探討α-internexin啟動子在離體與活體系統中的表現以及大豆甙元對鼠胚海馬回神經元的神經滋養作用及其機制

Abstract

第一部分 探討α-internexin啟動子在離體與活體系統中的表現 α-Internexin是一種分子量為66 kDa的神經中間絲蛋白，其會大量表現在發育早期的神經系統中。為了進一步了解小鼠α-internexin基因啟動子的功能，我們分別設計以0.7 kb和1.3 kb不同長度的小鼠α-internexin基因5端序列來驅動綠色螢光蛋白 (EGFP，enhanced green fluorescence protein) 基因之DNA質體 (分別為p0.7intfs-EGFP和p1.3intfs-EGFP)，並觀察螢光蛋白在細胞株中專一性表現的情形。將DNA質體以lipofectamine轉染至非神經性纖維母細胞株3T3及神經細胞株Neuro2A後，我們觀察到1.3 kb α-internexin基因啟動子驅動綠色螢光蛋白基因會顯著地選擇表現在Neuro2A細胞內。為了確定1.3 kb α-internexin基因啟動子的神經專一性，我們設計一由1.3 kb α-internexin啟動子驅動DNA重組酶Cre蛋白基因的DNA質體 (p1.3intfs-Cre)，並進一步以此質體架構建立基因轉殖鼠品系。先把p1.3intfs-Cre與ploxpLacZ質體共同轉染至Neuro2A細胞株確定Cre DNA重組酶蛋白在神經性細胞株內的表現活性後，我們將p1.3intfs-Cre基因轉殖鼠與具有ROSA26 reporter (R26R) 的小鼠交配，透過X-gal染色法標示β-galactosidase在新生鼠體內的表現，即可以檢視Cre DNA重組酶的表現活性與位置。在具有p1.3intfs-Cre基因與R26R基因表現的1天齡新生鼠體內，我們可以在周邊神經系統中偵測到β-galactosidase的活性，例如在支配舌及顏面的腦神經分支及分布到軀幹的脊神經分支內。更進一步地，我們證實在背根神經節內具有被X-gal染色標定的神經元，其細胞體具有內生性α-Internexin免疫染色活性；然而脊髓腹角內具有α-Internexin免疫染色活性的運動神經元則並沒有表現任何β-galactosidase的活性。因此，本研究所建立之α-internexin啟動子驅動DNA重組酶Cre蛋白表現的轉殖鼠品系可提供作為一動物模式，用以操控其他特定神經元基因的表現，研究這些基因在神經系統發育過程中所扮演的角色。 第二部份 大豆甙元對鼠胚海馬回神經元的神經滋養作用及其機制 大豆甙元 (Daidzein, Dz) 具有與動物雌激素 (estrogen or β-estradiol, E2) 相似的神經活性。在本研究中，我們以初級培養大鼠海馬回神經元 (hippocampal neuron) 為材料，探討大豆甙元的神經滋養效果 (neurotrophic effect) 及可能之機制。實驗結果發現，大豆甙元可增進軸突生長 (axonal outgrowth) 及促進生長錐 (growth cone) 的形成，大豆甙元亦可使在軸突生長早期生長錐內的生長相關蛋白43 (growth-associated protein 43, GAP-43) 免疫染色增加。更進一步地，我們發現大豆甙元會增加磷酸化GAP-43的含量而對GAP-43總含量沒有影響。這些大豆甙元促進軸突生長及GAP-43磷酸化的作用會被蛋白激酶C (protein kinase C, PKC) α/βI的抑制劑Gö6976所阻斷，但卻不受PKCε的抑制劑εV1-2或MEK/ERK的抑制劑PD89059影響。此外，大豆甙元可誘導PKCα的磷酸化和細胞膜轉位，而非PKCβ。我們亦發現在大豆甙元處理後，雌激素受器 (estrogen receptor, ER) β的免疫染色會集中在神經細胞體及生長中軸突的細胞膜上，而在細胞核的ERα免疫染色分布則不受影響。當投予ER的拮抗劑ICI182,780時，由大豆甙元引發的軸突生長以及PKCα和GAP-43的活化作用均會被抑制。綜合上述的研究結果，我們認為在軸突生長的早期，大豆甙元會引發ERβ的細胞膜轉位，接著造成PKCα的活化，因而使GAP-43被磷酸化，透過此一可能的機制，大豆甙元可以促進海馬回神經元的軸突生長。接著，我們進一步探討大豆甙元對樹突生長 (dendritic outgrowth) 與突觸形成 (synapse formation) 的神經滋養效果及可能之機制。我們發現，大豆甙元可增加神經元樹突的總長度、樹突末端總數、樹突分岔次數及二級以上樹突的數目，也會提高樹突微小管相關蛋白MAP2 (microtubule-asociated protein 2) 和神經中間絲蛋白NFL (neurofilament light) 的表現量；此外我們亦發現在海馬神經元生長後期，大豆甙元會增加GAP-43、突觸蛋白I (synapsin I)、突觸後緻密蛋白95 ( post-synaptic density protein 95, PSD95) 和嗜棘蛋白 (spinophilin) 的免疫染色。經由FM1-43染劑攝入測定法，我們觀察到大豆甙元會增加活化態突觸前末端的數量。這些結果證實大豆甙元具有促進樹突生長與突觸形成的效果。這些大豆甙元所促進的樹突生長和增加細胞骨架蛋白的表現主要會被ER的拮抗劑ICI182,780、MEK/ERK抑制劑PD98059、PKA抑制劑PKI和CaMKII抑制劑KN93所阻斷，但較不受JNK抑制劑SP600125所影響。進一步發現大豆甙元促進CaMKII和ERK1/2的活化 (磷酸化作用) 會被ICI182,780所抑制，這顯示CaMKII和ERK1/2是在ER的下游。我們亦發現大豆甙元會使CREB磷酸化而且轉位至細胞核內。綜合上述的研究結果，我們認為大豆甙元可能會透過ER引發CaMKII和ERK1/2磷酸化，並且使CREB活化，進而促進海馬回神經元的樹突生長並影響細胞間突觸的形成。由於大豆甙元的神經滋養作用，未來有可能運用作為修復受傷海馬神經元的藥物之ㄧ。

