Johansson Sara, Radesäter Ann-Cathrin, Cowburn Richard F, Thyberg Johan, Luthman Johan
Local Discovery Research Area CNS& Pain Control, AstraZeneca, S-151 85, Södertälje, Sweden.
Exp Brain Res. 2006 Jan;168(1-2):11-24. doi: 10.1007/s00221-005-0069-z. Epub 2005 Sep 21.
Pronounced neurodegeneration of hippocampal pyramidal neurons has been shown in Alzheimer's disease. The aim of this study was to establish an organotypic in vitro model for investigating effects of the amyloid beta (Abeta)-peptide on pyramidal neuron degeneration, glial cell activation and tau phosphorylation. Tissue cultures in a quasi-monolayer were obtained using roller-drum incubation of hippocampal slices from neonatal Sprague Dawley rats. Neuronal populations identified included N-methyl-D-aspartate (NMDA-R1) receptor immunoreactive pyramidal neurons, and neurons immunopositive for glutamic acid decarboxylase-65 (GAD65) or gamma amino butyric acid (GABA). Many neurons expressed phosphorylated tau as shown by pS(396), AD2 and PHF-tau immunostaining. Astrocytes, microglial cells and macrophages were also identified. The Abeta(25-35) peptide formed fibrillar networks within 2 days as demonstrated by electron microscopy. In the presence of the neurotoxic Abeta(25-35) peptide, but not Abeta(35-25), deposits developed in the tissue that were stainable with Thioflavine T and Congo red and showed the characteristic birefringence of Abeta plaques. Following Abeta(25-35) exposure, neurodegenerative cells were observed with Fluoro-Jade B staining. Further characterization of pyramidal neurons immunopositive for NMDA-R1 showed a decrease of cell number in the immediate surrounding of Abeta(25-35) deposits in a time- and concentration-dependent fashion. Similar effects on pyramidal neurons were obtained following exposure to the full-length, Abeta(1-40) peptide. Also, a loss of neuronal processes was seen with GAD65, but not GABA, immunohistochemistry after exposure to Abeta(25-35). Abeta(25-35)-exposed neurons immunopositive for phospho-tau showed degenerating, bent and often fragmented processes. Astrocytes showed increased GFAP-positive reactivity after Abeta(25-35) exposure and formation of large networks of processes. No obvious effect on microglial cells and macrophages could be seen after the Abeta(25-35) exposure. The developed in vitro system may constitute a useful tool for screening novel drugs against Abeta-induced alterations of tau and degeneration of hippocampal neurons.
在阿尔茨海默病中已显示出海马锥体细胞明显的神经退行性变。本研究的目的是建立一种体外器官型模型，用于研究β淀粉样蛋白（Aβ）肽对锥体细胞变性、胶质细胞活化和tau蛋白磷酸化的影响。使用新生Sprague Dawley大鼠海马切片的滚筒培养法获得准单层组织培养物。鉴定出的神经元群体包括N-甲基-D-天冬氨酸（NMDA-R1）受体免疫反应性锥体细胞，以及谷氨酸脱羧酶-65（GAD65）或γ-氨基丁酸（GABA）免疫阳性的神经元。如pS(396)、AD2和PHF-tau免疫染色所示，许多神经元表达磷酸化tau蛋白。还鉴定出了星形胶质细胞、小胶质细胞和巨噬细胞。电子显微镜显示，Aβ(25-35)肽在2天内形成纤维状网络。在神经毒性Aβ(25-35)肽存在的情况下，但Aβ(35-25)不存在时，组织中出现了用硫黄素T和刚果红可染色的沉积物，并显示出Aβ斑块的特征性双折射。在暴露于Aβ(25-35)后，用Fluoro-Jade B染色观察到神经退行性细胞。对NMDA-R1免疫阳性的锥体细胞的进一步表征显示，在Aβ(25-35)沉积物紧邻区域的细胞数量以时间和浓度依赖性方式减少。暴露于全长Aβ(1-40)肽后，对锥体细胞也获得了类似的影响。此外，在暴露于Aβ(25-35)后，用GAD65免疫组化观察到神经元突起减少，但用GABA免疫组化未观察到。对磷酸化tau蛋白免疫阳性的Aβ(25-35)暴露神经元显示出退化、弯曲且常断裂的突起。星形胶质细胞在暴露于Aβ(25-35)后显示出GFAP阳性反应性增加，并形成大的突起网络。在暴露于Aβ(25-35)后，未观察到对小胶质细胞和巨噬细胞有明显影响。所建立的体外系统可能构成一种有用的工具，用于筛选针对Aβ诱导的tau蛋白改变和海马神经元变性的新型药物。
