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國立成功大學「發展國際一流大學及頂尖研究中心計畫」
延攬優秀人才工作報告表
NCKU Project of Promoting Academic Excellence & Developing World Class Research Centers Work Report Form for Distinguished Scholars
□續聘 continuation of employment ■離職resignation
99 年 4 月 20 日更新 受聘者姓名
Name of the Employee
李 政 哲 ■男 女 Male Female 聘 期 Period of Employment from 98 年(y) 7 月(m) 1 日(d) to 99 年(y) 12 月(m) 31 日(d) 研究或教學或科技研發與 管理計畫名稱 The project title of research,
teaching, technology development and management
Psychosocial stress alters synaptic plasticity and neurogenesis in the hippocampus: a pathogenetic mechanism of major depressive disorder 計畫主持人 （申請單位主管） Project Investigator (Head of Department/Center) 許 桂 森 補助延聘編號
Grant Number HUA 99 - 03 - 07 - 165
一、 研究、教學、科技研發與管理工作全程經過概述。（由受聘人填寫）
Please summarize the entire research, teaching, or science and technology R&D and management work process (To be completed by the employee)
Research topic：The role of insulin in the regulation of dendritic spine and synapse formation
Insulin is a peptidergic hormone secreted by the pancreatic islets of Langerhans and plays an important role in regulating glucose homeostasis. Furthermore, insulin and its receptor are also broadly expressed throughout the brain with relatively high density located in the olfactory bulbs, cerebral cortex, hypothalamus, and hippocampus,where they are thought to serve as a number of functions including regulation of glucose metabolism, food intake and body weight, fertility and reproduction, learning, memory, and attention. Recently, various functions for insulin receptor signaling in the brain have been demonstrated in normal neurophysiology, and a dysfunction of insulin secretion or insulin receptor signaling has been reported in serious mental illnesses. Brain insulin receptors are expressed in particularly high concentrations in neurons, and in much lower levels in glia. Although the mRNA of insulin receptors is mainly localized in neuronal somata, abundant insulin receptors are found in both cell bodies and synapses. In recent years, several studies have shown that insulin could regulate the endocytosis of 3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, which causes long-term depression (LTD) of excitatory synaptic transmission. LTD in the mammalian brain is assumed to be a synaptic mechanism underlying learning during novel experiences, this insulin-induced LTD may therefore play a crucial role in brain information processing and synaptic plasticity. However, very little is known about the functional significance of synaptic insulin receptors in the neurons. Our previous studies has also indicated that brief insulin treatment substantially increases the synthesis of PSD-95 protein, a fundamental structural proteins within the PSD, in local dendritic compartments via activation of the PI3K-Akt-mammalian target of the rapamycin (mTOR) signaling pathway. Insulin activates mTOR and its downstream translation regulatory molecules, 4E-BP1 and p70S6K, to
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stimulate the translation of the dendritic spine scaffolding protein PSD-95. Besides, insulin receptor signaling may also regulate the extracellular concentration of Aβ -degrading Our recent data have further been shown that
insul , a characteristic clinical manifestation in
Alzheimer’s disease, and ameliorates Aβ-induced impairment of LTP in the CA1 region of rat hippocampal slices. In addition to the modulation of insulin on the synaptic plasticity, recent study has demonstrated that manipulation of insulin receptors upon visual system of Xenopus tadpole can regulate synapse number, dendritic plasticity and function of neuronal circuit, but the exactly effect that insulin and its receptor act on the dendritic spines formation of mammalian system is still unidentified.
Thus, I further want to identify whether insulin has potentiality to regulate structural plasticity, the predominant mechanisms for changes in the network architecture in the brain. The main subject is to address that the precise molecular mechanism of insulin and its receptor in the regulation of dendritic spine formation in which associated with synaptic function in the hippocampus.
Dendritic spines are small structure protruding from the dendrites that form the postsynaptic compartments for excitatory inputs. These small protrusions show actin-based rapid motility, dynamic turnover and morphological plasticity. Changes in the morphology and number of dendritic spines have been proposed to contribute to numerous physiological processes such as synapse development, function and plasticity. Recent evidences have been identified that manipulation of several key molecular regulators of actin cytoskeleton including Rac1, cdc42 and Rho family of small GTPases regulate dendritic spine morphogenesis and synapse formation. However, little detail mechanism is known about the upstream activators that stimulate these signaling mediators for dendritic spine regulation.
