Background
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]. Additionally, four unrelated ASD patients with a monoallelic deletion of
NBEA were reported[
4‐
7], and three novel CNVs were detected within the
NBEA gene in three unrelated individuals diagnosed with ASD[
8‐
10]. Moreover, a single nucleotide polymorphism (SNP) in intron 38 of
NBEA has been associated with ASD[
11]. The
NBEA gene contains a low-frequency common fragile site (
FRA13) linked to ASD and is located in a 19 cM region identified as a candidate region for ASD by a linkage study (MMLS/het score of 2.3 between markers D13S217 at 17.21 cM and D13S1229 at 21.51 cM)[
12‐
14].
NBEA, a brain-enriched multidomain scaffolding protein, is located at the tubulovesicular endomembranes near the
trans-Golgi network[
15,
16]. 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]. The C-terminal part of NBEA possesses a pleckstrin homology - beige and Chediak-Higashi (BEACH) - WD40 domain module which is thought to be implicated in vesicle trafficking[
16,
19]. NBEA and eight other human proteins contain the highly conserved BEACH domain, and thus belong to the family of BEACH proteins[
20].
Although the exact function of NBEA is currently unknown, a complete loss of Nbea in mice leads to a perinatal lethal phenotype due to a complete block of evoked neuromuscular transmission[
21]. By studying neuronal cultures derived from E18 Nbea
-/- mice, a role emerges for Nbea in trafficking important cargo to pre- and postsynaptic compartments, as these cultures have shown abnormal large clusters of actin in the soma, dendritic shafts and axons, and a reduced level of neurotransmitter receptors has been detected at the surface of the postsynaptic membrane[
22,
23]. Moreover, knockdown of Nbea in a neuroendocrine cell line (βTC3 cells) leads to enhanced secretion of dense-core secretory granules, the neuroendocrine counterpart of large dense-core vesicles (LDCVs) in neurons, making Nbea a negative regulator of the regulated secretion[
24].
Blood platelets are the first players to be activated upon vascular injury. They are essential for initiating platelet plug formation and do so by secreting the content of their secretory granules. Similar to neurons, platelets contain two types of secretory granules, namely the alpha and dense granules, corresponding to the small synaptic vesicles (SVs) and LDCVs in neurons, respectively[
25]. Blood platelet alpha granules have a heterogeneous cargo of polypeptides ranging from adhesion molecules to growth factors, whereas dense granules contain the small molecules ATP, ADP and serotonin, necessary for vasoconstriction. Due to the similar regulation of granule formation and transport, platelets were put forward as a model system to study the biology of granule formation, trafficking and secretion in neurons[
26,
27]. Preliminary
in vivo analysis unveiled an abnormal morphology of the dense granules in platelets of our reported patient with a chromosomal translocation in
NBEA[
24]. Moreover, similar platelet granule abnormalities were observed in ASD patients with chromosomal rearrangements in
Amisyn or
SCAMP5, and in an ASD patient with a deletion including
SHANK3[
24]. Interestingly, mutations in LYST and NBEA-like 2 (NBEAL2), two other BEACH proteins, are described in patients with Chediak-Higashi and Gray platelet syndrome and result in abnormal to absent platelet dense and alpha granules, respectively[
28,
29].
As platelets can easily be obtained from patients, further insights into platelet abnormalities might lead to the identification of biomarkers associated with ASD. We have characterized platelets from mice heterozygous for Nbea to substantiate the causality of NBEA haploinsufficiency for the abnormal platelet phenotype. The ultrastructure of the dense granules of murine platelets was analyzed and platelet function was investigated. Moreover, serotonin levels were determined in both serum and platelets, as hyperserotonemia is the only biochemical anomaly reported in approximately 30% of ASD patients. Serotonin is a hormone and monoamine neurotransmitter that can induce vasoconstriction and is implicated in neuron outgrowth, maturation, function and plasticity. It is synthesized in serotonergic neurons of the central nervous system and in the intestine, and more than 99% of whole blood serotonin is stored in blood platelets[
30]. To assess whether Nbea haploinsufficiency affects the protein and peptide content of platelets, a full proteomic and peptidomic analysis was performed and results were further validated in platelets and in total brain.
Methods
All experiments were approved by the ethical research committee of KU Leuven in accordance with the declaration of Helsinki (project number P182/2011).
