公募シンポジウム

公募シンポジウム12【情報トリガーである軸索起始部（AIS）の構築メカニズムとダイナミクス】

2021/10/1　13:00～15:00　ZOOM B会場 S12-1 神経活動依存的な選択的スプライシングによるAIS 可塑性制御 Neuronal activity-dependent alternative splicing underlies AIS plasticity ^○飯島崇利東海大学医学部医学科基礎医学系分子生命科学領域 ^○Takatoshi Iijima Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, Japan The axon initial segment (AIS), a specialized compartment of proximal axons, initiates action potentials and plays important role in determining neuronal outputs. It has been revealed that the structural and functional properties of AIS are dynamically changed by age, disease, and neuronal activity. However, the function and mechanisms underlying AIS plasticity remain unclear. Here, we identify a novel form of neuronal activity-dependent structural and functional plasticity at the cerebellar AIS and show these changes are controlled by activity-regulated alternative splicing (AS) programs. Depolarization dynamically changes the axonal distribution of AIS proteins in the cerebellar granule cells and these structural changes are late-onset, long-lasting, and reversible with the order of days through the L-type calcium channel. We revealed that the depolarization-induced structural changes are accompanied by a decreased density of voltage-gated sodium channels, which play a homeostatic role in network activity. Using RNA-seq analysis, we uncovered that AS of transcripts encoding major AIS proteins is altered upon neuronal activity. Notably, the activity-regulated insertion of specific micro exons at AIS proteins is responsible for the structural and functional changes of AIS. Thus, in the symposium, I will present and discuss novel aspects of axonal plasticity dynamic regulations by neuronal activity-dependent splicing programs.		2021/10/1　13:00～15:00　ZOOM B会場 S12-2 新規AIS タンパク質のハイスループット解析 High-throughput mapping of axon initial segment associated proteins by genome editing ^○小川優樹,ラズバンドマシューベイラー医科大学神経科学部門 ^○Yuki Ogawa, Matthew N. Rasband Department of Neuroscience, Baylor College of Medicine, Houston, TX The axon initial segment (AIS) and nodes of Ranvier are important for the generation and propagation of action potentials, and regulation of neuronal polarity. It is well established that AnkyrinG (AnkG) and β4 spectrin play key roles in the formation and maintenance of the AIS and nodes. Interestingly, AIS and nodes share a common molecular organization. However, some new AIS-specific and node-specific or -enriched proteins have been discovered (e.g. Trim46, NuMA1, TREK1) and the molecular mechanisms that stabilize the AIS and control neuronal polarity remain obscure. Recently, we identified candidate AIS-associated proteins using AnkG-knock out mice, proximity biotinylation, and proteomics. We further investigated these candidates using the AAV-based CRISPR-mediated genome editing method. This method allowed for high-throughput modiﬁcation of endogenous proteins for knock-out and knock-in, both in vivo and in vitro. These approaches will provide new insights into the mechanisms underlying AIS and node of Ranvier formation and stabilization.

