A Munc18-1 mutant mimicking phosphorylation by Down Syndrome-related kinase Dyrk1a supports normal synaptic transmission and promotes recovery after intense activity

Jessica Classen,Ingrid Saarloos,Marieke Meijer,Patrick F Sullivan,Matthijs Verhage

doi:10.1038/s41598-020-59757-y

Jessica Classen, Ingrid Saarloos + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-59757-y

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Abstract

Phosphorylation of Munc18-1 (Stxbp1), a presynaptic organizer of synaptic vesicle fusion, is a powerful mechanism to regulate synaptic strength. Munc18-1 is a proposed substrate for the Down Syndrome-related kinase dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a) and mutations in both genes cause intellectual disability. However, the functional consequences of Dyrk1a-dependent phosphorylation of Munc18-1 for synapse function are unknown. Here, we show that the proposed Munc18-1 phosphorylation site, T479, is among the highly constrained phosphorylation sites in the coding regions of the gene and is also located within a larger constrained coding region. We confirm that Dyrk1a phosphorylates Munc18-1 at T479. Patch-clamp physiology in conditional null mutant hippocampal neurons expressing Cre and either wildtype, or mutants mimicking or preventing phosphorylation, revealed that synaptic transmission is similar among the three groups: frequency/amplitude of mEPSCs, evoked EPSCs, paired pulse plasticity, rundown kinetics upon intense activity and the readily releasable pool. However, synapses expressing the phosphomimic mutant responded to intense activity with more pronounced facilitation. These data indicate that Dyrk1a-dependent Munc18-1 phosphorylation has a minor impact on synaptic transmission, only after intense activity, and that the role of genetic variation in both genes in intellectual disability may be through different mechanisms.

Highlights

Several potential phosphorylation sites of Munc[] have been found[1] or predicted[13]
We found that mimicking phosphorylation of Munc[] by dual-specificity tyrosine phosphorylation-regulate kinase 1a (Dyrk1a), and comparing it to a non-phosphorylatable Munc[] and a wildtype, did not affect basic synaptic transmission or short-term plasticity parameters such as release probability and the size of the readily releasable pool
This study shows that threonine 479 (T479) is among several phosphorylation sites in Munc[] that are constrained and can be phosphorylated in vitro

Summary

Introduction

Several potential phosphorylation sites of Munc[] have been found[1] or predicted[13]. Dyrk1a knockout mice are lethal because phosphorylation of Hip-1 blocks neuronal cell death in neural progenitor cells and promotes neurite outgrowth[36]. These similarities between Dyrk1a and Munc[] strengthen the idea that the phosphorylation of Munc[] by Dyrk1a might be important for synapse function and contributes to an explanation for the severe ID caused by mutations in the two genes. We hypothesized that phosphorylation of Munc[] by Dyrk1a regulates the role of Munc[] in neuronal survival, synaptic transmission, and plasticity. Mimicking phosphorylation of Munc[] by Dyrk1a is not essential for synaptic transmission but promotes recovery

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Results

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