Animals
The GH240B transgenic line described in Su
et al.[
21] was backcrossed for at least 10 generations with C57BL/6JRj mice (Janvier). Peripheral blood samples were obtained from adult (12-week-old) female mice.
Brains were dissected from 12-week-old mice and immediately put at -80°C. Tissue was homogenized in sucrose buffer (3.18 mM sucrose; 4 mM Tris–HCl pH 7.4) containing a protease and phosphatase inhibitor cocktail and a complete protease inhibitor cocktail (both from Roche Applied Science, Penzberg, Germany).
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). Platelet-rich plasma (PRP) was obtained after centrifugation at 3,000 rpm for 30 sec followed by 800 rpm for 5 minutes. The platelet count was adjusted to 250,000 platelets/μl with autologous plasma. Platelet aggregation and secretion were performed as described after stimulation with Horm collagen (5 μg/ml)[
31,
32]. Platelet secretion was determined by measuring the release of ATP using luciferin/luciferase reagent (Kordia, Leiden, The Netherlands). Electron microscopy analysis of murine platelets was performed as previously reported[
33]. Additional ultra-thin sections of 50 to 70 nm were cut, stained with uranyl acetate and lead citrate, and examined at 80 kV using a JEM1400 transmission electron microscope (JEOL, Tokyo, Japan). Micrographs were acquired on an SIS Quemesa camera (Olympus, Münster, Germany). The number of dense granules per platelet and dense granule dimension and morphology were further assessed with the ImageJ imaging system (National Institutes of Health, Bethesda, MD, USA) (n = 200 platelets/genotype). 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].
Determination of platelet size and distribution by flow cytometry
Integrin αIIbβ3 expression was measured in whole blood by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-CD41/61 monoclonal antibody (BD Biosciences, Bergen County, NJ, USA) for 15 minutes. Forward and side scatter and percentage platelets to total cell number were analyzed using FACSDiva version 6.1.2 software (BD Biosciences) on a FACSCalibur flow cytometer (BD Biosciences).
Platelet isolation for protein analysis
Peripheral blood samples were obtained from the retro-orbital sinus (anticoagulated with ACD pH6.5 (7 mM citric acid; 93 mM sodium citrate; 140 mM dextrose); 9:1). PRP was obtained as described above. Platelets were obtained by PRP centrifugation at 2,300 rpm for 10 minutes and washed twice with ACD pH6.5. For proteomic purposes, PRP of littermates with the same genotype was pooled to yield sufficient protein contents to prepare the platelet pellets.
Determination of serotonin levels in platelets and serum
Serum was obtained from blood coagulated for 30 minutes at 37°C in glass cuvettes followed by centrifugation at 2,300 rpm for 10 minutes. Serotonin content of platelets, isolated as mentioned above, and serum was calculated using the serotonin research ELISA (Labor Diagnostika Nord, Nordhorn, Germany) according to the protocol of the manufacturer (n = 8 mice/genotype).
Two dimensional-differential gel electrophoresis (2D-DiGE)
Platelet pellets (n = 4 samples/genotype) were lysed in DiGE lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 30 mM Tris pH 8.5 and a complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The samples were purified with the 2D Clean-Up Kit (GE Healthcare, Buckinghamshire, UK) and the concentration was determined using the 2D Quant Kit (GE Healthcare) according to the manufacturer’s guidelines. Proteins were labeled with carbocyanine (Cy) dyes as previously described[
31]. Briefly, 50 μg of each sample was labeled with 200 pmol of Cy3 or Cy5. To avoid possible bias due to labeling efficiency, two samples of each genotype were labeled with Cy3 and the other two with Cy5. The internal standard consisting of a pool of all samples was labeled with Cy2 allowing a quantitative comparison for a protein of two samples resolved on the same gel (ratio Cy3/Cy2 and Cy5/Cy2) and a quantitative comparison of multiple gels. Mixtures of Cy3-, Cy5- and Cy2-labeled samples were diluted 1:1 with lysis buffer containing 0.5% IPG buffer (pH 4 to 7) and 1.3% dithiothreitol (DTT) and applied by cup loading on rehydrated IPG strips (pH 4 to 7, 18 cm). The first dimension was carried out in an IPGphor system (GE Healthcare) with the following conditions: 1 h 30 minutes at 150 V, 2 h at 500 V, 5 h at 1,000 V, 3 h at 8,000 V in gradient and 5 h at 8,000 V. IPG strips were subsequently incubated in equilibration buffer (6 M urea, 30% glycerol, 2% SDS and 50 mM Tris pH 8.5) supplemented with 65 mM DTT for 20 minutes and followed by incubation in equilibration buffer supplemented with 200 mM iodoacetamide and 0.02% bromophenol blue for 20 minutes. The second dimension was performed on 11% polyacrylamide gels on the Hoefer DALT vertical system (GE Healthcare). Visualization and analysis of the images as well as the identification of differentially expressed proteins were executed as described previously[
31].
