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Expression of adhesion and degranulation promoting adapter protein (ADAP) in hippocampal neurons. (A) ADAP expression can be detected in the somata of primary hippocampal neurons and along their MAP2-positive dendritc and MAP2-negative/Tau-positive axonal structures. Scale bar, 100 μm. (B) Immunoblot analysis with two different ADAP antibodies confirms the expression in juvenile (postnatal day 9) and adult (postnatal day 90) hippocampus, as well as in neurally differentiated PC12 cells (after 4 days of NGF treatment). ADAP expression in murine CD3+ T-cells is shown for comparison. (C) The specificity of immunocytochemical ADAP labeling is demonstrated in HEK-293T cells, which do not express endogenous ADAP. Positive signals are strictly limited to those cells that have been transfected with His-tagged ADAP, whereas non-transfected cells marked by DAPI staining alone are negative for ADAP immunoreactivity. Scale bar, 100 μm. (D) In 7 days in vitro (DIV7) hippocampal neurons, SKAP-HOM is distributed in soma and proximal dendrites along with ADAP, but not in a MAP2-negative neurite (arrow). Scale bar, 100 μm. (E) Immunoprecipitation (IP) of hippocampal lysate identifies SKAP-HOM, RAPL and MST1, but not RIAM as binding partners of ADAP in neuronal tissue. WL, whole lysate.

Expression of adhesion and degranulation promoting adapter protein (ADAP) in hippocampal neurons. (A) ADAP expression can be detected in the somata of primary hippocampal neurons and along their MAP2-positive dendritc and MAP2-negative/Tau-positive axonal structures. Scale bar, 100 μm. (B) Immunoblot analysis with two different ADAP antibodies confirms the expression in juvenile (postnatal day 9) and adult (postnatal day 90) hippocampus, as well as in neurally differentiated PC12 cells (after 4 days of NGF treatment). ADAP expression in murine CD3+ T-cells is shown for comparison. (C) The specificity of immunocytochemical ADAP labeling is demonstrated in HEK-293T cells, which do not express endogenous ADAP. Positive signals are strictly limited to those cells that have been transfected with His-tagged ADAP, whereas non-transfected cells marked by DAPI staining alone are negative for ADAP immunoreactivity. Scale bar, 100 μm. (D) In 7 days in vitro (DIV7) hippocampal neurons, SKAP-HOM is distributed in soma and proximal dendrites along with ADAP, but not in a MAP2-negative neurite (arrow). Scale bar, 100 μm. (E) Immunoprecipitation (IP) of hippocampal lysate identifies SKAP-HOM, RAPL and MST1, but not RIAM as binding partners of ADAP in neuronal tissue. WL, whole lysate.

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Integrin-mediated cell adhesion and signaling is of critical importance for neuronal differentiation. Recent evidence suggests that an “inside-out” activation of β1-integrin, similar to that observed in hematopoietic cells, contributes to the growth and branching of dendrites. In this study, we investigated the role of the hematopoietic adaptor pro...

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... expression was tested in primary hippocampal cultures, as well as in hippocampal tissue during development and adulthood. Immunocytochemical staining revealed ADAP expression in somata, dendrites and axons of primary neurons (Figures 1A,D, 4), as various stages of neuronal differentiation, including DIV3 (Figure 4A), DIV7 (Figures 1A,D), DIV10 (Figure 4B), as well as DIV14, DIV18 and DIV21 (data not shown). ...
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... expression was tested in primary hippocampal cultures, as well as in hippocampal tissue during development and adulthood. Immunocytochemical staining revealed ADAP expression in somata, dendrites and axons of primary neurons (Figures 1A,D, 4), as various stages of neuronal differentiation, including DIV3 (Figure 4A), DIV7 (Figures 1A,D), DIV10 (Figure 4B), as well as DIV14, DIV18 and DIV21 (data not shown). ...
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... this time point, ADAP can be found along the core neurite microtubules and the microtubule network of growth cones and growth tips and MAP2-negative ADAP-positive filaments are rarely observed (Figure 4A). However, at later stages of development ADAP-positive axons without MAP2 labeling are frequently observed in addition to the generally double-labeled dendritic structures (Figures 1A,D). ...
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... kDa corresponding to the ADAP signal obtained in naive T-cells. ADAP was also found in neural differentiated PC12 cells ( Figure 1B). ...
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... specificity of immunocytochemical ADAP labeling was confirmed using heterologous expression of ADAP-His tagged protein in HEK-293T cells, which are devoid of endogenous ADAP expression. Only cells with detectable signal against the His-Tag (1:500; Santa Cruz) displayed immunoreactivity for ADAP antibodies (Figure 1C). ...
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... complex is known to mediate TCR-induced inside-out activation of integrins. Indeed, staining of hippocampal neurons for SKAP-HOM resulted in a distributed labeling of somata and dendrites, similar to ADAP ( Figure 1D). Moreover, using anti-ADAP antibodies, we were able to co-precipitate the SKAP55 homolog SKAP-HOM, as well as RAPL and MST1, but not RIAM from hippocampal tissue ( Figure 1E). ...
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... staining of hippocampal neurons for SKAP-HOM resulted in a distributed labeling of somata and dendrites, similar to ADAP ( Figure 1D). Moreover, using anti-ADAP antibodies, we were able to co-precipitate the SKAP55 homolog SKAP-HOM, as well as RAPL and MST1, but not RIAM from hippocampal tissue ( Figure 1E). ...

