Thus, the intracellular trafficking and synaptic targeting of NMDA receptors should be one of the prerequisites for NMDA receptor activity ( Lau and Zukin, 2007 and O’Brien et al., 1998). However, little is known about the precise roles and mechanisms of NMDA receptor transport in vivo. In the present study, we determined that KIF17 is critical to maintain NR2A/2B levels in different manners and that it Cobimetinib is required for multiple processes of long-term memory formation in the mammalian brain. Furthermore, we found that NR2B receptor supply is augmented on demand through a reciprocal interaction between CREB and NR2B/KIF17. Thus, our data

suggest that KIF17 is critical for a complex regulatory mechanism involving cargo transport, nuclear transcriptional signaling, and protein degradation that controls the levels of NR2A/2B.

We generated mice with a disrupted kif17 gene using a poly(A) trap strategy (see Figures S1A–S1C available online). Absence of KIF17 in kif17−/− mouse brain was verified by immunoblotting with a polyclonal antibody against KIF17 ( Figure S1D). The kif17−/− mice grew normally without any gross anatomical defects in brain ( Figures S1E–S1G). Immunoprecipitation demonstrated FG-4592 price that KIF17 interacts with NR2B but not with NR2A in hippocampal extracts (Figure 1A; Setou et al., 2000). The level of NR2B protein in the hippocampi of kif17−/− mice was decreased to 42% of that in kif17+/+ mice ( Figure 1B). There was also a reduction Idoxuridine in the levels of NR2A and Mint1 (a scaffolding protein that binds to KIF17 to mediate NR2B transport; Setou et al., 2000), but no change in the levels of other related proteins ( Figures 1B and S2A).

The levels of nr2b mRNA in the hippocampi of kif17−/− mice were decreased compared with those in control mice, but the levels of nr2a mRNA were not ( Figure 1C). An RNA stability assay, using cultured hippocampal neurons treated with actinomycin D (10 μg/ml, an inhibitor of mRNA transcription), revealed no difference in the half-lives of both NR2B mRNA and NR2A mRNA between kif17+/+ and kif17−/− neurons ( Figures 1D–1F). Immunoreactivities for NR2B and NR2A in the hippocampal CA1 region of kif17−/− mice were reduced compared with those in control mice ( Figure S2B, upper panel and middle panel). Biochemically, kif17 deletion reduced the levels of NR2B and NR2A in synaptosomal membrane fractions by 43% and 22%, respectively, compared with those in kif17+/+ mice ( Figures 1G, S2C, and S2D). These data indicate that the amounts of synaptic NR2B and NR2A are decreased in kif17−/− mouse hippocampal neurons. To study NR2B/2A localization in kif17+/+ and kif17−/− mouse neurons in detail, we performed immunocytochemistry using hippocampal cultures. The lack of KIF17 resulted not only in a decreased density of NR2B/2A-positive clusters along dendrites, but also in a reduced size of NR2B/2A clusters in neurons ( Figures 2A–2F).