Notably, the density of structures double positive for either presynaptic active zone marker (munc13-1, bassoon) and postsynaptic scaffolding proteins (homer, PSD95) was unaltered, indicating that mSYD1A loss in cultured neurons does not change synapse density but only presynaptic composition (Figure 2F). We tested whether function of mSYD1A is specifically required

in the presynaptic cell by introducing a human, siRNA-resistant form of SYD1A (hSYD1A) into the synaptophysin-mCherry-positive cells. Importantly, this was sufficient to rescue the presynaptic terminal Apoptosis Compound Library supplier density back to wild-type level (Figures 2C and 2D). Recording of miniature excitatory postsynaptic currents (mEPSCs) in mSYD1A knockdown cultures further supported a presynaptic phenotype. The mEPSC frequency in knockdown neurons was reduced by 43% ± 7% as compared to PLX-4720 cost controls (Figure 2G). This reduction was rescued by re-introduction of hSYD1A using lentiviral infection (see Figure S2 for re-expression level and further controls for the RNA interference experiments). In combination with the morphological effects on synaptic vesicle distribution these results demonstrate that mSYD1A controls presynaptic differentiation in cultured neurons and is required in the presynaptic cell. Some functions of invertebrate

SYD-1 proteins are thought to rely on a catalytically inactive Rho-GAP-like domain whereas others have been pinpointed to the PDZ-domain of the protein (Hallam et al., 2002 and Owald et al., 2012). Mammalian SYD1 proteins differ significantly from their invertebrate counterparts in that they lack PDZ-domains and contain Rho-GAP domains that may be active based on amino acid sequence analysis all (Figure S3A). We directly probed GAP activity of mSYD1A in intact cells using a FRET-based assay (Itoh et al., 2002 and Pertz et al., 2006; Figure 3A). Using a RhoA sensor,

we observed significant RhoA inactivation in cells expressing mSYD1A. The degree of RhoA inactivation was similar to that observed for p50rhoGAP, a well-characterized GAP (Figures 3B and 3C). Importantly, mutation of the arginine finger (Graham et al., 1999) in mSYD1A (R436A) strongly reduced mSYD1A activity observed in this assay and no change in FRET was observed when Lin-2/CASK, a protein lacking GAP domains, was introduced (Figures 3B and 3C). Similarly, the amino acid alterations from the Rho-GAP consensus seen in the C. elegans and Drosophila SYD-1 proteins strongly reduce activity toward RhoA ( Figure S3B). Finally, we used morphological changes of neuronal dendrites as a read-out for RhoA regulation in cerebellar granule cells. Overexpression of C-terminally Myc-tagged mSYD1A but not the R436A or ΔYRL mutants led to a significant increase in dendritic trees compared to GFP-transfected neurons ( Figure S3D).