Creation of transgenic mouse expressing C-terminus of APP or ERβ and its application for studying protective effect of antioxidant or correlation of ERβ with Alzheimer's disease

Abstract

(Part I) The amyloid protein precursor (APP) is cleaved in its intramembranous domain by γ-secrease to generate amyloid β and a free carboxyl-terminal intracellular fragment. The carboxyl-terminal of 105 amino acids of APP (APP-C105) plays a crucial role in the neuropathology of Alzheimer’s disease (AD), but it is incompletely understood how APPC105 overexpression interacts and regulates the brain function and Aβ42 levels, and whether or not it is associated with the expressions of glycogen synthase kinase (GSK3)β-binding proteins. To determine this, transgenic mice expressing neuron-specific enolase (NSE)-controlled APP-C105 were produced and tested for their phenotypes. A behavioral deficit was observed in the 9 months old transgenic mice, and western blot indicated that there was a predominant expression of APP-C105 in brains of the transgenic mice compared with those of non-transgenic mice. In parallel, APP-C105 overexpression resulted in the modulation of the Aβ42 level, γ-secretase activity, and GSK3β-binding proteins including presenilin (PS) 1, tau, and β-catenin in the brains of the transgenic mice relative to the non-transgenic mice. Altered expressions of these neuropathological phenotypes could be useful targets in developing new therapeutic treatments. Further study was performed using these transgenic mice, to test whether the treatment of transgenic mice with green tea catechin (GTC) improves the AD’s phenotypes. To determine this, 7-month old transgenic mice were treated with a low (0.02%) and high doses (0.2%) for 6 months, and was tested the AD’s phenotypes. Herein, the GTC-treated groups exhibited significant recovery in behavioral impairment, Aβ42, APP-C99/89, secretase activities, γ-secretase components, Wnt signal, and MAPK activations. In contrast, the levels of APP-C83 proteins, and α-secretase, neprilysin, and Pin1 activities were elevated in the GTC-treated groups. Moreover, GTC-treated groups showed lower total cholesterol (TC) and low-density lipoprotein-choesterol (LDL-C) level, but higher high-density lipoprotein-choesterol (HDL-C). These results provide first experimental evidences that GTC may lead to global improvements across the AD’s phenotypes, and thereby GTC might be good for prevention or treatment of AD patients. (Part Ⅱ) Estrogen action is mediated by ERs in activating MAPK-signaling pathway. To test whether the estrogenic effect on ER β-mediated MAPK activation in in vitro, hybrid genes containing each human ER β fused to the NSE promoter were constructed and were transfected to the neuronal SK-N-MC cells as a control, NSE/APP-C105 was transfected into some cell line. Western blots shows that the activation of JNK-signaling pathway, but not p38 and ERK, is dependent of ER β through estrogen treatment and APP-C105 is also mediated through estrogen in activating MAPK-signaling pathway. The results suggest that ER β and APP-C105 derived from APP are necessary for estrogenic effect in activating MAPK-signaling pathway. Otherwise, ERs are continuously shuttling between the nucleus and cytoplasm, and a shift was accompanied by a relative change of hyperphosphorylated tau protein (AT8) with sex