At first, in order to evaluate the regulation of insulin on dendritic spine formation in vitro, I utilize rat hippocampal primary cultured neurons transfected with the enhanced green fluorescence protein (EGFP) for visualizing the morphological changes in neurons before insulin addition. I use the fluorescent microscope to observe the labeled neurons after the hippocampal primary cultured neurons growing to 14-15 days in vitro, which is the suitable stage for assessing dendritic spine formation. Secondly, pharmacological inhibitors, lentiviral-based shRNA infection or transfection and biochemical methods are performed to address the downstream molecular mechanism of insulin-induced dendritic spine formation.
Recent work from our laboratory has been identified an important role of insulin receptor signaling in the regulation of dendritic spine and excitatory synapse formation. Use the specific inhibitors to block insulin signaling pathway (AG1024, LY294002, wortmannin, U0126 and rapamycin) or small-GTPase activity (NSC23766), we demonstrate that insulin acting on insulin receptors activates the PI3K-Akt-mTOR signaling pathway, not MEK/ERK signaling pathway, which in turn promotes Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent actin cytoskeletal rearrangement and
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dendritic spine formation. Besides, knockdown of Rac1 by specific shRNA also appears the inhibition of insulin-induced dendritic spine formation, whereas is similar with the Rac1 inhibitor treatment. Furthermore, this enhancement of insulin-induced spine formation is not only associated with synaptic activity but simultaneous with synapse formation. Conversely, as we sequester endogenous insulin by addition of anti-insulin antibody or downregulate insulin receptors by specific shRNA construct, the increase of insulin-induced dendritic spine formation is disturbed. These findings add to the growing list of functions for insulin receptor signaling in neuronal structure development and synaptic plasticity. Furthermore, these results suggest that insulin receptor signaling may regulate not only the processing of synaptic plasticity but also the structural plasticity by cytoskeletal rearrangement that is required for the incorporation of neurons into brain circuits.
二、研究或教學或科技研發與管理成效評估（由計畫主持人或單位主管填寫）
Please evaluate the performance of research, teaching or science and technology R&D and management Work: (To be completed by Project Investigator or Head of Department/Center)
(1)是否達到延攬預期目標？
Has the expected goal of recruitment been achieved?
The expected goal of recruitment has been achieved.
(2)研究或教學或科技研發與管理的方法、專業知識及進度如何？
What are the methods, professional knowledge, and progress of the research, teaching, or R&D and management work?
The results have been submitted for publication consideration.
(3)受延攬人之研究或教學或科技研發與管理成果對該計畫(或貴單位)助益如何？
How have the research, teaching, or R&D and management results of the employed person given benefit to the project (or your unit)?
The results of the employed person provide much benefit to the future projects of my research field. (4)受延攬人於補助期間對貴單位或國內相關學術科技領域助益如何？
How has the employed person, during his or her term of employment, benefited your unit or the relevant domestic academic field?
The results of the employed person are highly benefited relevant domestic academic field of brain insulin research.
(5)具體工作績效或研究或教學或科技研發與管理成果：
Please describe the specific work performance, or the results of research, teaching, or R&D and management work:
Dr. Lee has identified an important role of insulin receptor signaling in the regulation of dendritic spine and excitatory synapse formation. Insulin and its receptor are broadly expressed throughout the brain and have been postulated to play a crucial role in synaptic plasticity. Although structural remodeling of dendritic spines is associated with stable expression of synaptic plasticity, the role of insulin receptor (IR) signaling in the establishment and dynamic changes of dendritic spines remains unclear. Here Dr. Lee demonstrates that insulin promotes dendritic spine formation in primary cultures of rat hippocampal neurons. Conversely, downregulation of IR signaling using a blocking antibody or short hairpin RNAs (shRNAs) resulted in a decrease in number of dendritic spines and caused a significant reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) without affecting the distribution of their amplitudes. Pharmacological blockade of
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phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway and the small GTPase Rac1 specifically prevented the insulin-induced increase in dendritic spine density. In parallel, genetic ablation of Rac1 expression by lentiviral infection with shRNA abrogated the increase in dendritic spines induced by insulin. More importantly, the increase in dendritic spine density by insulin was accompanied by increasing in presynaptic marker staining density and displayed an increase in mEPSC frequency. Taken together, these results reveal a novel role for IR signaling in the regulation of dendritic spine formation and excitatory synapse development in hippocampal neurons through activation of the PI3K/Akt/mTOR and Rac1 signaling pathways.
(6)是否續聘受聘人? Will you continue hiring the employed person? ■續聘Yes □不續聘No
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