若手道場口演（Wakate Dojo）

Wakate Dojo Development and Regeneration 2 若手道場口演発生・発達・再生2

7月26日（金）9:30～9:50　第10会場（万代島ビル　6F　会議室） 2WD10am2-1 高齢ラット脳におけるプロテオミクス解析 Hamizah Shahirah Hamezah（Hamezah Hamizah Shahirah）¹，Lina Wati Durani（Durani Lina Wati）¹，Daijiro Yanagisawa（柳沢大治郎）¹，Nor Faeizah Ibrahim（Ibrahim Nor Faeizah）²，Wan Mohd Aizat（Aizat Wan Mohd）³，Jean Pierre Bellier（Bellier Jean Pierre）¹，Suzana Makpol（Makpol Suzana）²，Wan Zurinah Wan Ngah（Wan Ngah Wan Zurinah）²，Hanafi Ahmad Damanhuri（Damanhuri Hanafi Ahmad）²，Ikuo Tooyama（遠山育夫）¹ ¹滋賀医大分子神経科学研究センター、大津、日本 ²マレーシア国民大学、クアラルンプール、マレーシア ³マレーシア国民大学、システムバイオロジー研究所、サランゴ、マレーシア Background: Brain aging is a complex multifactorial process accompanied by physiological and cognitive deteriorations that ultimately lead to death. Our recent study reported that increased brain atrophy in the hippocampus, medial prefrontal cortex (mPFC), and striatum occurred between middle- to late-aged rats, which was accompanied by impairment in learning, memory, and locomotor activity. Hence, we carried out proteomics study on these rats to further our understanding of proteome profiles changes in the brain. Method: We studied the proteome profiling in the hippocampus, mPFC, and striatum of rats aged 14, 18, 23, and 27 months old. Proteins from the rat brain tissue were extracted and separated by SDS-PAGE, followed by in-gel digestion protocol. Mass spectrometry (MS) analysis was performed using a Q Exactive HF Orbitrap MS coupled to a Dionex Ultimate 3000 UHPLC instrument. The acquired MS data were processed using MaxQuant software. Perseus software was used to perform bioinformatics and statistical analysis. The proteins were subjected to pathway search against KEGG database. The proteins were validated by Western blotting. Results: The present study provides initial insights into understanding protein changes with aging in specific brain regions. We found that the proteins were not consistently changed between different brain regions. We identified 97, 25, and 5 proteins as age-related proteins in the hippocampus, mPFC, and striatum, respectively. The altered proteins with age were mostly categorized in oxidative phosphorylation (e.g. cytochrome c oxidase and ATP synthase), glutathione metabolism (e.g. peroxiredoxins, glutathione-s-transferase, and superoxide dismutase), and calcium signaling pathway (e.g. protein S100B, calmodulin, and glutamate receptor NMDA). Overall, these changes were mostly observed in the late-aged (27 months old) when compared with the middle-aged (14 months old) rats. This finding is supported by our previous study that demonstrated brain atrophy, as well as cognitive and locomotor deficits which were marked in the 27 months old rats compared with the 14 months. Conclusion: Aging-induced alterations in various protein characteristics implicate several vital cellular pathways such as oxidative phosphorylation, glutathione metabolism, and calcium signaling, that may mediate cognitive and locomotor impairments.		7月26日（金）9:50～10:10　第10会場（万代島ビル　6F　会議室） 2WD10am2-2 新規ネプリライシン活性制御因子に基づくアルツハイマー病予防法の開発 Naoto Watamura（綿村直人）¹，Naomsa Kakiya（垣矢直雅）¹，Nilsson Per（Per Nilsson）²，Takashi Saito（斎藤貴志）¹，Takaomi Saido（西道隆臣）¹ ¹国立研究開発法人理化学研究所 ²Division of Neurogeriatrics, Karolinska Institutet, Stockholm, Sweden Alzheimer's disease (AD) is the most common type of dementia among the elderly. One of the pathological hallmarks of AD is accumulation of senile plaques composed of amyloid-β peptides (Aβ) in the brain. Neprilysin (NEP) is potent Aβ degrading enzyme. Identification of the NEP activity control mechanism may leads to development of a fundamental preventive method for AD. Previously we showed that somatostatin (SST) regulates NEP activity. However the molecular mechanism by which SST regulates NEP in the brain is unclear. Recently, We found that cortex/hippocampus-basal ganglia communication is important to upregulate NEP upon SST stimulation. The aim of this study is elucidation of the molecular mechanism of NEP activity based on the cell-cell communication. Firstly proteomics using primary neurons treated with SST identified α-endosulfine (ENSA), an endogenous ligand for a potassium channel, as a novel NEP regulator. To investigate the function of ENSA in vivo, we generated ENSA knock out mice using CRISPR/Cas9 and found that ENSA deficiency increased NEP activity and accelerated translocation of NEP to presynaptic region. Furthermore, ENSA deficiency decreased Aβlevels in the brain. Finally administration of Diazoxide which is a potassium channel modulator decreased amyloid pathology in AD model mice mediated by activation of NEP. Thus, we found a novel preventive approach for AD based on the cell-cell communication. KATP channel would be a novel therapeutic target and Dz is a candidate drug to be preventive approach for preclinical AD.

7月26日（金）10:10～10:30　第10会場（万代島ビル　6F　会議室） 2WD10am2-3 認知症発症を加速する新規Myokineの解析 Tsukasa Nagase（長瀬綸沙），Chihiro Tohda（東田千尋）富山大学　和漢医薬学総合研究所　神経機能学分野 Several epidemiological and clinical studies show that exercise is positively associated with cognitive function and preventing Alzheimer's disease (AD). On the contrary, physical inactivity is known as one of risk factors for AD onset. Several myokines are reported to be secreted by exercise, so the positive contribution of those beneficial myokines to cognitive improvement is supposed. In contrast, unbeneficial myokines directly deteriorating cognitive function have not been identified. We hypothesized that unbeneficial myokines exist and accelerate AD onset, and those might be secreted from skeletal muscles at the physically inactivated state. Therefore, this study aimed to investigate the relationship between skeletal muscle atrophy and AD onset, and to identify new myokines involved in the phenomenon To prepare a disuse muscle atrophy model, bilateral hindlimbs were immobilized by putting into casts for 14 days. Young age of 5xFAD mice (3 months old, male) were used. 5xFAD mice usually reveal cognitive impairment after 4 - 5 months of age. Object recognition memory in casted 5xFAD mice was impaired after the 14-day casting although age matched wild-type mice and non-cast 5xFAD mice showed normal memory function. Numbers of amyloidβ plaques in the brain were not different between casted and non-casted 5xFAD groups. After the memory test, hindlimbs were isolated for organ culture. Conditioned medium of each muscle culture was separated on SDS-PAGE and silver stained. Increased proteins in the conditioned medium of casted 5xFAD were identified by nanoLC-MS/MS analysis. Transferrin was significantly increased in conditioned medium both of gastrocnemius and tibialis anterior muscles of casted 5xFAD mice. Source of increased transferrin was skeletal muscle, but not blood. These results suggest that atrophied skeletal muscles secrete transferrin. Transferrin has never been recognized as a myokine. We suppose that secreted transferrin from skeletal muscles reach to and affect the brain, specific function of transferrin against neurons was investigated. This study found a new myokine, transferrin that might be first identified as unbeneficial myokine for AD onset. Regulating secretion and/or signaling of the transferrin possibly leads to deceleration and inhibition of AD progress.