Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense ...mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.
The calcium-/calmodulin dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinases (MAGUK) family of proteins. It fulfils several different cellular functions, ...ranging from acting as a scaffold protein to transcription control, as well as regulation of receptor sorting. CASK functions depend on the interaction with a variety of partners, for example neurexin, liprin-alpha, Tbr1 and SAP97. So far, it is uncertain how these seemingly unrelated interactions and resulting functions of CASK are regulated. Here, we show that alternative splicing of CASK can guide the binding affinity of CASK isoforms to distinct interaction partners. We report seven different variants of CASK expressed in the fetal human brain. Four out of these variants are not present in the NCBI GenBank database as known human variants. Functional analyses showed that alternative splicing affected the affinities of CASK variants for several of the tested interaction partners. Thus, we observed a clear correlation of the presence of one splice insert with poor binding of CASK to SAP97, supported by molecular modelling. The alternative splicing and distinct properties of CASK variants in terms of protein-protein interaction should be taken into consideration for future studies.
Members of the Shank family of postsynaptic scaffold proteins (Shank1–3) link neurotransmitter receptors to the actin cytoskeleton in dendritic spines through establishing numerous interactions ...within the postsynaptic density (PSD) of excitatory synapses. Large Shank isoforms carry at their N-termini a highly conserved domain termed the Shank/ProSAP N-terminal (SPN) domain, followed by a set of Ankyrin repeats. Both domains are involved in an intramolecular interaction which is believed to regulate accessibility for additional interaction partners, such as Ras family G-proteins, αCaMKII, and cytoskeletal proteins. Here, we analyze the functional relevance of the SPN-Ank module; we show that binding of active Ras or Rap1a to the SPN domain can differentially regulate the localization of Shank3 in dendrites. In Shank1 and Shank3, the linker between the SPN and Ank domains binds to inactive αCaMKII. Due to this interaction, both Shank1 and Shank3 exert a negative effect on αCaMKII activity at postsynaptic sites in mice
in vivo
. The relevance of the SPN-Ank intramolecular interaction was further analyzed in primary cultured neurons; here, we observed that in the context of full-length Shank3, a closed conformation of the SPN-Ank tandem is necessary for proper clustering of Shank3 on the head of dendritic spines. Shank3 variants carrying Ank repeats which are not associated with the SPN domain lead to the atypical formation of postsynaptic clusters on dendritic shafts, at the expense of clusters in spine-like protrusions. Our data show that the SPN-Ank tandem motif contributes to the regulation of postsynaptic signaling and is also necessary for proper targeting of Shank3 to postsynaptic sites. Our data also suggest how missense variants found in autistic patients which alter SPN and Ank domains affect the synaptic function of Shank3.
Neurodevelopmental disorders arise due to malfunctions in brain development and often are genetically caused. The disease-associated genes encode three major classes of proteins: transcriptional ...regulators, synaptic proteins, and RNA-binding proteins. It was proposed that the pathomechanisms may converge in shared molecular pathways. I functionally analyzed and compared the pathomechanisms of insufficiency for SHANK1 and SHANK3, two genes associated with autism spectrum disorders, with a variant of the AGO2 gene, encoding the RNA-binding protein Argonaute-2 and causing Lessel-Kreienkamp syndrome.The SHANK genes encode Shank proteins, scaffolds of excitatory, glutamatergic synapses that indirectly connect postsynaptic glutamate receptors to F-actin via a protein network. The N-terminus of Shank3 interacts with Ras family G-proteins, connecting Shank to the MAPK pathway, which is involved in translational regulation. Altered translation has been implicated in the pathology of autism spectrum disorders. The hypothesis of this project was that loss of Shank may alter the regulation of translation. Omics were applied to test this hypothesis. The translatome and proteome of mouse models for Shank-associated autism spectrum disorders were investigated. Actively translated mRNAs were purified from hippocampal neurons of Shank3αβ knockout mice via RNA affinity purification and analyzed by RNA sequencing. Absence of Shank3αβ resulted in subtly altered translation of a subset of neuronal transcripts. Proteomics were performed with biochemically purified postsynaptic density fractions from hippocampi of Shank1 and Shank3αβ knockout mice. The loss of Shank1 and