丹羽 史尋

Astrocytes play key roles in the central nervous system and regulate local blood flow and synaptic transmission via intracellular calcium (Ca2+) signaling. Astrocytic Ca2+ signals are generated by multiple pathways: Ca2+ release from the endoplasmic reticulum (ER) via the inositol 1, 4, 5-trisphosphate receptor (IP3R) and Ca2+ influx through various Ca2+ channels on the plasma membrane. However, the Ca2+ channels involved in astrocytic Ca2+ homeostasis or signaling have not been fully characterized. Here, we demonstrate that spontaneous astrocytic Ca2+ transients in cultured hippocampal astrocytes were induced by cooperation between the Ca2+ release from the ER and the Ca2+ influx through store-operated calcium channels (SOCCs) on the plasma membrane. Ca2+ imaging with plasma membrane targeted GCaMP6f revealed that spontaneous astroglial Ca2+ transients were impaired by pharmacological blockade of not only Ca2+ release through IP(3)Rs, but also Ca2+ influx through SOCCs. Loss of SOCC activity resulted in the depletion of ER Ca2+, suggesting that SOCCs are activated without store depletion in hippocampal astrocytes. Our findings indicate that sustained SOCC activity, together with that of the sarco-endoplasmic reticulum Ca2+-ATPase, contribute to the maintenance of astrocytic Ca2+ store levels, ultimately enabling astrocytic Ca2+ signaling. (C) 2017 Elsevier Inc. All rights reserved.

Calcium (Ca2+) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca2+ signals, generated by the coordination of Ca2+ influx from the extracellular space and Ca2+ release from the intracellular Ca2+ store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca2+ signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca2+ signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca2+ indicator (GECI) targeted to the outer ER membrane, can monitor Ca2+ release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca2+ imaging of C elegans. We also found that the spontaneous Ca2+ elevation in cultured astrocytes reported by OER-GCaMP6f showed a distinct spatiotemporal pattern from that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f); less frequent Ca2+ signal was detected by OER-GCaMP6f, in spite of the fact that Ca2+ release from the ER plays important roles in astrocytes. These findings suggest that targeting of GECI5 to the ER outer membrane enables sensitive detection of Ca2+ release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca2+. Our results indicate that Ca2+ imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca2+ signals, by enabling dissection of Ca2+ signals at subcellular resolution. (C) 2016 Elsevier Inc. All rights reserved.

細胞膜の構成要素である脂質・タンパク質は流体としての性質を持ち，側方拡散運動により自由に動く．自由拡散運動により脂質・タンパク質は均一に分布すると予想されるが，実際の細胞膜では膜分子の分布は一様ではない．シナプス後膜には神経伝達物質受容体が高密度で局在し，効率の良い神経伝達を可能にしている．いかにして神経細胞は，自由拡散運動に逆らって受容体の密度勾配を形成・維持し，シナプス伝達の制御を行っているのか？ 我々は，ほ乳類の中枢神経系のGABA作動性シナプスに注目し，「量子ドット1分子イメージング」という技術で細胞膜上の受容体1分子のふるまいを「見る」ことにより取り組んで来た．神経細胞膜上のGABAA受容体の動きを1分子レベルで追跡したところ，小胞体からのIP3誘導カルシウム放出（IICR）が，GABAA受容体を動きにくくし，抑制性シナプスの中でのGABAA受容体の安定性を高めていることが分かった．さらに，GABAA受容体の安定性を高めるためには，IP3受容体に加えて代謝型グルタミン酸受容体（mGluR）とリン酸化酵素プロテインキナーゼCの活性化が必要であることも明らかになった．これまでの研究で，グルタミン酸はNMDA型グルタミン酸受容体（NMDAR）を活性化し，細胞外から細胞内へ大量のカルシウムを流入させることにより，GABAA受容体を動きやすくすることが知られていた．一方，今回解明したメカニズムでは，同じグルタミン酸とカルシウムというシグナル物質が，mGluRとIP3受容体という全く異なる受容体を介して，逆にGABAA受容体を動きにくくし，安定性を高める働きをしていることが明らかになった．これはグルタミン酸とカルシウムというGABA作動性シナプス（GABAA受容体が機能するシナプス）の制御に関与するシグナル物質が，従来知られていた役割とは正反対の役割も担っていることを示す．

GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABA(A) receptor (GABA(A)R) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABA(A)R clustering and GABAergic transmission. This GABA(A)R stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.

Metabotropic glutamate receptor (mGluR)-dependent calcium ion (Ca2+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca2+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca2+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca2+ responses. Immunocytochemistry indicated that these polarized Ca2+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca2+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca2+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

The activity-dependent modulation of GABA-A receptor (GABA(A)R) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABA(A)R clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABA(A)R declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABA(A)R declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABA(A)Rs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or N-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABA(A)Rs revealed that excitation-induced enhancement of GABA(A)R lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABA(A)R lateral diffusion on the cell surface and inhibition of a Ca2+ dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABA(A)R-dependent regulatory mechanism and a GABA(A)R-independent one. Our results also indicate that GABA(A)R mobility and clustering after sustained excitatory activity is independent of gephyrin.