2021/10/1　13:00～15:00　ZOOM B会場 S12-3 細胞骨格のリン酸化を介した軸索起始部の形成機構 Phosphorylation of cytoskeletal proteins regulates axon initial segment formation ^○吉村武大阪大学大学院連合小児発達学研究科分子生物遺伝学 ^○Takeshi Yoshimura Department of Child Development and Molecular Brain Science, United Graduate School of Child Development, Osaka University The axon initial segment (AIS) is a structurally and molecularly unique neuronal compartment of the proximal axon that functions as both a physiological and physical bridge between the somatodendritic and axonal domains. The AIS has two main functions: to initiate action potentials and to maintain neuronal polarity. The AIS has a specific cytoskeletal structure consisting of αII-spectrin, βIV-spectrin, ankyrin-G and actin. Super-resolution microscopy has revealed that these proteins form a remarkable periodic lattice lining the cytoplasmic face of the AIS membrane. AIS proteins have been implicated in a variety of human diseases. Pathogenic SPTAN1 (human gene encoding αII-spectrin) variants cause West syndrome. βIV-spectrin is associated with profound intellectual disability, congenital hypotonia and motor axonal neuropathy. Human mutations in ankyrin-G were reported and shown to be associated with severe intellectual disability, attention deficit hyperactivity disorder, and autism spectrum disorder. While recent studies have identified the AIS proteome, the molecular mechanism by which AIS formation is regulated remains unclear. In this study, we report that Cdk5 phosphorylates αII-spectrin. We identified the phosphorylation site of αII-spectrin by Cdk5, and made anti-phospho-specific αII-spectrin antibody. The phosphorylated αII-spectrin was localized in the AIS. Inhibition of Cdk5 impaired AIS formation and neuronal polarity. These results suggest that Cdk5 regulates AIS formation through the phosphorylation of αII-spectrin.		2021/10/1　13:00～15:00　ZOOM B会場 S12-4 神経活動依存的な軸索起始部の再編機構 Activity-dependent refinement of the axon initial segment in central auditory circuit ^○久場博司名古屋大学大学院医学研究科 ^○Hiroshi Kuba Nagoya University, Graduate School of Medicine The axon initial segment (AIS) is a proximal axonal domain that is responsible for action potential initiation. Evidences have accumulated that structural and biophysical characteristics of the AIS differ among cell types and/or brain regions, playing a critical role in determining excitability of the AIS, but the underlying mechanisms remain elusive. In this symposium, I will introduce our recent studies addressing this issue in avian nucleus magnocellularis, which encodes timing information of sound and is well known for the differentiations of the AIS in a manner dependent on neuronal characteristic frequency (CF). Using immunofluorescence and electrophysiological methods, we found that AIS shortening and accumulation of voltage-gated sodium channels progressed during development, with the shortening augmented and the accumulation suppressed in higher-CF neurons, which created the tonotopic differences in both AIS characteristics and neuronal excitability within the nucleus. Afferent input contributed to this adjustment by augmenting the AIS shortening specifically in higher-CF neurons. Moreover, super-resolution imaging revealed that the periodic submembranous cytoskeleton at the AIS remained intact during the AIS shortening, suggesting that the shortening occurred via disassembly of the AIS cytoskeleton at its distal end. Our results highlighted that the cytoskeletal and ion channel organization of the AIS are differentially regulated, but work synergistically in each neuron to optimize the sodium conductance and hence the neuronal signal processing.

2021/10/1　13:00～15:00　ZOOM B会場 S12-5 神経損傷に応答した軸索起始部のダイナミクス The dynamics of the AIS during neuronal injury ^○桐生寿美子,木山博資名古屋大学大学院医学系研究科機能組織学 ^○Sumiko Kiryu-Seo, Hiroshi Kiyama Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine Nerve-injured motor neurons can regenerate through proper proteostasis, whose impairment, in particular proteasomal dysfunction, has been implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, it remains elusive how proteasome-mediated stress responses impact damaged motor neurons at early time points. To address this issue, we have established a mouse model (Rpt3 CKO) in which a subunit of proteasome is ablated and mitochondria are labeled by GFP in injury-dependent manner. Injury-induced Rpt3-deficient motor neurons exhibit ALS-relevant degeneration despite having regenerative competence during the initial stage of nerve injury. Injured motor neurons of Rpt3 CKO mice fail to disassemble the axon initial segment (AIS), a specialized compartment between the soma and axon, while those of wild type mice disrupt the AIS. There is the accumulated evidence that the AIS changes its morphology in response to neuronal insults. It is likely that the AIS functions as a selective filter for axonal transport in addition to action potential generation under normal condition. However, the physiological significance for AIS disassembly following neuronal damage have not been well addressed. We have found using Rpt3 CKO mice that the proteasome-mediated disassembly of the AIS upon injury facilitates mitochondrial entry into the injured axons, perhaps to compensate for the increased energy expenditure associated with axon regeneration. ALS motor neurons vulnerable to pathological damage also fail to lose the AIS, causing the restricted number of mitochondria in axons, similar to injury-induced Rpt3-deficient motor neurons. In this symposium, I would like to introduce how damaged motor neurons receive a physiological benefit from AIS disassembly.