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... For GAD67, the oligonucleotide sequence 5 ′ -GCTGGAAGTGGTAGACATACT-3' (corresponding to NM 008077, base pairs 616-636 in mouse and NM017007 base pairs 531-551, in rat) was used in the same manner. A random sequence shRNA ((5 ′ -TCGTCATGACGTGCATAGG -3 ′ (Thiere et al., 2016), and an anti-luciferase shRNA (shLuc) from pMIR-mU6-Luc (Rehberg et al., 2014) were used as controls. All shRNA constructs under U6 promoter were cloned into pll3.7 vector (Rubinson et al., 2003) using Hpa1-Xho1 restriction sites. ...
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... Two kinases of the Hippo-NDR signaling pathway, that is, Trc and Wts work in concert to establish and maintain dendritic fields, respectively (Emoto, 2012). Dendritic development in Hippocampal neurons is regulated by β1-integrin-hematopoietic adaptor protein adhesion and degranulation promoting adapter protein (ADAP)-mediated activation of Hippo kinase MST1 (Thiere et al., 2016). Moreover, angiomotin (AMOT) tune dendritic morphogenesis in Purkinje cells and in hippocampal cells by forming a complex with YAP1. ...
Article
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... MST1 participates in long-term potentiation and spatial memory processes [112]. Further research shows that MST1 serves as an important component in the dendritic development of hippocampal neurons [113]. A recent study showed that MST1 might participate in the regulation of brain size, and an interrupted interaction between CDK5RAP2 and MST1 might be relevant in autosomal recessive primary microcephaly [114]. ...
Article
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... Primary cultures of hippocampal neurons were obtained from embryonic 18-19-d Sprague-Dawley rats (Jin and Selkoe, 2015;Thiere et al., 2016). The isolated hippocampus was dissociated with trypsin, and the cells were plated on a 24-well culture plate coated with poly-D-lysine and containing Neurobasal medium supplemented with B27 (Gibco, Waltham, MA, USA). ...
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The amygdala plays a key role in the pathophysiology of depression, but the molecular mechanisms underlying amygdalar hyperactivity in depression remain unclear. In this study, we used a chronic mild stress (CMS) protocol to separate susceptible and insusceptible rat subgroups. Proteomes in the amygdalae were analyzed differentially across subgroups based on labeling with isobaric tags for relative and absolute quantitation (iTRAQ) combined with mass spectrometry. Of 2,562 quantified proteins, 102 were differentially expressed. Several proteins that might be associated with the stress insusceptibility/susceptibility difference, including synapse-related proteins, were identified in the amygdala. Immunoblot analysis identified changes in VGluT1, NMDA GluN2A and GluN2B and AMPA GluA1 receptors, and PSD-95, suggesting that CMS perturbs glutamatergic transmission in the amygdala. Changes in these regulatory and structural proteins provide insight into the molecular mechanisms underlying the abnormal synaptic morphological and functional plasticity in the amygdalae of stress-susceptible rats. Interestingly, the expression level of CaMKIIβ, potentially involved in regulation of glutamatergic transmission, was significantly increased in the susceptible group. Subsequent in vitro experimentation showed that overexpression of CaMKIIβ increased the expression of PSD-95 and GluA1 in cultured hippocampal neurons. This result suggested that CaMKIIβ functions upstream from PSD-95 and GluA1 to affect LTP-based postsynaptic functional plasticity in the amygdalae of susceptible rats. Therefore, amygdalar CaMKIIβ is a potential antidepressant target. Collectively, our findings contribute to a better understanding of amygdalar synaptic plasticity in depression.
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The adhesion and degranulation-promoting adapter protein (ADAP) is expressed in T cells, NK cells, myeloid cells, and platelets. The involvement of ADAP in the regulation of receptor-mediated inside-out signaling leading to integrin activation is well characterized, especially in T cells and in platelets. Due to the fact that animal studies using conventional knock-out mice are limited by the overlapping effects of the different ADAP-expressing cells, we generated conditional ADAP knock-out mice (ADAP fl/fl PF4-Cre tg ). We observed that loss of ADAP restricted to the megakaryocytic lineage has no impact on other hematopoietic cells even after stimulation conditions. ADAP fl/fl PF4-Cre tg mice showed thrombocytopenia in combination with reduced plasma levels of PF4 and TGF-β1. In vitro, platelets from these mice revealed reduced P-selectin expression, lower TGF-β1 release, diminished integrin αIIbβ3 activation and decreased fibrinogen binding after stimulation with podoplanin, the ligand of the C-type lectin-like receptor-2 (CLEC-2). Furthermore, loss of ADAP was associated with impaired CLEC-2-mediated activation of PLCγ2 and Erk1/2. Induction of experimental autoimmune encephalomyelitis (EAE) in mice lacking ADAP expression in platelets caused a more severe disease. In vivo administration of TGF-β1 early after T cell transfer improved EAE severity in mice with loss of ADAP restricted to platelets. Our results reveal a regulatory function of ADAP in platelets in vitro and during autoimmune disease EAE in vivo.