学 位 記 番 号 理工博 第

氏 名 古澤

孝太郎

所 属 理工学研究科 生命科学専攻 学 位 の 種 類 博士（理学）

学 位 記 番 号 理工博 第

号 学位授与の日付 平成

年

月

日 課程・論文の別 学位規則第

条第

項該当

学 位 論 文 題 名

神経細胞の軸索伸長と

代謝を制御する新たな膜輸送機構の解析

（英文）

論 文 審 査 委 員 主査 教 授 久永 眞市 委員 教 授 川原 裕之 委員 教 授 岡本 龍史 委員 准教授 安藤 香奈絵

【論文の内容の要旨】

Neurons communicate with each other through their axons and dendrites. Neurite outgrowth requires the supply of membrane components for surface expansion.

However, the molecular mechanisms of membrane supply in growing neurite is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a neuron specific Ser/Thr protein kinase that is activated by the binding of either p35 regulatory subunit. It plays an important role in a variety of neuronal functions including neuronal migration during brain development, synaptic signaling, survival, and neuron death. Cdk5-p35 is also involved in membrane trafficking. For example, Cdk5-p35 regulates trafficking of Rab11A-positive recycling endosomes during neurite outgrowth through lemur tyrosine kinase 1 (LMTK1). In general, membrane trafficking is regulated by Rab GTPase family proteins. Rab GTPases are activated by guanine nucleotide exchange factors (GEFs) which stimulate the exchange of GDP to GTP. Rab8 and Rab11 are known as a regulator of neurite outgrowth.

In the first part of this thesis, I focused on a GRAB, a GEF for Rab8 but also a binding protein for Rab11, and investigated the role of Rab11-Rab8 cascade in axon

outgrowth. Herein, I found that GRAB is a novel regulator of axon outgrowth.

Knockdown of GRAB suppressed axon outgrowth of cultured mouse brain cortical neurons. GRAB mediates the interaction between Rab11A and Rab8A and this activity is regulated by phosphorylation at Ser169 and Ser180 by Cdk5-p35. The non-phosphorylatable GRAB mutant S169/180A promoted axonal outgrowth to a greater extent than did the phospho-mimic GRAB mutant S169/180D. Phosphorylation of GRAB suppressed its guanine nucleotide exchange factor activity and its ability to recruit Rab8A to Rab11A-positive endosomes. In vivo function of GRAB and its Cdk5-phophorylation was shown in migration and process formation of developing neurons in embryonic mouse brains. These results indicate that GRAB regulates axonal outgrowth via activation and recruitment of Rab8A to Rab11A-positive endosomes in a Cdk5-dependent manner. This is a novel regulatory mechanism of axonal outgrowth by Cdk5.

Secondary, I studied the role of CD2AP in APP metabolism in neurons. Cdk5 modulates the endocytosis of ApoER2 through the regulation of CIN85-Dab1 interaction by Dab1 phosphorylation. CIN85 is involved in the endocytic trafficking of amyloid precursor protein (APP) in yeast. CD2AP belongs to same family adaptor protein CIN85, which also regulates endocytosis. I hypothesized that CD2AP is involved in the endocytosis of APP under the control of Cdk5. Herein, I examined the effect of CD2AP on the subcellular localization of APP by using membrane traffic regulator, Rab GTPases as endosomal markers, and found that the overexpression of CD2AP decreased the colocalization of APP with Rab5. The expression of CD2AP decreased the APP protein and promoted the degradation of APP by the treatment with cycloheximide.

When the decrease of APP protein was induced by serum starvation, the reduction of APP was promoted by the expression of CD2AP. The serum starvation decreased the colocalization of APP with Rab5, and this phenotype was similar to the expression of CD2AP. These results suggested that CD2AP regulates APP metabolism through Rab5-mediated membrane traffic