Which site or sites of action are relevant for activity-induced new spine growth? We observed that expression of Rpt6-S120A in individual neurons inhibited activity-induced spine outgrowth. Because this genetic manipulation was carried

out in sparsely transfected RO4929097 datasheet neurons, and thus any nearby presynaptic neurons were untransfected, our results demonstrate that postsynaptic proteasomal function is necessary to facilitate new spine growth. In addition, because global pharmacological inhibition was not more effective at reducing spine outgrowth than overexpression of Rpt6-S120A in individual postsynaptic cells, our data also suggest BIBW2992 mw that independent presynaptic

and circuit-wide effects do not contribute significantly to the observed reduction in new spine growth. Finally, uncaging-induced spine outgrowth, which is independent of presynaptic activity, was also significantly reduced by blocking the proteasome, emphasizing the role of localized postsynaptic signaling. Our results strongly support a postsynaptic site of action for the proteasome in activity-induced new spine outgrowth. How might synaptic activity and the proteasome act together to facilitate new spine growth? One possibility is that synaptic activity enhances proteasome function to cause the emergence of new spines. Alternatively, the synaptic stimulus could be the primary cause of spine outgrowth, and normal steady-state levels of proteasomal degradation are required for activity-induced new spine growth. We think that the latter possibility is unlikely

because expression of Rpt6-S120A for 4 days does not produce any noticeable effects on cell health compared to untransfected neurons, suggesting that general proteasomal function Idoxuridine is not significantly disrupted. In addition, the Rpt6-S120A mutation does not interfere with normal steady-state levels of proteasome-mediated protein degradation in heterologous cells (Djakovic et al., 2012). Instead, because the Rpt6-S120A mutation blocks CaMKII-mediated enhancement of proteasomal degradation (Djakovic et al., 2012), our data suggest that locally enhanced proteasomal degradation, probably through CaMKII phosphorylation of Rpt6 at S120, is required for activity-induced new spine growth. How might neural activity translate to enhanced local proteasomal degradation? Changes in neuronal activity have been shown to alter both proteasome activity (Bingol and Schuman, 2006 and Djakovic et al., 2009) and localization (Bingol and Schuman, 2006 and Bingol et al., 2010).