Regulation of dendritic spine morphogenesis and synapse formation by copines

Primary rat hippocampal culture is a well established system to study molecular mechanisms occurring during synapse formation and maturation. By closer analysis of the temporal appearance of presynaptic Bassoon and the postsynaptic proteins SynGAP, PSD-95 and GluR2 we were able to dissect synapse formation into distinct modules. Cultured neurons first show axonal clusters of the presynaptic protein Bassoon in the previously described 80 nm dense core vesicles, mobile aggregates of presynaptic proteins. In a second phase, neurons start to form PSD-95 and SynGAP positive synaptic structures that are absent for AMPA receptors. These “silent synapses” appear first in the somato-dendritic region and extend during time into more distal dendritic regions. In the same extend as the synapses appear at more distal regions, also the number, density, size and the colocalization of pre- and postsynaptic markers increases. Finally, in a third step, the number of synapses with incorporated AMPA receptors starts to rise, suggesting active synapses.
In a screen, aimed to detect genes upregulated during initial synapse formation in primary hippocampal culture we detected various copine family members. We then investigated the role of copine family members Knockdown of endogenous copines by RNAi during the period of synaptogenesis unveiled opposing roles on synapse formation. Loss of copine 3 reduced dendritic protrusions and caused a collapse of the dendritic tree during synapse formation. In contrast, knockdown of endogenous copine 6 triggered ectopic polymerisation of the actin cytoskeleton on dendritic filopodia and increased synapse size, number and activity. Thus, copine 6 appears to act as a synapse-suppressor. Interestingly, copines also affect mature spines in adult cultures. Copine 6 is expressed exclusively in the brain and within the brain mainly in the amygdala, hippocampus and the olfactory bulb. On the level of a single neuron, copine 6 is localized exclusively in the somato-dendritic compartment and therein it is enriched in spines.
Furthermore, by co-immunoprecipitation, we show that copine 3 and copine 6 interact with each other and with the actin-modulating small GTPases Rac 1 and Pak 1. Moreover, a knockdown of Pak 1 revert the effect of copine 6 on spine formation. These data suggests that copines contribute in morphological synaptic plasticity by regulating the actin cytoskeleton trough direct interaction with small Rho GTPases.