JNK activity modulates postsynaptic scaffold protein SAP102 and kainate receptor dynamics in dendritic spines.

Synapse formation depends on the coordinated expression and regulation of scaffold proteins. The JNK family kinases play a role in scaffold protein regulation, but the nature of this functional interaction in dendritic spines requires further investigation. Here, using a combination of biochemical methods and live-cell imaging strategies, we show that the dynamics of the synaptic scaffold molecule SAP102 are negatively regulated by JNK inhibition, that SAP102 is a direct phosphorylation target of JNK3, and that SAP102 regulation by JNK is restricted to neurons that harbor mature synapses. We further demonstrate that SAP102 and JNK3 cooperate in the regulated trafficking of kainate receptors to the cell membrane. Specifically, we observe that SAP102, JNK3, and the kainate receptor subunit GluK2 exhibit overlapping expression at synaptic sites and that modulating JNK activity influences the surface expression of the kainate receptor subunit GluK2 in a neuronal context. We also show that SAP102 participates in this process in a JNK-dependent fashion. In summary, our data support a model in which JNK-mediated regulation of SAP102 influences the dynamic trafficking of kainate receptors to postsynaptic sites, and thus shed light on common pathophysiological mechanisms underlying the cognitive developmental defects associated with diverse mutations.

Extending the allelic spectrum at noncoding risk loci of orofacial clefting.

Genome-wide association studies (GWAS) have generated unprecedented insights into the genetic etiology of orofacial clefting (OFC). The moderate effect sizes of associated noncoding risk variants and limited access to disease-relevant tissue represent considerable challenges for biological interpretation of genetic findings. As rare variants with stronger effect sizes are likely to also contribute to OFC, an alternative approach to delineate pathogenic mechanisms is to identify private mutations and/or an increased burden of rare variants in associated regions. This report describes a framework for targeted resequencing at selected noncoding risk loci contributing to nonsyndromic cleft lip with/without cleft palate (nsCL/P), the most frequent OFC subtype. Based on GWAS data, we selected three risk loci and identified candidate regulatory regions (CRRs) through the integration of credible SNP information, epigenetic data from relevant cells/tissues, and conservation scores. The CRRs (total 57 kb) were resequenced in a multiethnic study population (1061 patients; 1591 controls), using single-molecule molecular inversion probe technology. Combining evidence from in silico variant annotation, pedigree- and burden analyses, we identified 16 likely deleterious rare variants that represent new candidates for functional studies in nsCL/P. Our framework is scalable and represents a promising approach to the investigation of additional congenital malformations with multifactorial etiology.

Nanoscale synapse organization and dysfunction in neurodevelopmental disorders.

Neurodevelopmental disorders such as those linked to intellectual disabilities or autism spectrum disorder are thought to originate in part from genetic defects in synaptic proteins. Single gene mutations linked to synapse dysfunction can broadly be separated in three categories: disorders of transcriptional regulation, disorders of synaptic signaling and disorders of synaptic scaffolding and structures. The recent developments in super-resolution imaging technologies and their application to synapses have unraveled a complex nanoscale organization of synaptic components. On the one hand, part of receptors, adhesion proteins, ion channels, scaffold elements and the pre-synaptic release machinery are partitioned in subsynaptic nanodomains, and the respective organization of these nanodomains has tremendous impact on synaptic function. For example, pre-synaptic neurotransmitter release sites are partly aligned with nanometer precision to postsynaptic receptor clusters. On the other hand, a large fraction of synaptic components is extremely dynamic and constantly exchanges between synaptic domains and extrasynaptic or intracellular compartments. It is largely the combination of the exquisitely precise nanoscale synaptic organization of synaptic components and their high dynamic that allows the rapid and profound regulation of synaptic function during synaptic plasticity processes that underlie adaptability of brain function, learning and memory. It is very tempting to speculate that genetic defects that lead to neurodevelopmental disorders and target synaptic scaffolds and structures mediate their deleterious impact on brain function through perturbing synapse nanoscale dynamic organization. We discuss here how applying super-resolution imaging methods in models of neurodevelopmental disorders could help in addressing this question.

Colony-stimulating factor-1 mediates macrophage-related neural damage in a model for Charcot-Marie-Tooth disease type 1X.

Previous studies in our laboratory have shown that in models for three distinct forms of the inherited and incurable nerve disorder, Charcot-Marie-Tooth neuropathy, low-grade inflammation implicating phagocytosing macrophages mediates demyelination and perturbation of axons. In the present study, we focus on colony-stimulating factor-1, a cytokine implicated in macrophage differentiation, activation and proliferation and fostering neural damage in a model for Charcot-Marie-Tooth neuropathy 1B. By crossbreeding a model for the X-linked form of Charcot-Marie-Tooth neuropathy with osteopetrotic mice, a spontaneous null mutant for colony-stimulating factor-1, we demonstrate a robust and persistent amelioration of demyelination and axon perturbation. Furthermore, functionally important domains of the peripheral nervous system, such as juxtaparanodes and presynaptic terminals, were preserved in the absence of colony-stimulating factor-1-dependent macrophage activation. As opposed to other Schwann cell-derived cytokines, colony-stimulating factor-1 is expressed by endoneurial fibroblasts, as revealed by in situ hybridization, immunocytochemistry and detection of ß-galactosidase expression driven by the colony-stimulating factor-1 promoter. By both light and electron microscopic studies, we detected extended cell-cell contacts between the colony-stimulating factor-1-expressing fibroblasts and endoneurial macrophages as a putative prerequisite for the effective and constant activation of macrophages by fibroblasts in the chronically diseased nerve. Interestingly, in human biopsies from patients with Charcot-Marie-Tooth type 1, we also found frequent cell-cell contacts between macrophages and endoneurial fibroblasts and identified the latter as main source for colony-stimulating factor-1. Therefore, our study provides strong evidence for a similarly pathogenic role of colony-stimulating factor-1 in genetically mediated demyelination in mice and Charcot-Marie-Tooth type 1 disease in humans. Thus, colony-stimulating factor-1 or its cognate receptor are promising target molecules for treating the detrimental, low-grade inflammation of several inherited neuropathies in humans.