Immunoblot analysis
Platelet pellets were resolved in lysis buffer (1% Igepal; 0,015% DTT; 1 mM ethylene diamine tetraacetic acid (EDTA) in PBS supplemented with a complete protease inhibitor cocktail) (Roche Applied Science). Protein concentration of the platelet and brain lysates was quantified with a bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA). Depending on the molecular weight of the protein of interest, 25 μg of platelet or brain lysates was loaded on a 10% Bis-Tris gel (Bio-Rad, Hercules, CA, USA) or 3 to 8% Tris-Acetate gel (Life technologies, Paisley, UK). Proteins were transferred to Protan Nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) and incubated with antibodies against Munc13-4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1/1,000), Rab27b (homemade antibody, 1/1,000), Calmodulin (kindly provided by Professor H Desmedt, KU Leuven; 1/1,000), Talin-1 (Cell Signaling Technology, Danvers, MA, USA; 1/500), Calpain-2 large subunit (Cell Signaling Technology; 1/1000), Calpain-4 regulatory subunit (Santa Cruz Biotechnology; 1/1,000), phospho-(Ser/Thr) PKA substrate antibody (Cell Signaling Technology; 1/1,000), and actin (Sigma-Aldrich, St Louis, MO, USA; 1/5,000), used for normalization. Equal amounts of actin protein expression were verified after incubation with an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Abcam, Cambridge, UK; 1/15,000). Afterwards, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1/2,000; DAKO, Glostrup, Denmark) and proteins were visualized with western blotting ECL detection reagent. Quantification was performed using the Kodak Imager software (Kodak, Rochester, NY, USA).
Immunoprecipitation
Per sample, 20 μl of 50% bead slurry of protein A agarose beads (GE Healthcare) was used and washed twice with PBS before use. All incubations were performed at 4°C on a mechanical rotator. Prior to immunoprecipitation, pre-clearing of the platelet or brain sample was performed as follows. Protein A agarose beads were incubated with rabbit serum (ImmuniBioScience, Mukiltoe, WA, USA) for 1 h after which 60 μg of platelet or brain sample was added for 1 h. The immunoprecipitation was subsequently performed with the pre-cleared supernatant by adding phospho-(Ser/Thr) PKA substrate antibody (Cell signaling technology; 1/100) for overnight incubation, followed by an additional 2 h incubation with protein A agarose beads. Beads were washed five times with PBS and proteins were harvested by resuspension of the beads in sample buffer (50 mM Tris–HCl pH 7; 10% glycerol; 2% SDS; bromophenol blue) compatible with immunoblot analysis.
Peptidomics
Platelets were isolated as described above with modification of the final wash buffer, which was now replaced by PBS. The procedure for processing the platelet pellets (n = 5 mice/genotype) for mass spectrometry analysis was performed as reported[
36]. DeCyder MS 2.0 (GE Healthcare) is a differential analysis software tool that also allows for easy visualization of liquid chromatography mass spectrometry (LC-MS) runs by creating artificial two-dimensional maps with the
m/z values and retention times in the first and second dimension, respectively. Time alignment, intensity normalization and statistics were performed using this software. Peptides were identified in additional LC-MS/MS runs of the pooled samples as reported in[
36] using LC quadrupole time-of-flight (Q-TOF) MS.
Statistical analysis
Data are presented as mean ± standard error of the mean (SEM). Significance of differences was analyzed using (where appropriate) the two-tailed t-test, t-test for single means, Mann–Whitney U-test (MWU) or Pearson Chi-square test using Statistica version 9.0 (StatSoft Inc., Tulsa, OK, USA). All statistical tests were performed with 0.05 as the α-level of significance (*P <0.05, **P <0.01 and ***P <0.001).
