| Summary: | 博士 === 臺灣大學 === 解剖學暨生物細胞學研究所 === 96 === 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.
|
|---|
