Abstract
BackgroundNeurobeachin (NBEA) has been identified as a candidate gene for autism spectrum disorders (ASD) in several unrelated patients with alterations in the NBEA gene. The exact function of NBEA, a multidomain scaffolding protein, is currently unknown. It contains an A-kinase anchoring protein (AKAP) domain which binds the regulatory subunit of protein kinase A (PKA) thereby confining its activity to specific subcellular regions. NBEA has been implicated in post-Golgi membrane trafficking and in regulated secretion. The mechanism of regulated secretion is largely conserved between neurons and platelets, and the morphology of platelet dense granules was found to be abnormal in several ASD patients, including one with NBEA haploinsufficiency. Platelet dense granules are secreted upon vascular injury when platelets are exposed to for instance collagen. Dense granules contain serotonin, ATP and ADP, which are necessary for platelet plug formation and vascular contraction.MethodsTo further investigate possible roles for NBEA in secretion or dense granule morphology, platelets from Nbea+/- mice were analyzed morphometrically, functionally and biochemically. A differential proteomics and peptidomics screen was performed between Nbea+/- and Nbea+/+ mice, in which altered Talin-1 cleavage was further investigated and validated in brain samples. Finally, the phosphorylation pattern of PKA substrates was analyzed.ResultsPlatelet dense granules of Nbea+/- mice had a reduced surface area and abnormal dense-core halo, but normal serotonin-content. Nbea haploinsufficiency did not affect platelet aggregation and ATP secretion after collagen stimulation, although the platelet shape change was more pronounced. Furthermore, peptidomics revealed that Nbea+/- platelets contain significantly reduced levels of several actin-interacting peptides. Decreased levels were detected of the actin-binding head and rod domain of Talin-1, which are cleavage products of Calpain-2. This is most likely due to increased PKA-mediated phosphorylation of Calpain-2, which renders the enzyme less active. Analysis of other PKA substrates revealed both increased and reduced phosphorylation.ConclusionOur results show the pleiotropic effects of alterations in PKA activity due to Nbea haploinsufficiency, highlighting the important function of the AKAP domain in Nbea in regulating and confining PKA activity. Furthermore, these results suggest a role for Nbea in remodeling the actin cytoskeleton of platelets.
Highlights
Neurobeachin (NBEA) has been identified as a candidate gene for autism spectrum disorders (ASD) in several unrelated patients with alterations in the NBEA gene
Platelet function analysis and platelet counts Murine blood was anticoagulated with 3.2% trisodium citrate (9:1) and mean platelet volume (MPV) and platelet count were determined on an automated cell counter (Cell-Dyn 1300 Abbott laboratories, Abbott Park, IL, USA)
Dense granules were classified as different types: type 1, solid core occupying more than 50% of the granule; type 2, solid core occupying less than 50% of the granule; type 3, fragmented core; or type 4, empty granule/no visible core [34,35]
Summary
Neurobeachin (NBEA) has been identified as a candidate gene for autism spectrum disorders (ASD) in several unrelated patients with alterations in the NBEA gene. The genetic architecture of autism spectrum disorders (ASDs) is highly heterogeneous and to date more than 100 genes have been reported to be deleted, duplicated, mutated or disrupted by a translocation breakpoint in ASD patients [1,2] One of these candidate genes, Neurobeachin (NBEA) [MIM: 604889] was identified in a patient with a de novo balanced chromosomal translocation t(5;13)(q12.1; q13.2) with one breakpoint in intron 2 of NBEA resulting in a NBEA haploinsufficient status [3]. The N-terminal region contains a Concanavalin A-like lectin domain flanked by armadillo repeats suggested to play a role in intracellular trafficking [17,18] Distal from these regions, an A-kinase anchoring protein (AKAP) domain is present, recruiting cAMP-dependent protein kinase A (PKA) by high-affinity binding to its regulatory RIIα subunit [16]. NBEA and eight other human proteins contain the highly conserved BEACH domain, and belong to the family of BEACH proteins [20]
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