Discussion
In this study we have confirmed a causal link between Nbea haploinsufficiency and abnormal granule morphology, similar to our previous observation in ASD patients[
24]. Dense granules of Nbea
+/- platelets were significantly smaller and showed a more pronounced shape change than Nbea
+/+ platelets. However, none of the 1,432 proteins identified in a proteomic screen was differentially expressed. Differential peptidomics of platelets did identify four actin-interacting peptides with a reduced presence in platelets of Nbea
+/- mice. Validation of the peptidomic screen revealed reduced cleavage of Talin-1, most likely due to increased phosphorylation of Calpain-2 by PKA. The reduced cleavage of Talin-1 was confirmed in Nbea
+/- total brain, but phosphorylation of Calpain-2 was decreased. The importance of Nbea as a regulator of PKA activity was further substantiated, as haploinsufficiency of Nbea positively and negatively affects PKA-mediated phosphorylation of a multitude of proteins in resting platelets and in brain.
Normal serotonin levels were detected in platelets and serum of Nbea
+/- mice, which is consistent with the similar amount of dense granules per platelet in Nbea
+/+ and Nbea
+/- mice as serotonin is taken up by platelets and stored in the dense granules[
37]. Hyperserotonemia is one of the few biochemical anomalies reported in ASD patients and several possible causes have been suggested, such as increased synthesis or altered release of serotonin from enterochromaffin cells[
49,
50]. However, only 30% of ASD patients have been classified as being hyperserotonemic and increased levels of serotonin have also been detected in relatives of ASD patients[
30,
51]. These findings highlight the controversy regarding hyperserotonemia as a biomarker for ASD.
Several lines of evidence point to alterations in the cytoskeleton of platelets of Nbea
+/- mice. First, an increased shape change upon collagen stimulation was observed for platelets of Nbea
+/- mice. Interestingly, platelets from patients with a dense granule secretion defect also presented with a more pronounced shape change[
31]. A similar shape change was noticed in platelets of patients with Duchenne muscular dystrophy, a disease characterized by a disturbed cytoskeletal organization[
52]. Second, all peptides that have a significantly different level in platelets of Nbea
+/- mice are actin-interacting peptides. Transgelin-2, also named SMβ22, was identified as an actin-associated protein with an unknown function[
53]. Thymosin β4 and β10 are the main intracellular G-actin sequestering peptides present in most mammalian cells. Binding of Thymosin β to G-actin prevents the polymerization to F-actin and reduced levels of Thymosin β4 and β10 lead to excessive formation of F-actin[
54]. A decreased presence of Thymosin β4 and β10 in platelets of Nbea
+/- mice might be indicative for a decreased expression in neurons as well. This could contribute to the excessive F-actin clusters detected in the soma, dendritic shafts and axons of hippocampal neuronal cultures of Nbea
-/- mice and in the soma of cortical neurons of Nbea
+/- mice, although the exact mechanism remains to be determined[
22]. Of note, a proteomic study using platelets from patients with a dense granule secretion defect also revealed actin-binding proteins as the major change in differentially expressed proteins compared to control platelets[
31].
Multiple F-actin binding sites have been identified in Talin-1, both in the head and rod domain[
55,
56]. The peptide of Talin-1 identified in the peptidomic screen is likely to be a degradation product of the rod domain. The diminished presence of this peptide in platelets of Nbea
+/- mice correlates with the reduced presence of the rod domain detected on western blot. Talin-1 is cleaved by Calpain-2 after amino acid 432, yielding the head and rod domain of Talin-1[
44]. Calpain-2 activity is regulated by phosphorylation of PKA; more specifically, phosphorylation of Ser369 or Thr370 results in decreased activity[
46,
47]. The observed increase in PKA-specific phosphorylation of Calpain-2 is therefore a likely cause of the reduced cleavage of Talin-1 in blood platelets.
It is known that the F3 subdomain in the head domain of Talin-1 interacts with the cytoplasmic tail of β-integrin during the platelet activation process[
57]. Therefore, alterations in platelet function might be expected based on the reduced presence of the head domain of Talin-1. However, in contrast to the conditional-knockout mouse models generated to study the molecular function of Talin-1, no complete ablation of Talin-1 was detected in platelets of Nbea
+/- mice. Of note is the finding that heterozygous conditional Talin-1-knockout mouse models do not present alterations in platelet function[
58].
