Oral Session

Oral Session 4 一般口演4

O4-1 Inhibition of psychotomimetic-induced hyperactivity by a selective calcium permeable AMPA receptor antagonist, IEM 1460, in the mouse マウスにおける選択的なカルシウム透過型AMPA受容体遮断のIEM1460による精神異常発現薬誘発性の活動性増加の抑制 Umino Masakazu（海野真一），海野麻未，西川徹 Department of Psychiatry and Behavioral Sciences Graduate School of Medical and Dental Sciences Tokyo Medical and Dental University Blockers and autoantibodies of NMDA-type glutamate receptor (NMDAR) cause positive and negative symptoms, and cognitive dysfunction which are indistinguishable from those of schizophrenia. These observations indicate that NMDAR dysfunction may be involved in the pathophysiology of this disorder and that facilitation of NMDAR function could ameliorate negative symptoms and cognitive dysfunction that are resistant to current therapeutic drugs, antipsychotics, which mainly block the D2 dopamine receptor. A compensatory increase in glutamate release following the reduced NMDAR-mediated transmission could further attenuate NMDAR function in the brain, because we have shown that overactivation of the calcium permeable AMPA type glutamate receptor (CP-AMPAR) decreases the extracellular levels of an NMDAR coagonist, D-serine, in the prefrontal cortex. Therefore, blockade of the CP-AMPAR could improve overall symptoms of schizophrenia by preventing the attenuation of NMDAR function through the retain of the D-serine levels. In this study, we have first demonstrated that the inhibition of the CP-AMPAR results in amelioration of pharmacological animal models of schizophrenia. Thus, an acute subcutaneous administration of IEM 1460, a selective antagonist for the CP-AMPAR, reduced hyperactivity induced by an acute injection of either of NMDAR antagonists, phencyclidine or dizocilpine (MK801), and an indirect dopamine agonist, methamphetamine, which produces schizophrenia-like positive symptoms. These results indicate that excessive activation of the CP-AMPAR might be associated with impairment of glutamate transmission and glutamate-dopamine interaction in schizophrenia, and the CP-AMPAR antagonists could be candidates relevant to a novel treatment for this intractable disorder.		O4-2 Lysosomal ubiquitin ligase RNF182 regulates neuronal differentiation through upregulation of mTORC1 signaling ライソゾームに局在するRNF182はmTORC1シグナルの増強を介して神経分化に関与する Kaneko Masayuki（金子雅幸），郭暁鵬，今泉和則 Department of Biochemistry, Institute of Biomedical and Health Sciences, Hiroshima University We identified 37 ubiquitin ligases containing RING finger and transmembrane domains. Of these, we found that RNF182, a central nervous system-specific gene, is the most highly expressed during neuronal differentiation in P19 cells and that it is predominantly expressed in lysosomes and late endosomes. In the present study, we investigated the role of RNF182 in neural differentiation. We performed shotgun proteomics techniques and identified lysosomal-associated transmembrane protein (LAPTM) 4A and 4B as its substrates. We found that RNF182 preferentially poly-ubiquitylates LAPTMs via K63-linked chains, suggesting that RNF182 does not promote the K48 chain-mediated degradation of LAPTMs. LAPTM4B reportedly participates in the recruitment of the large neutral amino acid transporter LAT1 to lysosomes, leading to leucine uptake into lysosomes and mammalian target of rapamycin complex 1 (mTORC1) activation. We showed that the RNF182-mediated poly-ubiquitylation of LAPTMs facilitated the interaction of LAPTMs with LAT1 and the recruitment of LAT1 to lysosomes. Furthermore, RNF182-deficient P19 cells exhibited abnormal neurite outgrowth. RNF182-knocked down P19 cells displayed decreased mTORC1 signaling. In contrast, we found that RNF182 expression was induced by the lysosome inhibitor chloroquine and the transcription factor TFEB, which is activated by lysosomal stress. Based on these results, we speculate that RNF182 upregulates mTORC1 signaling via LAPTM ubiquitination during neuronal differentiation. As TFEB is inactivated by mTORC1, RNF182 may reciprocally regulate the mTORC1 pathway.

O4-3 Deletion of Class II ARFs in mice causes tremor by inhibiting Nav1.6 trafficking to cerebellar Purkinje cell axon initial segments ARFタンパク質と軸索起始部へのナトリウムチャネルの輸送 Sadakata Tetsushi（定方哲史）¹，細井延武²，柴崎貢志³，今野歩²，平井宏和²，石崎泰樹³，古市貞一⁴ ¹Education and Research Support Center, Gunma University Graduate School of Medicine ²Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine ³Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine ⁴Department of Applied Biological Science, Tokyo University of Science ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3); class II (ARF4 and 5); and class III (ARF6). In contrast to class I and III ARFs, which are known to be the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4^+/-/ARF5^-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4^+/-/ARF5^-/- mice exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4^+/-/ARF5^-/- mice. A severe and selective decrease of pore-forming voltage-dependent Na+ channel subunit Nav1.6, important for maintaining repetitive action potential firing, was identified in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein expression and the consequent tremors in ARF4^+/-/ARF5^-/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Taken together, our data demonstrate that the decreased expression of total class II ARF proteins in ARF4^+/-/ARF5^-/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, causes the disorganization of Nav1.6-containg AIS and hence the reduction of membrane excitability in PCs, resulting in the ET-like movement disorder. Thus, we suggest that class II ARFs play roles in targeting specific proteins, such as Nav1.6, to the AIS of neurons.