein k interacts with Abi-1 at postsynaptic sites and modulates dendritic spine morphology.

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.

Developmental and Functional Nature of Human iPSC Derived Motoneurons

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