In a complementary, shRNA-independent approach to assess the role of LRRTM4 in synapse development, we treated hippocampal neurons with excess LRRTM4-Fc to FRAX597 solubility dmso competitively disrupt the trans-synaptic interaction of LRRTM4 with presynaptic receptors.

Neurons were treated for 6 days and the density of VGlut1/PSD-95-positive puncta in Prox1-positive cells was analyzed at DIV14. LRRTM4-Fc treatment reduced excitatory synapse density by 40% compared to cells treated with Fc control protein, similar to treatment with LRRTM2-Fc ( Figure 5O). These results are in agreement with the effects of LRRTM4 knockdown, supporting a role of LRRTM4 in regulating excitatory synapse development. LRRTM2 and LRRTM4 share a similar synaptogenic activity in hippocampal neurons, but LRRTM4 is distinct from LRRTM2 in that it has two

presynaptic binding partners, neurexin and glypican. To begin assessing the role of these two LRRTM4 receptors in synapse development, we tested whether excess GPC4-Fc or Nrx1β(-S4)-Fc could block excitatory synapse formation in Prox1-positive neurons. In agreement with previous results (Chih et al., 2006), 6-day treatment with Nrx1β(-S4)-Fc caused a reduction in excitatory synapse density in DIV14 hippocampal neurons (Figures S6A and S6B). However, GPC4-Fc did not affect excitatory synapse density at this time point nor did 3-day treatment with GPC4-Fc in immature neurons see more (Figures

S6A–S6D). Possibly, neurexin can compensate when the glypican-LRRTM4 interaction is blocked. Alternatively, since LRRTM4-Fc decreases excitatory synapse density (Figure 5O), but GPC4-Fc does not (Figure S6), it could be that GPI-anchored glypican is only part of a functional presynaptic LRRTM4 receptor and requires a yet unidentified transmembrane signaling coreceptor. Such signaling might be required for the development of synaptic contacts between neurons. We next analyzed whether excess HS could interfere with LRRTM4-mediated synapse formation onto heterologous cells. HEK293T cells already expressing myc-LRRTM2 or myc-LRRTM4 were cocultured with DIV7 hippocampal neurons for 12 hr in the presence of 0.5 mg/ml HS. Exogenous HS did not affect LRRTM2-mediated presynaptic differentiation but abolished the synaptogenic activity of LRRTM4 (Figures 6A–6D). To test whether LRRTM4-mediated presynaptic differentiation requires endogenous HS, we treated DIV7 neurons with heparinase III (2 hr, 1 U/ml), washed and cocultured them for an additional 8 hr with 293T cells expressing myc-LRRTM. Staining with the 3G10 HS stub antibody confirmed the efficiency of hepIII treatment in hippocampal neurons (data not shown). Enzymatic removal of HS did not affect LRRTM2’s ability to instruct presynaptic differentiation (Figures 6E and 6F) but strongly reduced LRRTM4’s synaptogenic activity (Figures 6G and 6H).