Dendritic spines have two major structural elements: postsynaptic densities (PSDs) and actin cytoskeletons. PSD proteins are proposed to regulate spine morphogenesis. However, other molecular mechanisms should govern spine morphogenesis, because the initiation of spine morphogenesis precedes the synaptic clustering of these proteins. Here, we show that synaptic clustering of drebrin, an actin-binding protein highly enriched in dendritic spines, governs spine morphogenesis. We immunocytochemically analyzed developing hippocampal neurons of low-density cultures. Filopodia-like dendritic protrusions were classified into two types: diffuse-type filopodia, which have diffuse distribution of drebrin, and cluster-type filopodia, which have drebrin clusters with filamentous actin (F-actin). Most cluster-type filopodia were synaptic filopodia. Postsynaptic drebrin clusters were found in both most synaptic filopodia and spines. Postsynaptic PSD-95 clusters, however, were found in only one-half of synaptic filopodia but in most spines. These data indicate that cluster-type filopodia are not mature spines but their precursors. Suppression of the upregulation of drebrin adult isoform (drebrin A) by antisense oligonucleotides against it attenuated synaptic clustering of PSD-95, as well as clustering of drebrin and F-actin. Furthermore, the restoration of drebrin A expression by injection of the expression vectors of drebrin A tagged with green fluorescent protein into the neurons treated with the antisense oligonucleotides induced synaptic reclustering of PSD-95 on clusters of the labeled drebrin A. These data indicated that the synaptic clustering of drebrin is necessary for that of PSD-95 in developing neurons. Together, these data suggest that synaptic clustering of drebrin is an essential step for spine morphogenesis.

SignificanceBrain-derived neurotrophic factor (BDNF) is a neurotrophin that elicits biological effects on synaptic plasticity. BDNF is initially synthesized as precursor proBDNF, and then the BDNF pro-peptide is simultaneously produced from the precursor protein. However, the physiological functions of the pro-peptide are largely unknown. Here, we demonstrate that the BDNF pro-peptide is a facilitator of hippocampal long-term depression (LTD), requiring the activation of GluN2B-containing NMDA-type receptors and the pan-neurotrophin receptor p75NTR. Second, a common BDNF polymorphism substitutes valine for methionine at amino acid position 66 (Val66Met) in the pro-peptide of BDNF and impairs memory function. Unexpectedly, the pro-peptide with Met mutation completely inhibits hippocampal LTD. These findings provide insights into the physiological role of the BDNF pro-peptide in the brain.

Drebrin is a major F-actin-binding protein in the brain. We have recently demonstrated that drebrin A (neurone-specific isoform) clusters at synapses and governs targeting of the post-synaptic density 95 protein to synapses during development. To determine the role of drebrin A on excitatory synapse formation, we analysed whether the suppression of drebrin A expression affects filopodia-spine morphology and synaptic targeting of NMDA receptors in cultured hippocampal neurones. Suppression of developmentally programmed up-regulation of drebrin A by antisense treatment significantly decreased the density and width of filopodia-spines.Immunocytochemistry showed that the antisense treatment did not attenuate synaptic clustering of NMDA receptors under conditions that permitted spontaneous activities but inhibited the accelerated targeting of NMDA receptors into synapses by its antagonist D-(-)-2-amino-5-phosphonopentanoic acid. These results indicate that drebrin A up-regulation plays a pivotal role in spine morphogenesis and activity-dependent synaptic targeting of NMDA receptors. Keywords: actin cytoskeleton, antisense oligonucleotide, dendritic spine, drebrin, NMDA receptor, synaptic targeting. Drebrin A is a neurone-specific F-actin-binding protein and its expression is increased in parallel with synapse formation (see review, Shirao 1995). Drebrin A is found exclusively in dendrites and is particularly concentrated in dendritic spines (Aoki et al. 2005). Overexpression of drebrin A in mature neurones induces elongation of spines (Hayashi and Shirao 1999). Down-regulation of drebrin A expression during the process of spine morphogenesis suppresses the accumulation of spinal F-actin, the most prevalent cytoskeletal element in spines (Takahashi et al. 2003). These observations suggest that spine morphogenesis depends on the drebrin A accumulation in the spine.Further, drebrin is necessary for synaptic clustering of a post-synaptic density (PSD) scaffold protein, PSD-95 (Takahashi et al. 2003). PSDs contain high concentrations of NMDA receptors (NMDARs) as well as PSD-95 (Kennedy 2000). As PSD-95 binds to the NMDAR subunits NR2A and NR2B (Niethammer et al. 1996), drebrin A might also regulate synaptic targeting of NMDARs via PSD-95.In this study, we analysed whether down-regulation of drebrin A expression affects spine morphogenesis and synaptic targeting of NMDARs in developing hippocampal neurones. Our results indicate that developmentally programmed up-regulation of drebrin A expression is involved in the spine morphogenesis and activity-dependent synaptic targeting of NMDAR. Materials and methodsHippocampal cell culture and antisense treatment Low-density cell cultures of hippocampal neurones were prepared by Banker's methods (Takahashi et al. 2003 Address correspondence and reprint requests to Tomoaki Shirao, Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, 3-39-22, Showamachi, Maebashi, Gunma, 371-8511, Japan. E-mail: tshirao@med.gunma-u.ac.jpAbbreviations used: ...