Part I Neural Expression of mouse α-Internexin Promoter In Vitro and In Vivo α-Internexin is a 66 kDa neuronal intermediate filament protein found most abundantly in the neurons of the nervous systems during early development. To characterize the function of mouse α-internexin promoter, we designed two different expression constructs driven by 0.7 kb or 1.3 kb of mouse α-internexin 5'-flanking sequences; one was the enhanced green fluorescent protein (EGFP) reporter for monitoring specific expression in vitro (p0.7intfs-EGFP and p1.3intfs-EGFP respectively), and the other was the cre for studying the functional DNA recombinase in transgenic mice. After introducing DNA constructs into non-neuronal 3T3 fibroblasts and a neuronal Neuro2A cell line by lipofectamine transfection, we observed that the expression of p1.3intfs-EGFP was in a neuron-dominant manner. To establish a tissue-specific pattern in the nervous system, we generated a transgenic mouse line expressing Cre DNA recombinase under the control of 1.3 kb α-Internexin promoter (p1.3intfs-Cre). The activity of the Cre recombinase was examined by mating the cre transgenic mice to ROSA26 reporter (R26R) mice with knock-in Cre-mediated recombination. Analyses of postnatal day 1 (P1) newborns showed that β-galactosidase activity was detected in the peripheral nervous system (PNS), such as cranial nerves innervating the tongue and the skin as well as spinal nerves to the body trunk. Furthermore, X-gal-labeled dorsal root ganglionic (DRG) neurons showed positive for α-Internexin in cell bodies but negative in their spinal nerves. The motor neurons in the spinal cord did not exhibit any β-galactosidase activity. Therefore, the cre transgene driven by mouse α-internexin promoter described here provides a useful animal model to specifically manipulate genes in the developing nervous system. Part II Signaling Mechanisms of Daidzein-induced Neurotrophic Efficacy in Rat Cultured Embryonic Hippocampal Neurons Daidzein (Dz) has an estrogen-like neuroactive effect. In this study, we aim to study the mechanisms underlying the neurotrophic effect of Dz in hippocampal neurons. First, we found that Dz enhanced axonal outgrowths and increased immunostaining intensity of growth-associated protein 43 (GAP-43) in growth cones of neurons at day in vitro (div) 1. Consistent with this, Dz increased GAP-43 phosphorylation and its membrane translocation without affecting total GAP-43 levels. In the presence of Dz, significant increase in the immunoreactivity for estrogen receptor (ER) β, but not ERα, was observed on the membrane of cell bodies and growing axons. Dz also induced the activation of protein kinase C α (PKCα), which was inhibited by the ICI182,780 pretreatment. Similarly, Dz-promoted axonal elongation was blocked by ICI182,780 and Gö6976. Moreover, Dz-stimulated activation of GAP-43 was specifically abolished by Gö6976, suggesting PKCα being the upstream effector of GAP-43. Therefore, Dz triggers an ERβ/PKCα/GAP-43 signaling cascade to promote axonal outgrowths in cultured hippocampal neurons. We further examined the neurotrophic effect of Dz on dendritic outgrowth and synapse formation of hippocampal neurons, and the possible underlying mechanisms. Dz increased total dendritic length by increasing dendritic branch order, and upregulated protein levels of microtubule-associated protein 2 (MAP2) and neurofilament light (NFL) in neurons at div 3. This promoted neurite outgrowth was completely inhibited by the ICI182,780 pretreatment. The immunostaining intensities for several synaptic proteins, GAP-43, synapsin I, PSD95, and spinophilin, were increased on the cell bodies and processes of neurons at div 5 after Dz treatment. Additionally, Dz-increased dendritic growth and MAP2 expression were effectively blocked by the MEK/ERK inhibitor PD98059, the PKA inhibitor PKI, and the CaMKII inhibitor KN93, but not by the JNK inhibitor SP600125. In agree with this, Dz increased CaMKII and ERK phosphorylation, which was abolished by ICI182,780 pretreatment. Dz also enhanced CREB phosphorylation and its nuclear translocation. These data suggest that Dz induced ER-dependent activation of CaMKII and MEK/ERK, which led to the activation and nuclear translocation of CREB and mediated dendritic outgrowth and synaptogenesis. Taken collectively, these results suggest that Dz triggers diverse neurotrophic effects on cultured rat hippocampal neurons via multiple signaling cascades.