論 文 内 容 の 要 約(イン タ ーネッ ト 公 開用)
Internet Summary of thesis contents
Title: Structural and functional analysis of cytoplasmic region of synapse organizer Neurexin-1 that mediates intracellular signals for synapse formation
ナ プ ス 形 成 グ ナ ル の 伝 達 に 関 わ る ナ プ ス オ ー ガ ナ イ ー Neurexin-1 の細胞 内領域 の 構 造 機 能 解 析
Author: Chang Wen Hsin 張文欣 1,2 Affiliation:
1Department of Physiological Sciences, School of Life Science
SOKENDAI University (The Graduate University for Advanced Studies ),Japan
2National Institute for Physiological Sciences (N IPS), Okazaki, Japan
Content
-Introduction, -methods & Results -Conclusions
-Reference List
-Introduction
Chemical synapses are highly specialized inter-neuronal junctions that consist of pre- and postsynaptic parts where signals are released and received, respectively. Cell adhesion molecules are known to be required for recognition and connection between pre- and postsynapses. Neurexins are a family of synaptic cell adhesion molecules that bridge pre- and postsynapses and considered to be involved not only in synapse formation, but also maturation of synapses [1]. Neurexins are originally isolated as receptors for α-Latrotoxin, a widow spider venom, which attacks presynaptic terminals and induces massive neurotransmitter release resulting in dysfunction of synapses [2, 3].
Mammals possess three neurexin genes (Nrx1, Nrx2, and Nrx3), each of which has two independent promoters that produce longer α- and shorter β-form [4, 5]. α-Neurexin proteins contain six LNS (laminin, neurexin, sex-hormone globulin)
domains and three EGF (epidermal growth factor) -like repeats in their extracellular regions. α-Neurexins share the last LNS-domain to cytoplasmic region with β-Neurexins. Cytoplasmic tails of Neurexins are short, with ~ 50 amino acids, and they contain PDZ binding sequences at carboxyl terminals, where they bind a synaptic scaffolding protein, CASK [6].
All three neurexin genes are subjected to alternative splicing at five sites, all in their extracellular coding regions [5]. Among these, the effect of splice site 4 (s s4) has been best characterized. ss4 is located within the last LNS domain and the insertion affects interaction with Neuroligins or LRRTM2, postsynaptic ligands expressed predominantly in the cerebral cortex and the hippocampus [7 -9]. On the other hand, the insertion of this exon confers the binding affinity to Cbln1, the soluble protein secreted from granule cells in the cerebellum [10, 11].
Neuroligins are the first identified postsynaptic ligands for Neurexins [12]. They are also single transmembrane proteins with relatively long extracellular regions and short cytoplasmic tails. Extracellular region of Neuroligins contains acetylcholinesterase-like domain at which they bind to the last LNS domain of Neurexins in the presence of calcium. Neurexin-Neuroligin interaction has been demonstrated to induce synapse formation by artific ial synapse formation assay. Cultured neurons form synapses only onto surface of neurons, but not on non -neuronal cells. However, when non-neuronal cells, such as COS or HEK293 cells, were transfected with Neuroligins and co-cultured with neurons, neurons form presynaptic structures onto Neuroligin expressing non-neuronal cells. An addition of recombinant extracellular domain of Neurexin in this culture blocks the formatio n of presynaptic structures onto non-neuronal cells, suggesting that Neuroligins induce presynaptic formation via the interaction with Neurexins.
LRRTM2 are also enriched in postsynaptic terminals in pyramidal neurons in the
hippocampus and the cerebral cortex and induce presynaptic differentiation through interaction with Neurexins. Cbln1 links between Neurexin at parallel fiber terminals and GluR2 at dendritic spines of the Purkinje neurons in the cerebellum and induce the synapse formation [10]. These findings suggest that Neurexins may function as receptors for presynapse formation and maturation. But the mechanisms by which Neurexins transmit those signals remain unknown.
In my PhD work, I studied Neurexin functions at intracellular signaling. For this, I first studied subcellular localization and function of Neurexins in synapses using electron microscopy and electrophysiology, respective ly. Then I analyzed the structure of cytoplasmic tail of Neurexin -1. I also studied lipid binding with Neurexins because this may be critical for the signals beneath plasma membrane. Based on the results, I searched the mechanism for signal transduction required for synapse formation by focusing on phosphorylation and molecular interaction.
-Methods & Results
To study the subcellular localization of Neurexins, I first generated antibodies for Neurexins. I immunized peptide with last nine amino acid sequence of mouse Neurexins that conserved among all three Neurexins in rabbits and guinea pigs and obtained those antisera. After confirmation of the specificity of these antibodies by western blotting, I performed pre-embedding immuno electron microscopy in molecular layer of the cerebellum of mice. In this experiment, I detected Neurexin signals specifically in the presynaptic terminals of parallel fibers, suggesting that Neurexins are presynaptic.
To study the Neurexin function in synapses, I knock ed down all isoforms of neurexin genes by introduction of shRNAs in cultured mouse hippocampal neurons. I analyzed spontaneous synaptic activity using patch clamp technique in the presence
of tetrodotoxin and observed that frequency, but not amplitude, of miniature excitatory postsynaptic currents (mEPSCs) was selectively reduced in neurexin knocked down neurons, suggesting that the function of Neurexins was also presynaptic.
