Pre- and postsynaptic localization of RC3/neurogranin in the adult rat spinal cord: an immunohistochemical study.

RC3 (neurogranin; BICKS) is a neuron-specific calmodulin-binding protein kinase C substrate. Thus far, immunohistochemical studies on the localization of RC3 revealed its presence in all neuronal phenotypes, which were restricted to specific areas in the neostriatum, the neocortex, and the hippocampus. RC3 was mostly found in cell bodies and dendrites, with some infrequent presence in axonal profiles, i.e. in the internal capsule. Until now, RC3 expression was reported to be absent in the adult rat spinal cord. RC3 might, however, act as an intermediate of protein kinase C-mediated signaling pathways during synaptic development and plasticity. We hypothesized a role for this 78-amino-acid protein in dendritic plasticity occurring after spinal cord injury. To our surprise, an immunohistological analysis of the uninjured adult rat spinal cord revealed the presence of RC3-positive cell bodies and dendrites in specific regions in the gray matter. Interestingly, axon-containing structures, such as the dorsal and ventral corticospinal tract, were also found to be RC3-positive. This axonal labeling was confirmed by preembedding electron microscopy. In conclusion, we demonstrate here that RC3 is present in the adult rat spinal cord in pre- and postsynaptic structures.

B-50/GAP-43 potentiates cytoskeletal reorganization in raft domains.

B-50 (GAP-43) is a neural, membrane-associated protein that has been implicated in neurite outgrowth and guidance. Following stable transfection of Rat1 fibroblasts with B-50 cDNA we observed a dispersed distribution of B-50 immunoreactivity in flattened resting cells. In contrast, motile cells exhibited high concentrations of B-50 at the leading edge of ruffling membranes, coinciding with actin polymerization. Time-lapse studies on Rat1 fibroblasts transiently transfected with B-50/EGFP revealed that large vesicles originated from the ruffling membranes. These large vesicles (pinocytes) were found positive for Thy-1, a GPI-anchored protein, but negative for rab-5, an early endosome marker. In primary hippocampal neurons B-50 also colocalized completely with the raft marker Thy-1. Antibody-mediated cross-linking of Thy-1 in hippocampal neurons resulted in a redistribution of the intracellular protein B-50 to Thy-1-immunopositive membrane patches, whereas syntaxin was mainly excluded from the patches, showing that B-50 is associated with rafts. Academic Press.

AlphaCaMKII binding to the C-terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post-synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C-terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S-transferase (GST)-NR2A(1349-1464) binds native CaMKII from solubilised hippocampal PSDs in 'pull-out' and overlay experiments and this binding is competed by recombinant alphaCaMKII(1-315). The longer GST-NR2A(1244-1464), although containing the CaMKII phosphosite Ser-1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349-1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.