Symposium

Symposium 16 Dynamics and significance of subcellular distribution and turnover of crucial molecules in the spine シンポジウム16 樹状突起スパイン内の蛋白質動態とその制御

SY16-1 Regulation of intra-spine distribution of myosin is associated with morphology of spine スパイン形態とミオシン分布制御機構 Yagi Hideshi（八木秀司）¹，佐藤真^2,3 ¹Dept. Anat. & Cell Biol., Hyogo Col. Med.. Nishinomiya, Japan ²Dept Anatomy and Neuroscience, Grad Sch Med, Osaka Univ, Osaka, Japan ³United Grad Sch of Child Development, Osaka Univ, Kanazawa Univ, Hamamatsu Univ Sch of Med, Chiba Univ and Univ of Fukui, Osaka, Neuronal morphology is controlled by the cytoskeleton. Dendritic spines are small protrusions that contain actin fibers. As dendritic spines are actin-rich protrusions, several actin-binding proteins and myosin-activating proteins have important roles in the morphological control of spines. Non-muscle myosin heavy chain type IIb (NMHC IIb), which interacts with actin fibers plays an important role in spine morphology through its contractile properties via interacting with actin fibers. FILIP is one of regulatory proteins of neuronal migration in the embryonic brain. FILIP colocalizes with actin fibers in cultured cells. In the adult brain, FILIP was expressed in the subgroup of excitatory neurons, especially piriform cortex neurons. FILIP mutated mice had shorter spines on the dendrites of piriform neurons than control mice. There was a tendency that cultured piriform cortex neurons had more elongated spines on their dendrites that those on cultured hippocampal neurons that do not express FILIP. While NMHC IIb was localized to the neck portion of the spine in neurons that did not express FILIP, it was diffusely distributed in the spines in the presence of FILIP. In normal cultured cells, FILIP was bound to NMHC IIb and altered its intracellular distribution. These results indicate that removed NMHC IIb from the dense actin-myosin network in the spine neck by FILIP lead to the alteration of the spine morphology. Control of NMHC IIb via its binding protein is critical for the morphology of dendritic spines.		SY16-2 Neuronal activity-dependent formation of ER-PM contact sites in spines regulates spine formation and dendritic extension via local manipulation of unfolded protein response 神経活動依存的な小胞体膜-細胞膜コンタクトサイト形成による小胞体ストレス応答の調節を介したスパイン形成と樹状突起の伸長制御 Saito Atsushi（齋藤敦）¹，今泉和則² ¹Dept. of Stress Protein, Inst. of Biomed. Health Sci., Hiroshima Univ., Hiroshima ²Dept. of Biochem., Inst. of Biomed. Health Sci., Hiroshima Univ., Hiroshima Endoplasmic reticulum (ER) stress transducers IRE1, PERK and ATF6 recognize the alternation of ER luminal environments and transduce signals from ER to cytoplasm or nucleus (unfolded protein response: UPR) to maintain ER functions. The dendritic ER network is complexly extended from cell soma to distal dendrites of neurons, indicating that dendritic ER functions and UPR signaling may orchestrate local events contributing to dendritic capabilities. To assess the induction of UPR in response to neuronal activities, primary cultured mouse hippocampal neurons were pretreated with tetrodotoxin subsequently the washout to induce spontaneous excitatory synaptic activities. The phosphorylation levels of IRE1 and PERK were transiently upregulated at spines after the washout. The phosphorylation levels were reduced by inhibiting calcium ion (Ca²⁺) release from ER, suggesting that Ca²⁺ release and its depletion in ER lumen by the excitatory synaptic activation triggers the induction of UPR at spines. The ER in the spines formed contact sites with plasma membrane (PM) (ER-PM contact sites) after the excitatory synaptic activation. These contact sites were composed of Stim1 localizing at ER membrane and Orai1 localizing at PM, which are responsible for Ca²⁺ entry from extracellular spaces to ER lumen. The knockdown of Stim1 extended the UPR activation after the excitatory synaptic activation followed by inhibiting spine formation and dendritic extension. These results suggest that the synaptic activation-dependent ER-dynamics including the formation of ER-PM contact sites in spines may fine-tune the development of dendritic spines and intricately branched dendrites through the regulation of UPR signaling derived from spines.

