**3. Signaling events during spine formation and maintenance**

Motility of dendritic spines is regulated by the dynamic balance between G-actin and F-actin [39]. F-actin consists of two pools, a large dynamic pool in the tip of the spine head and a small stable pool in the base of spine [6] [40]. With LTP induction, the stable F-actin is severed into short segments and reorganized to expand the spine [41]. Thus, the dynamics of actin cytoskeleton controls dendritic spine morphological remodeling and plenty of signaling molecules participate in this process [42–44].

Spine morphology is regulated by actin binding and cytoskeleton proteins. Drebrin was the first identified to modify dendritic spines since overexpression of drebrin in cultured neurons increased the length of spines [45]. Spines in the drebrin knockout mice exhibited normal morphology but altered plasticity [46]. Takahashi et al. reported that drebrin entered filopodia and formed an actin filament cluster to recruit postsynaptic components (including scaffolding protein PSD95), and this process enables the transition from filopodia to mature spines. Based on this observation, filopodia are classified into two types, an immature diffuse-type and a mature cluster-type. A filopodium with a drebrin cluster, whose maximum intensity was higher than twice the average intensity of the filopodium, was classified as a cluster-type filopodium. Otherwise, it was classified as a diffuse-type filopodium. The clustertype filopodia were likely to be converted to mature spines [47]. In addition, overexpression of drebrin in neurons caused F-actin to accumulate in the growth cone, whereas knockdown of drebrin reduced F-actin level [48]. Drebrin binds to F-actin to generate thick bundles of F-actin [49], and drebrin also competes with other actin binding proteins such as ADF/cofilin which depolymerizes F-actin [50, 51].

Besides drebrin, other actin-binding proteins including myosin II, Abi-1 and spinophilin regulate actin polymerization in the dendritic spines. Myosin II belongs to the family of molecular motors which is highly expressed in dendritic spines, and regulates dendritic spine morphology and synaptic plasticity [52]. Blockade of myosin II with shRNA suppressed the formation of mushroom-like spines and increased the presence of filopodia [53]. Abi-1 is a member of the c-Abl tyrosine interactor (Abi) protein family, which interacts with scaffolding proteins and F-actin in the spines [54]. Knocking down of Abi-1 by RNAi shifted spines to an immature form [55]. Spinophilin has an actin-binding domain at its N terminus and can bundle F-actin filaments [56]. Knockout of spinophilin in mice increased the presence of filopodia [57].

Actin polymerization is regulated by actin binding proteins, whose active and inactive states are regulated by small GTPases. Of the Rho family of small GTPases, three are most actively involved in spine morphogenesis, RhoA, Rac1 and Cdc42. These three GTPases are distinguished by two opposite activities: RhoA inhibits whereas Rac1 and Cdc42 promote spine growth. Tashiro et al. reported that in hippocampal neurons, Rac1 increased spine density but reduced spine length, while RhoA decreased both spine density and spine length [58]. Interestingly, RhoA and Cdc42 play opposite roles in stress fiber formation by controlling the phosphorylation of myosin light chain. RhoA inhibits myosin phosphatase via the Rho kinase while Rac1 and Cdc42 activate it via the serine/threonine kinase PAK [59, 60]. Thus, Rac1 and RhoA might have opposite effects on the same target proteins and hence opposite effects in regulating spine density. Similarly, Nakayama et al. found that Rac1 is essential for the maintenance of dendritic spines while enhanced RhoA activity led to significant simplification of dendrites [61].

Receptor tyrosine kinases also regulate spine morphology. Among them, the erythropoietinproducing hepatocellular carcinoma (Eph) receptors have unique activity on synapse. They consist of type A and type B receptor subclasses based on their binding capability to Ephrin A and Ephrin B ligands. Moeller et al. reported that activation of EphB2 in the cultured hippocampal neurons led to shortening of filopodia [62]. Furthermore, activation of EphB likely phosphorylates guanine exchange factors (GEFs) such as kalirin7, which further stimulates Rho family GTPases Rac1 and Cdc42 [63]. Opposite to EphB2, activation of EphA4 by its ligand, ephrin-A3, located in the perisynaptic processes of astrocytes, decreased spine length and density. Loss of EphA4 led to spine elongation and disorganization [64]. Similarly, in the hippocampus of ephrin-A3-null mice, EphA4 phosphorylation was decreased and abnormal spine elongation was observed [65]. Thus, either loss of EphA4 or ephrin-A3 induces identical dendritic spine deficits.

In summary, we have reviewed three types of important and representative signaling molecules in spine function. The first signaling pathway is mediated by actin binding proteins, the second is the family of small GTPases (including Rac1, RhoA and Cdc42, which determine the activity states of actin binding proteins), and the third is receptor tyrosine kinases. The absence or malfunctioning of the above three signaling pathways leads to altered spine morphogenesis and function.
