Bead-induced aggregation of YFP-TrkCTK- or YFP-TrkCTK+ on the dendrite surface resulted in significant coclustering

of PSD-95 and NR1 but not of gephyrin (Figures 5A–5F). Aggregation of YFP-CD4 negative control had no significant effects on clustering of either excitatory or inhibitory postsynaptic proteins. These results indicate that surface aggregation of TrkC on dendrites is sufficient to mediate glutamatergic postsynaptic differentiation. We also tested whether TrkC-induced aggregation of PTPσ on axons is a primary signal for triggering coclustering of presynaptic components. In axons of hippocampal neurons transfected with YFP-PTPσ, bead-induced aggregation of YFP-PTPσ, but not of YFP-PTPσΔICD, resulted in significant coclustering of synapsin (Figures 5G and 5H). Thus, PTPσ mediates presynaptic differentiation via its intracellular region. To obtain direct buy Bafilomycin A1 evidence about the involvement of the TrkC-PTPσ interaction in excitatory synapse development,

we explored the ability of TrkC antibodies to neutralize this interaction. The rabbit monoclonal antibody C44H5 against TrkC recognizes a part of the LRRCC region (data not shown) within the synaptogenic PTPσ-binding BVD-523 manufacturer domain of TrkC (Figure 1 and Figure 2). Thus, first we tested whether TrkC antibody C44H5 blocks TrkC-PTPσ interaction. We mixed soluble TrkC-Fc with C44H5, or rabbit polyclonal anti-TrkA antibody as a negative control, in a range of concentrations and then applied the mixture to PTPσ-expressing COS cells. C44H5 blocks the binding of TrkC-Fc to PTPσ in a dose-dependent manner (Figure 6A) with a half-maximum neutralizing dose (ND50) of about PDK4 2.2 μg/ml. Next, we tested whether C44H5

blocks synaptogenic activity of TrkC. We applied ∼10 μg/ml C44H5 or nonimmunized rabbit control IgG to cocultures of hippocampal neurons with COS cells expressing TrkCTK- or control neuroligin-2. TrkC antibody C44H5 almost completely blocked synapsin clustering induced by TrkCTK- but had no effect on synapsin clustering induced by neuroligin-2 (Figures 6B and 6C). We also tested whether C44H5 blocks synaptogenic activity of PTPσ. We applied ∼10 μg/ml C44H5 or control IgG to hippocampal neurons treated with PTPσ-Fc-coated beads or neurexin1β-Fc-coated control beads. C44H5 significantly yet partially suppressed PSD-95 clustering induced by PTPσ-Fc beads but had no effect on PSD-95 clustering induced by neurexin1β-Fc beads (Figures 6D and 6E). Thus, C44H5 is a neutralizing antibody that inhibits bidirectional synaptogenic activity of the TrkC-PTPσ complex. Finally, we tested by using C44H5 whether the endogenous TrkC-PTPσ interaction is essential for synapse formation in cultured hippocampal neurons. We treated cultured hippocampal neurons with ∼10 μg/ml of C44H5 or control IgG every day from DIV 9 to DIV 12, and then analyzed synapse markers at DIV12.5.