若手道場

若手道場6

2021/9/30　17:00～18:00　ZOOM 若手道場 WD6-1 ポストシナプスへのTRPV4局在化は神経成熟化により制御されている Post-synaptic clustering of TRPV4 is regulated by neuronal maturation ^○松元智子, Akane Egoshi, Miho Kusano, Koji Shibasaki 長崎県立大学看護栄養学部細胞生化学研究室 ^○Tomoko Matsumoto, Akane Egoshi, Miho Kusano, Koji Shibasaki Lab Neurochem, Univ of Nagasaki Physiological brain temperature is an important determinant for neuronal functions, and it is well established that changes in temperature have dynamic influences on brain neuronal excitabilities. We previously revealed that a thermo-sensor TRPV4 (activated above 34°C) is activated by physiological temperature in cultured hippocampal neurons and thereby controls their excitability (J. Neurosci. 2007, Shibasaki et al.). Compartmentalization of neuronal function is achieved via specifically localized clustering of ion channels in discrete subcellular membrane domains. TRPV4 channel exhibits highly variable cellular and subcellular patterns of expression. Here, we examined synaptic clustering of TRPV4 in developing hippocampal neurons. We found that TRPV4 accumulated in the soma of immature hippocampal neurons, and did not localize to post-synaptic locations although PSD-95-labeled post-synaptic structures were evident. During the maturation of neurons, TRPV4 was targeted to dendrites and also clustered at post-synaptic locations. Taken together, we reveal that TRPV4 localizes to post-synaptic sites and the post-synaptic targeting is strictly regulated in a neuronal maturation-dependent manner.		2021/9/30　17:00～18:00　ZOOM 若手道場 WD6-2 ヒト多能性幹細胞から誘導される抑制性神経細胞のサブタイプごとの可視化の試み Visualization of subtype-specific GABAergic interneurons derived from human pluripotent stem cells ^○銭映美, 吉松祥, 石川充, 岡野栄之 Department of Physiology, Keio University School of Medicine ^○Emi Qian, 祥吉松, 石川充, 栄之岡野 Department of Physiology, Keio University School of Medicine Evolutionary differences in the spatial distribution and composition of GABAergic interneuron (IN) subtypes between primates and rodents have been revealed recently(Krienen et al. 2020), which emphasizes the importance using functional neurons derived from induced pluripotent stem cells(hiPSCs). INs can be classified into several subtypes based on distinct functional, morphological and electrophysiological properties. Especially, parvalbumin+(PV+) and somatostatin+ (SST+) neurons are both derived from progenitor cells in medial ganglionic eminence(MGE), but it remains unclear how the distinct types are emerged, and separated from the same origin. In neuropsychiatry diseases, the neuronal vulnerability is different among IN subtypes. Therefore, it is crucial to discriminate IN subtypes derived from hiPSCs and analyze INs individually for developmental and pathophysiological research. In this study, we generated an hiPSC line harboring PV and SST double reporters. The mNeonGreen and tdTomato genes were respectively inserted into the direct downstream of PV and SST coding regions by using a robust KI gene targeting tool based on the Multisite Gateway technology(Yoshimatsu et al. 2019). To confirm the functionality of reporter genes, the iPSCs were treated with CRISPR-dCas9 forced gene expression system (Chavez et al. 2015). To verify the reporter gene expression in neuronal cells, a previously reported methods to induce INs were applied to the iPSCs(Yang et al. 2017). This iPSC line, which can easily distinguish whether it has been differentiated into PV+ or SST+ neurons, will be a useful tool for investigating efficient methods to induce subtype-specific INs from hiPSCs, or for addressing the mechanisms of PV+ and SST+ neurons lineage segregation using organoids.

2021/9/30　17:00～18:00　ZOOM 若手道場 WD6-3 E3ユビキチンタンパク質リガーゼUBR4は、微小管親和調節キナーゼ（MARK4）活性およびタウの神経変性を増強する E3 ubiquitin-protein ligase UBR4 enhances Microtubule Affinity Regulating Kinase (MARK)4 activity and tau-induced neurodegeneration ^○中嶋翔¹, Joma Ono¹, Akiko Asada^1,2, Taro Saito^1,2, Kanae Ando^1,2 1.東京都立大学　理学部　生命学科, 2.Department of Biological Sciences, Faculty of Science, Tokyo Metropolitan University ^○Sho Nakajima¹, Joma Ono¹, Akiko Asada^1,2, Taro Saito^1,2, Kanae Ando^1,2 1.Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 2.Department of Biological Sciences, Faculty of Science, Tokyo Metropolitan University Accumulation of microtubule-associated protein tau is believed to cause neuronal death in Alzheimer’s disease (AD). Tau is abnormally phosphorylated in AD brain, and accumulating evidence suggests that dysregulation of Microtubule Affinity Regulating Kinase 4 (MARK4) may underlie. Tau phosphorylation via MARK stabilizes tau and trigger further phosphorylation. MARK4 activity colocalizes with tau lesions. A gain-of-function mutation in MARK4 is associated with elevated risks of AD. These findings suggest that elevated MARK4 activity may contribute to AD pathogenesis. However, the mechanism that regulates MARK4 activity is not fully understood. Here we show that UBR4 (Ubiquitin protein ligase E3 component n-recognin 4) positively regulates MARK4 protein levels. From a genetic screen with a Drosophila model of tau toxicity, we identified poe (purity of essence) as an enhancer. MARK4 belongs to an evolutionary conserved family Par-1/MARK. In Drosophila, Par-1 exists as a single family member of Par-1/MARK and phosphorylates tau. We found that poe knockdown reduced, and overexpression of poe increased, Par-1 protein levels. Next, we analyzed the role of the human homolog of Poe, UBR4, on MARK4 levels in mammalian cultured cells. We found that the knockdown of UBR4 via siRNA reduced MARK4 levels. We also found that knockdown of Thyroid Hormone Receptor Interactor 12 (TRIP12) increased MARK4 levels. Since TRIP12 is a client of UBR4, these results suggest that UBR4 positively regulates MARK4 levels via degradation of TRIP12. Our study revealed an evolutionarily conserved pathway that regulates the degradation of Par-1/MARK4. Since MARK4 is emerging as a promising drug target for AD, these findings can lead to the development of therapy in the future.