Chauhan Neelima B, Siegel George J
Department of Anesthesiology, University of Illinois at Chicago 60612, and Neurology Service (127), Edward Hines, Jr., VA Hospital, Hines, IL 60614, USA.
Neuroscience. 2007 Apr 25;146(1):143-51. doi: 10.1016/j.neuroscience.2007.01.008. Epub 2007 Feb 14.
Misprocessing of beta-amyloid precursor protein (APP) leading to the formation of elevated quantities of beta-amyloid peptide (Abeta), derived by a cleavage at the beta-secretase site (N-671/673aa) and by a cleavage at the gamma-secretase site (C-711/713aa) of APP, is considered a key event in the pathogenesis of Alzheimer disease (AD). Point mutations near the beta-secretase site in the human gene for APP, such as in the Swedish mutation-KM670/671NL, lead to a form of dominantly inherited AD. These mutations are known to promote beta-site cleavage and to increase levels of Abeta. Abeta has been shown previously to increase acetyl cholinesterase (AChE) activity in vitro. We wished to test whether translational blocking of APP-mRNA at the mutated beta-site by antisense (AS) oligodeoxynucleotides (ODNs) directed to the mutated site will reduce cerebral amyloid in the Swedish transgenic mouse model (Tg2576). Mice were injected i.c.v. with AS-ODNs directed at the mutated beta-site (AS-beta site) or with AS-ODNs directed at the normal gamma-site (AS-gamma site) of human APP-mRNA, and compared with procedural controls that received i.c.v. injections of sense ODNs at the beta-site (S-beta site), sense ODNs at the gamma-site (S-gamma site) or mismatched ODNs, and with untreated littermates (Lt) and untreated transgenic mice (Tgs). ODNs were injected into the 3rd ventricle once a week for 4 weeks. Brains were processed for enzyme-linked immunosorbent assay analysis of beta- and gamma-cleaved soluble Abeta40 (sAbeta40), beta- and gamma-cleaved soluble Abeta42 (sAbeta42) and alpha-cleaved soluble beta-amyloid precursor protein (sAPPalpha). The physiological relevance of AS ODNs was tested by evaluating the cerebral distribution of AChE before and after the treatment. AChE was found increased about fivefold in Tg cortex as compared with control brain. Results show that compared with untreated and procedural controls, AS-beta increased cerebral levels of sAPPalpha by 43% and reduced sAbeta40/42 by approximately 39%; while simultaneously reducing the cortical density of AChE by approximately fourfold in the treated Tg animals, almost to the level found in the control brain (all values P<0.0001, analysis of variance, unpaired two-tailed Student's t-test), while AS-gamma did not have any effect. These results indicate that AS directed to the mutated beta-site may be an effective approach to treat familial AD.
β-淀粉样前体蛋白（APP）的错误加工导致大量β-淀粉样肽（Aβ）形成，该过程通过APP在β-分泌酶位点（N-671/673aa）的切割以及在γ-分泌酶位点（C-711/713aa）的切割产生，被认为是阿尔茨海默病（AD）发病机制中的关键事件。人类APP基因中β-分泌酶位点附近的点突变，如瑞典突变-KM670/671NL，会导致一种显性遗传性AD。已知这些突变会促进β位点的切割并增加Aβ水平。此前已表明Aβ在体外可增加乙酰胆碱酯酶（AChE）活性。我们希望测试针对突变β位点设计的反义（AS）寡脱氧核苷酸（ODN）对APP-mRNA的翻译阻断是否会减少瑞典转基因小鼠模型（Tg2576）中的脑淀粉样蛋白。给小鼠脑室内注射针对人类APP-mRNA突变β位点的AS-ODN（AS-β位点）或针对正常γ位点的AS-ODN（AS-γ位点），并与在β位点注射正义ODN（S-β位点）、在γ位点注射正义ODN（S-γ位点）或错配ODN的程序对照组，以及未处理的同窝小鼠（Lt）和未处理的转基因小鼠（Tgs）进行比较。ODN每周注射到第三脑室一次，共注射4周。对大脑进行处理，用于酶联免疫吸附测定分析β和γ切割的可溶性Aβ40（sAβ40）、β和γ切割的可溶性Aβ42（sAβ42）以及α切割的可溶性β-淀粉样前体蛋白（sAPPα）。通过评估治疗前后AChE的脑部分布来测试AS ODN的生理相关性。与对照脑相比，Tg皮质中的AChE增加了约五倍。结果表明，与未处理组和程序对照组相比，AS-β使sAPPα的脑水平增加了43%，使sAβ40/42降低了约39%；同时在治疗的Tg动物中使皮质AChE密度降低了约四倍，几乎降至对照脑中的水平（所有值P<0.0001，方差分析，非配对双尾学生t检验），而AS-γ没有任何效果。这些结果表明，针对突变β位点的AS可能是治疗家族性AD的有效方法。