SY16-3 A postsynaptic dysregulation in hippocampal granule cells that underlies spatial discrimination defect 空間弁別障害をもたらす海馬歯状回顆粒細胞のシナプス後部調節異常 Kinoshita Makoto（木下専）¹，上田-石原奈津実¹，深澤有吾²，宮川剛³，高雄啓三⁴，尾藤晴彦⁵ ¹Dept. of Mol. Bio., Nagoya Univ. Grad. Sch. Sci. ²Dept. of Brain Struct. & Funct., Fukui Univ. Sch. Med. ³Systems Med. Sci., Inst. Comp. Med. Sci., Fujita Health Univ. ⁴Center for Promotion of Res., Toyama Univ. ⁵Dept. of Neurochem., Univ. of Tokyo Grad. Sch. of Med. Discrimination among distinct spatial contexts depends on neural circuits connecting the entorhinal cortex and hippocampus. The low-firing property of glutamatergic synapses between the perforant path (pp) and dentate gyrus (DG) granule cells is thought to play a major role to implement sparse coding, a theoretical requisite for the spatial pattern separation. Despite the theory/circuit-level understanding of the information processing, molecular mechanism underlying the unique property of the pp-DG synapses remains unclear. Here we show that mice that lack a DG-enriched subunit of the septin cytoskeleton perform normally in a systematic behavioral screening. Notably, maze task results indicate that hippocampus-dependent spatial orientation and spatial working/long-term memories are largely intact. However, they underperform selectively in discrimination between cubic vs. cylindrical chambers with the same floor area and texture, indicating a selective cognitive impairment in differentiating distinct spatial contexts. To define brain regions and neuronal populations responsible for the phenotype, and to exclude possible developmental anomalies, we conduct local, subacute septin depletion from bilateral DGs of wildtype mice, and local septin supplementation into bilateral DGs of septin-null mice, which recapitulates and rescues the spatial discrimination defect, respectively. In the septin-null mice, the pp-DG synapse morphology is normal in LM and 3D-EM (dendritic arbor of granule cells; asymmetrical synapse density, PSD area, spine volume, etc.), except for a unique postsynaptic defect, which is recapitulated by septin depletion from primary cultured rat granule cells. This unique mutant will illuminate a novel postsynaptic regulatory mechanism of the pp-DG synapses.		SY16-4 The role of an actin-binding protein drebrin and its isoform-specific dynamics in dendritic spines 樹状突起スパインのアクチン結合タンパク質ドレブリンの役割とアイソフォーム特異的な動態の制御メカニズム Hanamura Kenji（花村健次）¹，山崎博幸¹，児島伸彦²，安田浩樹^3,4，白尾智明¹ ¹Dept. of Neurobiol. & Behav. Gunma Univ., Grad. Sch. Med. ²Faculty Life Sci., Toyo Univ., Itakura, Japan ³ERSC, Gunma Univ., Grad. Sch. Med. ⁴Dept. Physiol., Saga Univ. Sch. Med. Drebrin is an actin-binding protein in dendritic spines. Drebrin has two major isoforms, drebrin E (DE) and drebrin A (DA). Neuron-specific isoform conversion from the embryonic isoform DE to the adult isoform DA occurs in parallel with synapse formation. To examine the physiological role of the drebrin isoform conversion in synapse formation and function, we generated knockout mice in which a DA-specific exon was deleted. The knockout mice did not show any overt morphological deficits of brain structure, but had longer dendritic spines in CA1 pyramidal neurons. In addition, these knockout mice showed impaired LTP and abnormal induction of LTD in CA1 synapses. Hippocampus-dependent fear learning was also imapaired. To understand the molecular basis of these abnormalities, we measured the dynamics of GFP-tagged DE and DA in cultured hippocampal neurons by fluorescence recovery after photobleaching (FRAP) analysis. We found that stable fraction of GFP-DA is larger than GFP-DE. FRAP analysis of mutant GFP-DA demonstrated that the position of the DA-specific region is important for isoform-specific DA stability in spines. In the absence of stable actin filaments, drebrin stability was similar between the two isoforms indicating preferential binding of drebrin A to F-actin than drebrin E causes higher stable fraction of drebrin A in dendritic spines. Therefore, we suggest that conversion of DE to DA alters the amount of drebrin-bound stable F-actin in dendritic spines, which may contribute to synapse formation and function.