or age differences in patients of AD. Further study was to determine how human ER β (hER β) expression in cytosol and nucleus of 3 months and 20 months females and males of transgenic mice (ER β-tg) regulates and interacts the phosphorylation of tau-binding proteins. This was then compared with those in AD- transgenic mice expressing C-terminus of APP (APP/C105)(APP/C105-tg). Herein, we concluded that i) transgenic mice expressing neuron-specific enolase-controlled human ERb (hERβ) were successfully established. ii) the hERβ transgene was expressed at high levels in the variety of tissues including brain, liver, ovary, uterus, cervix, vagina, prostate, S.V, testis, LABC compared to those in nontransgenic littermates (Non-tg). iii) increases of nuclear ERβ were observed in 20 months females of ERβ-tg and APP/C105-tg groups, and its increases were significant in 3 months and 20 months males of ERβ-tg and APP/C105-tg groups, but decreased in 3 months females of ERβ-tg and APP/C105-tg groups. iv) the patterns of nuclear tau phosphorylations [nuclear AT8 and p-tau (Serine 396, and Serine 404)] were similar to those in nuclear p-GSK3β and cytosolic p-β-catenin, and their patterns almost paralles the nuclear ERβ pattern in 3 months and 20 months females or 20 months males of ERβ-tg and APP/C105-tg groups, but not in 3 months males of ERβ-tg and APP/C105-tg groups. v) the levels of nuclear β-catenin Wnt protein were decreased in 20 months males of ERβ-tg and APP/105-tg groups. The results suggest that the level of nuclear ERβ expression was decreased at high concentration of estrogen and its decreased level is correlated with an inhibition in phosphorylations of tau-binding proteins including GSK3β and β-catenin, but increased at low concentration of estrogen, leading to increase in their phosphorylations. Moreover, phosphorylation of tau-binding proteins was inhibited at high concentration of testosterone in nuclear ERβ-independent manner.;아밀로이드 전구체 단백질은 r-secretase에 의해 intramembrane domain이 베타 아밀로이드와 카르복실 말단 부분으로 processing 되며, 아밀로아드 카르복실 말단의 105개 아미노산은 알츠하이머 질환 뇌병변에 핵심적인 역할을 한다고 알려져 있다. 그러나 APP-C105 과다 발현이 뇌 기능과 Aβ42 수준사이의 상호관련 및 조절 방법과 GSK3 β 결합 단백질의 발현, 그리고 상호관련 여부에 대해서는 아직까지 연구가 부족한 실정이다. 따라서, 본 연구에서는 뉴론 특이적인 프로모터로 조절되는 APP-C105를 과다 발현시킨 형질전환 마우스를 개발하여, 이 형질전환 마우스를 대상으로 표현형의 변화양상을 살펴보고자 하였다. 본 연구결과, 인지 및 학습 능력을 평가할수 있는 행동이상실험에서 9개월령의 형질전환 마우스에서 비정상적인 행동이상을 나타내었다. 또한, 정상 마우스에 비해 형질전환 마우스에서 APP-C105의 과다 발현을 관찰할 수 있었으며, 이와같은 형질전환 마우스에서의 APP-C105 과다발현은 Aβ42, r-secretase 활성화, 그리고 PS1, tau, β-catenin을 포함하는 GSK3 β-결합단백질의 수준 조절에 관여하는 것으로 나타났다. 그러므로, 뇌질환 표현형의 변화는 알츠하이머 질환을 위한 새로운 치료제 개발에 유용한 지표로 활용될 수 있을 것으로 사료된다. 활성 유리기의 제거제인 녹차의 카테킨 (Green Tea Catechin; GTC)은 퇴행성 뇌병변의 진행과정을 억제시키는 효과를 가지고 있다고 알려져 있다. 그러므로, 본 연구에서는 알츠하이머 형질전환 마우스를 대상으로 녹차 카테킨을 섭취시킨 다음 알츠하이머 질환의 표현형의 개선유무를 확인하고자 하는데 본 연구의 목적을 두었다. 7개월령의 형질전환 마우스를 3 그룹으로 나누어 대조군 및 녹차 카테킨을 0.02% 및 0.2%로 구분하여 6개월 동안 매일 신선하게 만들어 음용수 상태로 자유롭게 섭취하도록 하였다. 실험 결과, 녹차 카테킨을 섭취시킨 그룹에서는 행동학적 이상, Aβ42, APP-C99/C89, r-secretase activity, r-secretase components, Wnt signal, and MAPK activation 등이 유의하게 감소하였다. 이에 반해, APP-C83, r-secretase, neprilysin 그리고, Pin1 활성은 녹차 카테킨 공급으로 인하여 상승되는 것으로 나타났다. 또한, 녹차 카테킨을 공급한 그룹에서 총 콜레스테롤과 LDL-콜레스테롤의 농도가 낮은 반면 HDL-콜레스테롤 농도는 높게 나타났다. 그러므로, 본 연구는 녹차 카테킨 섭취는 알츠하이머 질환의 표현형을 광범위한 부분에서 개선시킬 수 있다는 최초의 의의있는 실험적인 접근이며, 녹차 카테킨은 알츠하이머 질환을 위한 훌륭한 예방 및 치료제가 될 수 있을 것이라고 판단된다.