4 B). Open in a separate window Figure 4. Syndapin I interacts with ProSAP1 in vivo. to be important spatial cues and organizing platforms, shaping dendritic membrane areas into synaptic compartments. Introduction Brain development and function relies on remodeling of neuronal membranes. The postsynapses of glutamatergic neurons often protrude from dendrites as dendritic spines and contain neurotransmitter receptors and signaling components interconnected by scaffold proteins, such as ProSAP/Shanks, which additionally interact with F-actin 3-Methoxytyramine binding proteins (Hering and Sheng, 2003; Qualmann et al., 2004). Receptor clustering and linkage to F-actin thus represent two prominent mechanisms in synaptogenesis (Tada and Sheng, 2006). ProSAP/Shank deficiencies led to reduced synapse or spine densities and seem related to autism spectrum disorders (Grabrucker et al., 2011; Pe?a et al., 2011; Berkel et al., 2012; Schmeisser et al., 2012). Yet, the mechanisms that shape dendritic spines during formation and help to coordinate membrane remodeling, local actin nucleation, and postsynaptic scaffold formation remain elusive. Membrane shaping can be mediated by cytoskeletal causes and membrane-associated proteins. Syndapins (PACSINS) belong to the F-BAR subfamily of BAR domain proteins that are thought to shape membranes by scaffolding and/or partial insertion into one membrane leaflet (Qualmann et al., 2011). Syndapins have the potential to combine cytoskeletal and membrane shaping mechanisms. They interact with proteins promoting actin filament formation, interconnect SH3 domain binding partners via F-BAR domainCmediated self-association, and bind to membranes via their F-BAR domain (Qualmann et al., 1999; Itoh et al., 2005; Kessels and Qualmann, 2006; Dharmalingam et al., 2009; Schwintzer et al., 2011). The first loss-of-function analyses have indeed revealed a role for syndapin I in membrane shaping processes. Syndapin I knockout 3-Methoxytyramine (KO) mice had defects in retrieval and shaping synaptic vesicles in presynapses (Koch et al., 2011). Syndapin I was furthermore found to be crucial for early neuromorphogenesis and for ciliogenesis (Dharmalingam et al., 2009; Schwintzer et al., 2011; Schler et al., 2013). Considering its molecular and functional properties, we addressed a potential role of syndapin I in postsynapse formation by gene KO and by RNAi at specifically postsynaptic sides of evaluated excitatory synapses. Our biochemical and functional studies demonstrated that syndapin I plays a crucial role in dendritic spine and synapse formation based on SH3 domainCmediated associations with ProSAP1/Shank2 and F-BAR domainCmediated membrane binding. Consistently, syndapin I RNAi led to impairments in synaptic activity similar to ProSAP1/Shank2 KO and to defects in ProSAP2/Shank1 organization. Imaging of specifically membrane-associated, endogenous syndapin I at ultra-high resolution revealed that it preferentially 3-Methoxytyramine occurred in spines and formed clusters at membrane subareas of spines. Membrane-bound syndapin I nanodomains thereby can provide spatial cues and molecular organizing platforms during the formation of postsynapses. Results Neurons of syndapin I KO mice have reduced densities of dendritic spines Syndapin I KO mice suffer from generalized seizures correlating with altered neuronal network activity (Koch et al., 2011). To evaluate whether defects in synaptic organization may contribute to this phenotype, we analyzed Golgi-stained 3-Methoxytyramine CA1 hippocampal sections. The density of spines protruding from syndapin I KO neurons was decreased by 15% when compared with wild type (WT; Fig. 1, A and B). Open in a separate window Figure 1. Significant reduction of dendritic spine density in neurons of syndapin I KO mice. (A) Maximal intensity projections of inverted brightfield images of dendrites of Golgi-stained hippocampal CA1 neurons from adult WT (+/+) and syndapin I KO mouse (?/?) brain sections (top) and corresponding reconstruction and classification of dendritic spines with Imaris software showing stubby (red), thin (blue), and mushroom spines (green) as well as filopodia-like spines (magenta; bottom). Thin, white lines are Imaris evaluation grids. Bar, 4 m. (BCF) Quantitative Rabbit Polyclonal to MRIP analyses of 3-Methoxytyramine all types of spiny protrusions. **, P 0.01; ***, P 0.001. Data represent mean SEM (error bars). More detailed analyses revealed that filopodia-like spine density was not changed (Fig. 1 C). Instead, the density of thin spines declined by 18%, and the densities of stubby and mushroom spines declined by 25% and 27%, respectively (Fig. 1, DCF). Synapse formation requires postsynaptic SH3 domainCdependent syndapin I functions.