title: Platelets of mice heterozygous for neurobeachin, a candidate gene for autism spectrum disorder, display protein changes related to aberrant protein kinase A activity.
abstract: BACKGROUND
  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 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.
  
  
  METHODS
  To 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.
  
  
  RESULTS
  Platelet 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Department of Human Genetics, Laboratory for Biochemical Neuroendocrinology, KU Leuven, 3000 Leuven, Belgium.|Leuven Autism Research Consortium (LAuRes), KU Leuven, 3000 Leuven, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Nuytens
authors.affiliation: Department of Human Genetics, Laboratory for Biochemical Neuroendocrinology, KU Leuven, 3000 Leuven, Belgium.|Leuven Autism Research Consortium (LAuRes), KU Leuven, 3000 Leuven, Belgium.
authors.forename: Krizia
authors.initials: K
authors.lastname: Tuand
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Michela
authors.initials: M
authors.lastname: Di Michele
authors.affiliation: Department of Human Genetics, Laboratory for Biochemical Neuroendocrinology, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Kurt
authors.initials: K
authors.lastname: Boonen
authors.affiliation: Department of Cellular and Molecular Medicine, Laboratory of Protein Phosphorylation and Proteomics, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.affiliation: Leuven Autism Research Consortium (LAuRes), KU Leuven, 3000 Leuven, Belgium.|Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, 3000 Leuven, Belgium.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Freson
authors.affiliation: Department of Human Genetics, Laboratory for Biochemical Neuroendocrinology, KU Leuven, 3000 Leuven, Belgium.|Leuven Autism Research Consortium (LAuRes), KU Leuven, 3000 Leuven, Belgium.
authors.forename: John Wm
authors.initials: JW
authors.lastname: Creemers