THE ROLE OF THE ACTIN CAPPING PROTEIN EPS8 IN EXCITATORY SYNAPSE FORMATION AND FUNCTION

Our group has previously demonstrated the role of Eps8 (epidermal growth factor receptor pathway substrate 8) in regulating the formation of synapse-precursor structures in cultured hippocampal neurons. Indeed, neurons lacking Eps8 showed an increased number of axonal and dendritic filopodia, whereas the overexpression of this protein induced the formation of flat structures resembling lamellipodia. Moreover, we have demonstrated that the capping activity of Eps8 is regulated by BDNF. Indeed, upon BDNF stimulation, Eps8 is phoshorylated by MAPK, pEps8 detaches from the barbed ends of actin filaments, thus allowing the growth of new filopodia. Accordingly, Eps8 null neurons are no longer able to increase filopodia density upon BDNF stimulation [Menna et al., 2009]. Within the mouse brain Eps8 acts mainly as a capping protein [Menna et al., 2009]. Its role in the formation of filopodia –synapse-precursor structures- and in the BDNF pathway prompted us to further investigate the involvement of Eps8 in synaptic contact formation and function. To this aim, wt and null animals were subjected to learning and memory tasks. Eps8 KO mice showed a serious impairment in spatial, episodic memory and social behavior; these defects were associated also with an increased density of excitatory synapses and alteration of dendritic spine morphology of CA1 hippocampal pyramidal neurons. Moreover, the formation of new dendritic spines which normally occurs during memory formation was completely absent in mutated mice. In order to elucidate the molecular and cellular basis of such defects, cultured hippocampal neurons were established from both wt and Eps8 KO mice. Null cultures displayed an increase density of excitatory synapses and dendritic spines, which also presented immature features when compared with aged-matched wt cultures. In addition Eps8 null cultures were not able to undergo synaptic potentiation upon stimulation with a chemical LTP protocol. Transfection of neurons with cDNAs encoding for Eps8 wt or for a capping mutant (named Eps8H1) allowed us to dissect the involvement of capping activity in the process of synapse formation and maturation. In fact neurons expressing wt protein displayed an increased number of synapses and bigger spines; conversely, such modifications were not induced by the H1 capping mutant expression. We also showed that the capping activity of Eps8 is necessary for the expression of LTP. Indeed, the injection of a synthetic peptide which inhibits the Eps8 capping activity into the postsynaptic neuron via the patch pipette prevented synaptic potentiation. Finally, we found that Eps8 levels were significantly decreased in the brain of autistic patients suggesting that Eps8 may be involved in the pathogenesis of autism [Menna and Zambetti et al., 2013].