Shank3αβ severely altered the postsynaptic proteome. The abundance of active, phosphorylated CaMKIIα was increased in Shankknockout mice, which may contribute to misregulated neuronal signaling.Argonaute-2 functions in RNA interference. To execute translational silencing, Argonaute-2 associates with microRNA and forms the RNA induced silencing complex. It was hypothesized that patient derived Argonaute-2 mutants may bind different microRNAs, altering RNA interference. To test this hypothesis, primary cortical neurons were infected with adeno-associated viruses to induce expression of the Argonaute-2 mutant L192P. Expression of the L192P variant induced increased occurrence of dendritic processing bodies, shown by immunocytochemistry and confocal microscopy. MicroRNA affinity purification was applied, and microRNAs were sequenced. The expressed and Argonaute-2-bound microRNAs were identified. Four different aspects of microRNA-dependent RNA interference were altered in neurons that expressed the Argonaute-2 mutant. A subset of microRNAs was differentially expressed, indicating an affected miRNome. Several microRNAs exhibited altered association to the RNA induced silencing complex. The Argonaute-2 mutant showed altered strand selectivity, which resulted in arm switching events between guide and passenger strands of microRNAs. The patient variant induced enhanced incorporation of isomers of microRNAs, termed isomiRs. A subset of isomiRs was exclusively bound to Argonaute-2 L192P, whereas none of the isomiRs associated exclusively with the wildtype protein. The Argonaute-2 variant resulted in deregulation of the miR379-409 genomic cluster. Deregulation of this cluster likely affects neurogenesis, neuronal migration, and synaptic function.Analyses of translatome, proteome, miRNome, and Argonaute-2-bound microRNAs demonstrated variable deregulations contributing to the molecular defects in model systems for neurodevelopmental disorders. This suggests that the pathomechanisms of Shank-associated forms of autism spectrum disorders and Lessel-Kreienkamp syndrome do not converge in common pathways, but rather result in broad and gene-specific alterations of cellular functions.
Mutations in the X‐linked gene coding for the calcium‐/calmodulin‐dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in ...males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post‐synaptic NMDA and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK’s molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co‐expression and co‐immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse‐associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK‐interacting nucleosome assembly protein (CINAP) and T‐box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD‐95/discs large/ZO‐1 (PDZ) domains affect binding of CASK to the pre‐synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre‐synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin‐induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.
The pathogenic mechanism underlying several patient‐derived missense mutations of the MAGUK CASK remains unclear. MAGUKs have been shown to oligomerize when binding to their PDZ‐ligand through a conformational change in their PDZ, SH3 and GK domains. We demonstrate here that WT CASK also oligomerizes when bound to its PDZ‐ligand Neurexin, but that several of the 10 studied CASK mutant variants fail to bind to Neurexin and/or to oligomerize. This suggests that the loss of Neurexin‐induced oligomerization may be a key pathogenic mechanism underlying CASK missense mutations.
CASK is a unique membrane-associated guanylate kinase (MAGUK) because of its Ca
2+
/calmodulin-dependent kinase (CaMK) domain. We describe four male patients with a severe neurodevelopmental disorder ...with microcephaly carrying missense variants affecting the CaMK domain. One boy who carried the p.E115K variant and died at an early age showed pontocerebellar hypoplasia (PCH) in addition to microcephaly, thus exhibiting the classical MICPCH phenotype observed in individuals with
CASK
loss-of-function variants. All four variants selectively weaken the interaction of CASK with Liprin-α2, a component of the presynaptic active zone. Liprin-α proteins form spherical phase-separated condensates, which we observe here in Liprin-α2 overexpressing HEK293T cells. Large Liprin-α2 clusters were also observed in transfected primary-cultured neurons. Cluster formation of Liprin-α2 is reversed in the presence of CASK; this is associated with altered phosphorylation of Liprin-α2. The p.E115K variant fails to interfere with condensate formation. As the individual carrying this variant had the severe MICPCH disorder, we suggest that regulation of Liprin-α2–mediated phase condensate formation is a new functional feature of CASK which must be maintained to prevent PCH.
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