The dissociation constant, and and shows that Arc mRNA is compartmentalized in the cytosolic and translocates in the polyribosomal fraction after reelin addition

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The dissociation constant, and and shows that Arc mRNA is compartmentalized in the cytosolic and translocates in the polyribosomal fraction after reelin addition. receptors (4) but also to 3,1 integrin receptors with high affinity (5) thereby ERK5-IN-2 activating a signal transduction pathway that induces the adapter function of the DAB1 protein (4C6). It is therefore possible that in the adult mammalian brain integrin receptors initiate a signal transduction cascade between ECM reelin and pyramidal neuron DSPSD. In rat hippocampal slices, integrin receptors may contribute to synapse maturation (7) or to long-term potentiation (LTP) consolidation by transducing ECM signals, leading to a cytoskeleton reorganization via the activation of extrasomal mRNA translation (8C10). This view is supported by inhibition of LTP consolidation by the addition of function-blocking antibodies to 5/1 or 3/1 integrin receptors or by integrin receptor antagonists including an RGD (Arg-Gly-Asp) motif (9, 10). Hence, an integrin-mediated signal transduction may be critical to the stabilization of the LTP-induced plasticity at central excitatory synapses. The application of reelin to adult rat hippocampal slices enhances LTP induction (11). Moreover, in heterozygous reeler mice (HRM) that contain reduced reelin gene dosage, cortical neuropil and dendritic spine density are reduced (12). These considerations suggest that reelin, acting at integrin receptors, could stabilize DSPSD, providing a molecular scaffold for the assembly of cytoskeletal proteins that facilitate dendrite resident mRNA translation and provide the increased protein synthesis required for LTP consolidation and memory trace formation. This hypothesis is consistent with the deficit in memory acquisition and the increased dizocilpine amnestic action characteristic of HRM (13, ?). The synthesis of (Arc) is encoded by dendritic resident mRNAs. This rapidly inducible protein (14) functions as an immediate early gene product that very likely is involved in spine skeleton formation during LTP stabilization (15). The reduction of Arc synthesis elicited by antisense oligonucleotides curtails LTP duration and the associated long-term spatial memory consolidation without affecting task acquisition or short-term performance (16). In frontal cortex and hippocampus of reeler and heterozygous reeler mice, Arc expression is curtailed. To determine whether reelin can modulate dendritic Rabbit Polyclonal to MRGX3 resident Arc mRNA translation, we studied whether reelin can change Arc and total protein synthesis in synaptoneurosomes (SNSs). Our results are consistent with the view that recombinant reelin acting at integrin receptors activates Arc biosynthesis in a manner inhibited by echistatin, a competitive antagonist at integrin receptors (10, 17), and rapamycin, a blocker of the (mTOR) kinase (18). Materials and Methods Preparation of SNSs. SNSs were prepared according to the flotation-sedimentation density gradient centrifugation method described by Jones and Matus (19). Briefly, mouse forebrains were homogenized in 10% sucrose. The crude mitochondrial pellet fraction (P2) was brought to 34% sucrose by the addition of 48% sucrose. A sucrose (28.5%) was overlaid on the P2 phase, and a small volume of 10% sucrose was overlaid onto the to obtain a gradient of 36 ml. The gradients were centrifuged at 60,000 for 110 min in a Surespin 630 Sorvall rotor, and three fractions were separated. The middle fraction (fraction 2) contained purified SNSs, which were then treated with 0.01% Triton X-100 ERK5-IN-2 for 30 min and washed three times with buffer to remove native adhering reelin. SNS membranes were prepared by resuspending SNSs in 5 mM Tris?HCl buffer (pH 8.1) at 0C for 30 min, and sonicating for 15 s. Label-Fracture. SNSs were fixed with a solution of ERK5-IN-2 cold 4% paraformaldehyde in PBS for 1 h and washed in PBS, osmicated, embedded in EPON812, and processed for electron microscopy. Other fixed SNSs were incubated with an anti-3 integrin receptor subunit antiserum diluted at 1:250 (20 h at 4C and 2-h incubation at 22C). Samples were then incubated for 2 h with a secondary antibody conjugated with colloidal gold particles 10 nm in size. Immunolabeled SNS samples were then cryoprotected by 30 min incubation in a solution of 30% glycerol, frozen in Freon 22 cooled by liquid nitrogen, fractured, and replicated in a Balzers 400D freeze-fracture unit. The replicas were cleaned in distilled water (for at least 6 h) before being transferred onto formvar-coated grids. Western Blot Analysis. Western blot immunostaining was performed with G10 anti-reelin (1:5,000; a generous gift of A. M. Goffinet, University of Namur, Brussels), anti-integrin 3 and 1 (1:250; Chemicon), anti-synaptophysin.