There was no clue to solve the intracellular signaling for Neurexins. For this , I first studied the structure of cytoplasmic tail (C -tail) of mouse Neurexin-1. A computer program, called “PSIPRED”, predicted that this region was intrinsically disordered. I confirmed this with a mouse recombinant Neurexin -1 C-tail protein using circular dichroism spectroscopy. Intrinsically Disordered Proteins (IDPs) are known to exhibit molecular interaction based on the property of amino acids constituting the region, and phosphorylation tends to affect the interaction. The C-tail of Neurexin-1 is enriched by basic amino acids. I examined the interaction with phospholipids constituting plasma membrane by dot blot using GST-fusion Neurexin-1 C-tail protein as a probe. In this experiment, I found that Neurexin C-tail bound negatively charged phospholipids including PI(4,5)P2. There are eight serines in Neurexin-1 C-tail. To test the effect of phosphorylation of these serines, I examined the phospholipid interaction with phospho -mimic mutant of Neurexin-1 C-tail of which serines were mutated to aspartic acids. In this experiment, I found that the phospho-mimic mutation in the last three serines sufficiently abolished the interaction. To identify the kinase responsible for this phosphorylation, I transfected HEK293T cells with mouse Neurexin constructs and treated with chemicals to induce kinase activities. I monitored the phosphorylation by western blotting with phos -tag gels in which phosphorylation was detected by mobility shift of Neurexin -1 band. In this experiment, I found that Protein kin ase C (PKC), but not Protein kinase A (PKA) nor Ca2+/calmodulin-dependent protein kinase II (CaMKII), phosphorylated these serines. I performed the same experiment in mouse cultured cortical neurons and
confirmed that this phosphorylation was conserved in neurons.
To study the effect of this phosphorylation on synapse formation, I employed HEK293T-neuron co-culture assay. Neurons do not form synapses onto non -neuronal cells, such as HEK293T cells. But when HEK293T cells were transfected with mouse Neuroligin-1, co-cultured neurons form synapses onto HEK293T cells. This artificial synapse formation was abolished when neurexins were knocked down in co-cultured neurons. Superinfection of lentivirus expressing wild -type mouse Neurexin-1 in this co-cultured neurons rescued the artificial synapse formation. But infection with phospho-mimic mutant Neurexin-1 failed to rescue the synapse formation. These results suggest that phosphorylation of Neurexin -1 negatively regulates synapse formation.
-Conclusion
In conclusion, by using multidisciplinary approaches including immuno electron microscopy, electrophysiology, biochemistry and molecular biology, I determined the localization and function of Neurexins and the regulatory mechanism relevant to synapse formation. I found that Neurexins were specifically localized at presynaptic terminals and they contributed to the neurotransmitter release from presynaptic terminals. The C-tail of Neurexin-1 bound phosphatidylinositol and this binding was abolished by the PKC phosphorylation of the last three serines. And this reduced the synaptogenic activity of Neurexin-1.
Reference list
1. Sudhof, T.C., Neuroligins and neurexins link synaptic function to cognitive disease. Nature, 2008. 455(7215): p. 903-11.
2. Ushkaryov, Y.A., et al., Neurexins: synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin. Science, 1992. 257(5066): p. 50-6.
3. Geppert, M., et al., Neurexin I alpha is a major alpha-latrotoxin receptor that cooperates in alpha-latrotoxin action. J Biol Chem, 1998. 273(3): p. 1705-10. 4. Missler, M., R. Fernandez-Chacon, and T.C. Sudhof, The making of neurexins.
J Neurochem, 1998. 71(4): p. 1339-47.
5. Tabuchi, K. and T.C. Sudhof, Structure and evolution of neurexin genes: insight into the mechanism of alternative splicing. Genomics, 2002. 79(6): p. 849-59.
6. Hata, Y., S. Butz, and T.C. Sudhof, CASK: a novel dlg/PSD95 homolog with an N-terminal calmodulin-dependent protein kinase domain identified by interaction with neurexins. J Neurosci, 1996. 16(8): p. 2488-94.
7. Nguyen, T. and T.C. Sudhof, Binding properties of neuroligin 1 and neurexin 1beta reveal function as heterophilic cell adhesion molecules. J Biol Chem, 1997. 272(41): p. 26032-9.
8. Boucard, A.A., et al., A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to alpha- and beta-neurexins. Neuron, 2005. 48(2): p. 229-36.
9. Belichenko, P.V., et al., Excitatory-inhibitory relationship in the fascia dentata in the Ts65Dn mouse model of Down syndrome. J Comp Neurol, 2009. 512(4): p. 453-66.
10. Uemura, T., et al., Trans-synaptic interaction of GluRdelta2 and Neurexin through Cbln1 mediates synapse formation in the cerebellum. Cell, 2010. 141(6): p. 1068-79.
11. Matsuda, K. and M. Yuzaki, Cbln family proteins promote synapse formation by regulating distinct neurexin signaling pathways in various brain regions. Eur J Neurosci, 2011. 33(8): p. 1447-61.
12. Ichtchenko, K., et al., Neuroligin 1: a splice site-specific ligand for beta-neurexins. Cell, 1995. 81(3): p. 435-43.
