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ADAP is required for dendritic outgrowth. (A) The complexity of dendritic structures is reduced upon ADAP knock down and recovered upon expression of the rescue construct. Dendrite formation was analyzed in primary hippocampal neurons based on the reconstruction of transfected cells via the EGFP signal. Scale bar, 100 μm. (B) Quantification of dendrite branches using Sholl’s method revealed a significant reduction in the total number of dendritic intersections (n = 25–29 per condition). (C) Reduction of intersections is evident in knock-down cells along the entire dendritic arbor, whereas the rescue construct enhances growth over the first 150 μm. Data are Mean ± SEM. *p < 0.01.

ADAP is required for dendritic outgrowth. (A) The complexity of dendritic structures is reduced upon ADAP knock down and recovered upon expression of the rescue construct. Dendrite formation was analyzed in primary hippocampal neurons based on the reconstruction of transfected cells via the EGFP signal. Scale bar, 100 μm. (B) Quantification of dendrite branches using Sholl’s method revealed a significant reduction in the total number of dendritic intersections (n = 25–29 per condition). (C) Reduction of intersections is evident in knock-down cells along the entire dendritic arbor, whereas the rescue construct enhances growth over the first 150 μm. Data are Mean ± SEM. *p < 0.01.

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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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... of β1-integrin is critical for dendritic development in neuronal cells ( Schlomann et al., 2009;Rehberg et al., 2014); therefore we next examined the potential effect of ADAP knock down on dendrite formation in primary hippocampal neurons (Figure 3). Here we observed that shRNA-mediated ADAP knock-down induces a significant reduction of neurite growth, which can be recovered by co-expression of an shRNA- resistant ADAP expression construct. ...

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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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... 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 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.