In Wistar rats, Talin is detected at the membranes of granules and dense compartments in the megakaryocytes[
59,
60]. Wistar Furth rats, a rat model with abnormal thrombocytopoietic phenotype associated with morphologically aberrant megakaryocytes lack these dense compartments. It is hypothesized that the lack of cytoskeletal proteins such as Talin may be responsible for the absence of the dense compartments in megakaryocytes of Wistar Furth rats. Therefore, it is possible that the observed alterations in Talin-1 in Nbea
+/- platelets contribute to the morphological differences of dense granules resulting in the reduced size of these dense granules.
Decreased cleavage of Talin-1 was also observed in total brain of Nbea
+/- mice. However, PKA-mediated phosphorylation of Calpain-2 was decreased, leading to increased activity, in contrast to blood platelets. A possible explanation for this discrepancy between platelets and brain tissue is that platelets represent a pure cell population, whereas the brain is a mixed-cell population. Therefore, it is possible that cells with reduced phosphorylation of Calpain-2 are not the cells with high Talin-1 expression. For instance, the brain tissue consists of both excitatory and inhibitory neurons and earlier studies using neuronal cultures derived from either adult Nbea
+/- mice or E18 Nbea
-/- mice revealed an imbalance in excitatory and inhibitory signaling with a more affected inhibitory neurotransmission[
22,
23,
39]. Likewise, the phosphorylation status of Calpain-2 might be influenced by the excitatory/inhibitory imbalance present in the brain of Nbea
+/- mice. In addition, the expression level of Nbea varies in different brain regions[
16], which is likely to contribute to the alterations in PKA mediated phosphorylation on Calpain-2 upon Nbea haploinsufficiency.
The altered level of PKA-phosphorylated Calpain-2 is in line with the reported increase in PKA-mediated phosphorylation of the cAMP response element-binding protein (CREB) in a neuroendocrine cell line after knockdown of Nbea, and indirectly, the increased level of brain-derived neurotrophic factor (BDNF), a target of phospho-CREB, in Nbea
+/- mice[
40]. As well as Calpain-2, the phosphorylation status of other proteins is affected in Nbea
+/- mice, as indicated by the increased or decreased intensity of several proteins detected in a western blot of lysates of resting platelets or brain tissue with anti-phospho-(Ser/Thr) PKA substrate antibody. Although the alterations in PKA-mediated phosphorylation in total brain tissue from Nbea
+/- mice are more subtle, this might also be the result of the mixed-cell population and variable Nbea expression levels. The concurrence of increased, unaltered and decreased PKA phosphorylation of different proteins caused by Nbea haploinsufficiency can be explained by the compartmentalizing role of AKAPs. Because of its AKAP domain, Nbea belongs to the AKAP family of proteins, which is known to scaffold PKA near its target proteins in a distinct subcellular compartment. Different AKAPs lead to different PKA compartmentalizations[
48]. Haploinsufficiency of Nbea will lead to an altered subcellular distribution of PKA, which will cause a higher, lower or unchanged PKA-mediated phosphorylation of target proteins, depending on their subcellular presence. With a continuously expanding list of ASD candidate genes, there is increasing interest in signaling pathways linking ASD genetics with biological functions, such as growth and neurite outgrowth of developing neurons and synaptic function[
61,
62]. There is ample genetic evidence for the involvement of AKAPs in the etiology of ASD, and proteins encoded by 10 ASD-linked AKAP genes were shown to functionally integrate signaling cascades within and between several biological functions[
63]. Haploinsufficiency of
NBEA, one of the 10 ASD-linked AKAP genes, is thereby suggested to affect multiple pathways, most likely via the pleiotropic effect of altered PKA activity.
Competing interests
The authors have no competing interests to declare.
Authors’ contributions
KN and KT conducted experiments, analyzed and interpreted the data and drafted the manuscript. KB and EW conducted and analyzed experiments. MDM conducted experiments and assisted in the interpretation of the data. KF designed and conducted experiments, interpreted the data and helped to draft the manuscript. JC designed experiments, interpreted the data and helped to draft the manuscript. All authors read and approved the final manuscript.