Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling, Behaviors, and Short Term Plasticity

Yumi Watanabe,Norikazu Katayama,Kosei Takeuchi,Tetsuya Togano,Rieko Itoh,Michiko Sato,Maya Yamazaki,Manabu Abe,Toshiya Sato,Kanako Oda,Minesuke Yokoyama,Keizo Takao,Masahiro Fukaya,Tsuyoshi Miyakawa,Masahiko Watanabe,Kenji Sakimura,Toshiya Manabe,Michihiro Igarashi

doi:10.1074/jbc.m113.504050

Abstract

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

Highlights

Roles of the syntaxin-1A1⁄7CaMKII interaction are not physiologically understood in vivo
Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins
Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal calmodulin-dependent protein kinase II (CaMKII)/syntaxin interaction

Summary

Conclusion

The CaMKII/syntaxin-1A interaction is essential for maintenance of neuronal plasticity. Our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo. We reasoned that interactions between syntaxin-1A and CaMKII may play an important role in the regulation of STP; we generated syntaxin-1A (R151G) knock-in (KI) mice that carry a single point mutation in syntaxin-1A. SNARE complex formation itself and basal transmission were normal in these KI mice These phenotypes, together with abnormal behaviors indicative of excessive brain activity, may have resulted from insufficient CaMKII activity in presynaptic termini. Knock-in of this single point mutation (syntaxin-1A (R151G)) attenuated the interaction between syntaxin-1A and CaMKII and resulted in abnormal regulation of exocytosis, STP, SV recycling, and higher brain functions that regulate behaviors. We conclude that the action of CaMKII in the presynaptic terminal is closely related to the regulation of STP because of an interaction between CaMKII and syntaxin-1A and the consequent action of complexin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION