N‐terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors

Goodman, Lucy; Baddeley, David; Ambroziak, Wojciech; Waites, Clarissa L.; Garner, Craig C.; Soeller, Christian; Montgomery, Johanna M.

doi:10.1002/hipo.22723

“…2A). As a reflection of PSD size, we quantified the endogenous Homer content in mature spines (DIV 21) 22,23 . Blinded analysis of the Homer immunofluorescence signal area using the "Analyze Particles" tool (FIJI/ImageJ) 24 enabled a quantitative comparative analysis of Homer immunofluorescence signal intensity and area in neurons expressing either CNK2 shRNA or control shRNA (Fig.…”

Section: Loss Of Cnk2 Influences the Size Of The Postsynaptic Densitymentioning

confidence: 99%

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

Zieger

¹

,

Kunde

²

,

Rademacher

³

et al. 2020

Sci Rep

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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. Scaffold proteins are multi-domain proteins that typically lack enzymatic activity. However, they are crucial in regulating signal transduction cascades: through multiple protein-protein interactions, they organise protein complex formation and ensure spatiotemporal organisation of signalling processes and signal propagation. The scaffold protein connector enhancer of kinase suppressor of Ras (CNK/CNKSR) was first discovered in Drosophila, where it was shown to regulate Ras/MAPK signalling by binding to the Ras effector RAF and thereby play an essential role in eye and wing development 1,2. Subsequent studies on CNKs in various organisms showed that CNK homologues are present across species, ranging e.g. from C. elegans 3 to humans 1 , and that they likewise influence signalling by acting as scaffolds downstream of Ras 1. CNKs possess multiple protein interaction domains, including e.g. a sterile alpha motif (SAM), a conserved region in CNK (CRIC), a PSD-95/DLG-1/ZO-1 (PDZ) domain, and a pleckstrin homology (PH) domain, and the domain architecture is essentially conserved throughout the CNK family proteins and also across species. Since the original discovery of the D-CNK protein in Drosophila, it has become clear that CNK family ...

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“…Primary dissociated hippocampal cultures were prepared from postnatal day 0 (P0) Wistar rats of either sex using a modified Banker protocol (Kaech & Banker, ), as described in our previous studies (Arons et al, , ; Goodman et al, ). The handling of all animals was performed following strict regulations approved by the University of Auckland Animal Ethics Committee.…”

Section: Methodsmentioning

confidence: 99%

Autism‐associated Shank3 mutations alter mGluR expression and mGluR‐dependent but not NMDA receptor‐dependent long‐term depression

Lee

¹

,

Vyas

²

,

Garner

³

et al. 2019

Synapse

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SHANK3 is a postsynaptic structural protein localized at excitatory glutamatergic synapses in which deletions and mutations have been implicated in patients with autism spectrum disorders (ASD). The expression of Shank3 ASD mutations causes impairments in ionotropic glutamate receptor‐mediated synaptic responses in neurons, which is thought to underlie ASD‐related behaviors, thereby indicating glutamatergic synaptopathy as one of the major pathogenic mechanisms. However, little is known about the functional consequences of ASD‐associated mutations in Shank3 on another important set of glutamate receptors, group I metabotropic glutamate receptors (mGluRs). Here, we further assessed how Shank3 mutations identified in patients with ASD (one de novo InsG mutation and two inherited point mutations, R87C and R375C) disrupt group I mGluR (mGluR1 and mGluR5) expression and function. To identify potential isoform‐specific deficits induced by ASD‐associated Shank3 mutations on group I mGluRs, we surface immunolabeled mGluR1 and mGluR5 independently. We also induced mGluR‐dependent synaptic plasticity (R,S‐3,5‐dihydroxyphenylglycine [DHPG]‐induced long‐term depression [LTD]) as well as N‐methyl‐D‐aspartate receptor (NMDAR)‐dependent LTD. ASD‐associated mutations in Shank3 differentially interfered with the ability of cultured hippocampal neurons to express mGluR5 and mGluR1 at synapses. Intriguingly, all ASD Shank3 mutations impaired mGluR‐dependent LTD without altering NMDAR‐dependent LTD. Our data show that the specific perturbation in mGluR‐dependent synaptic plasticity occurs in neurons expressing ASD‐associated Shank3 mutations, which may underpin synaptic dysfunction and subsequent behavioral deficits in ASD.

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“…2A). As a reflection of PSD size, we quantified the endogenous Homer content in mature spines (DIV 21) 20,21 . Blinded analysis of the Homer immunofluorescence signal area using the "Analyze Particles" tool (FIJI/ ImageJ) 22 enabled a quantitative comparative analysis of Homer immunofluorescence signal intensity and area in neurons expressing either CNK2 shRNA or control shRNA (Fig.…”

Section: Loss Of Cnk2 Influences the Size Of The Postsynaptic Densitymentioning

confidence: 99%

Disease-associated scaffold protein CNK2 modulates PSD size and influences trafficking of new synaptic binding partner TNIK

Zieger

¹

,

Kunde

²

,

Rademacher

³

et al. 2019

Preprint

0

View full text Add to dashboard Cite

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 at desirable levels at synaptic sites.In summary, our data indicate that CNK2 expression is critical for modulating PSD morphology; moreover, our study highlights a role for CNK2 in directing 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 these proteins play complementary roles in the regulation of dendritic spine growth and maintenance.

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N‐terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors

Cited by 18 publications

References 74 publications

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

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

Autism‐associated Shank3 mutations alter mGluR expression and mGluR‐dependent but not NMDA receptor‐dependent long‐term depression

Disease-associated scaffold protein CNK2 modulates PSD size and influences trafficking of new synaptic binding partner TNIK

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