Prioritization of non-coding elements involved in non-syndromic cleft lip with/without cleft palate through genome-wide analysis of de novo mutations.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a highly heritable facial disorder. To date, systematic investigations of the contribution of rare variants in non-coding regions to nsCL/P etiology are sparse. Here, we re-analyzed available whole-genome sequence (WGS) data from 211 European case-parent trios with nsCL/P and identified 13,522 de novo mutations (DNMs) in nsCL/P cases, 13,055 of which mapped to non-coding regions. We integrated these data with DNMs from a reference cohort, with results of previous genome-wide association studies (GWASs), and functional and epigenetic datasets of relevance to embryonic facial development. A significant enrichment of nsCL/P DNMs was observed at two GWAS risk loci (4q28.1 (p = 8 × 10-4) and 2p21 (p = 0.02)), suggesting a convergence of both common and rare variants at these loci. We also mapped the DNMs to 810 position weight matrices indicative of transcription factor (TF) binding, and quantified the effect of the allelic changes in silico. This revealed a nominally significant overrepresentation of DNMs (p = 0.037), and a stronger effect on binding strength, for DNMs located in the sequence of the core binding region of the TF Musculin (MSC). Notably, MSC is involved in facial muscle development, together with a set of nsCL/P genes located at GWAS loci. Supported by additional results from single-cell transcriptomic data and molecular binding assays, this suggests that variation in MSC binding sites contributes to nsCL/P etiology. Our study describes a set of approaches that can be applied to increase the added value of WGS data.

High-resolution imaging and manipulation of endogenous AMPA receptor surface mobility during synaptic plasticity and learning.

Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPARs) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminal. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP-GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

Disease-associated synaptic scaffold protein CNK2 modulates PSD size and influences localisation of the regulatory kinase TNIK.

Scaffold proteins are responsible for structural organisation within cells; they form complexes with other proteins to facilitate signalling pathways and catalytic reactions. The scaffold protein connector enhancer of kinase suppressor of Ras 2 (CNK2) is predominantly expressed in neural tissues and was recently implicated in X-linked intellectual disability (ID). We have investigated the role of CNK2 in neurons in order to contribute to our understanding of how CNK2 alterations might cause developmental defects, and we have elucidated a functional role for CNK2 in the molecular processes that govern morphology of the postsynaptic density (PSD). We have also identified novel CNK2 interaction partners and explored their functional interdependency with CNK2. We focussed on the novel interaction partner TRAF2- and NCK-interacting kinase TNIK, which is also associated with ID. Both CNK2 and TNIK are expressed in neuronal dendrites and concentrated in dendritic spines, and staining with synaptic markers indicates a clear postsynaptic localisation. Importantly, our data highlight that CNK2 plays a role in directing TNIK subcellular localisation, and in neurons, CNK2 participates in ensuring that this multifunctional kinase is present in the correct place at desirable levels. In summary, our data indicate that CNK2 expression is critical for modulating PSD morphology; moreover, our study highlights that CNK2 functions as a scaffold with the potential to direct the localisation of regulatory proteins within the cell. Importantly, we describe a novel link between CNK2 and the regulatory kinase TNIK, and provide evidence supporting the idea that alterations in CNK2 localisation and expression have the potential to influence the behaviour of TNIK and other important regulatory molecules in neurons.

A Discrete Presynaptic Vesicle Cycle for Neuromodulator Receptors.

A major function of GPCRs is to inhibit presynaptic neurotransmitter release, requiring ligand-activated receptors to couple locally to effectors at terminals. The current understanding of how this is achieved is through receptor immobilization on the terminal surface. Here, we show that opioid peptide receptors, GPCRs that mediate highly sensitive presynaptic inhibition, are instead dynamic in axons. Opioid receptors diffuse rapidly throughout the axon surface and internalize after ligand-induced activation specifically at presynaptic terminals. We delineate a parallel regulated endocytic cycle for GPCRs operating at the presynapse, separately from the synaptic vesicle cycle, which clears activated receptors from the surface of terminals and locally reinserts them to maintain the diffusible surface pool. We propose an alternate strategy for achieving local control of presynaptic effectors that, opposite to using receptor immobilization and enforced proximity, is based on lateral mobility of receptors and leverages the inherent allostery of GPCR-effector coupling.

Protein kinase C regulates AMPA receptor auxiliary protein Shisa9/CKAMP44 through interactions with neuronal scaffold PICK1.

Synaptic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors are essential mediators of neurotransmission in the central nervous system. Shisa9/cysteine-knot AMPAR modulating protein 44 (CKAMP44) is a transmembrane protein recently found to be present in AMPA receptor-associated protein complexes. Here, we show that the cytosolic tail of Shisa9/CKAMP44 interacts with multiple scaffold proteins that are important for regulating synaptic plasticity in central neurons. We focussed on the interaction with the scaffold protein PICK1, which facilitates the formation of a tripartite complex with the protein kinase C (PKC) and thereby regulates phosphorylation of Shisa9/CKAMP44 C-terminal residues. This work has implications for our understanding of how PICK1 modulates AMPAR-mediated transmission and plasticity and also highlights a novel function of PKC.