Identification of novel APP-specific alpha secretase in human umbilical cord blood sera

项目摘要 我们最近发现，SAPPα直接抑制APP的β-分泌酶(BACE1)蛋白分解，从而 减少Aβ产量。此外，我们最新的研究表明，Sappα可以减少阿尔茨海默氏症 (Ad)通过抑制Gsk3β而导致tau过度磷酸化。此外，虽然多次小剂量输注 人脐血单核细胞(CB-M)减轻AD大鼠病理和行为障碍 PSAPP小鼠，部分通过吞噬Aβ，衰老的单核细胞表现出Aβ吞噬功能受损，这可能是 被萨普α逆转。这些结果表明，CB-M具有自身的α分泌或激活内源性α- PSAPP小鼠体内的分泌酶。因此，通过改变淀粉样变原来恢复大脑中SAPα的水平 朝向非淀粉样蛋白(α-分泌酶)途径可以改善AD相关的淀粉样蛋白和tau病理， 神经元丢失和认知障碍。因此，增加α分泌酶的活性是一个有吸引力的策略。 治疗阿尔茨海默病。在关于α-分泌酶的描述性报道几年后，负责的酶 已被鉴定为去整合素和金属蛋白酶(ADAM)家族的成员。其中三个 膜锚定的锌依赖金属蛋白酶ADAM10、ADAM17和ADAM9具有α- 分泌酶活性。然而，在过去的几年里，许多其他底物都与这三种物质联系在一起。 脱落酶，其中一些可能通过激活细胞因子或趋化因子而促进AD的发病。确实如此 又称肿瘤坏死因子α转换酶(TACE)，可释放促炎因子肿瘤坏死因子α。 细胞因子。也有报道称CD40L通过ADAM10从T细胞释放，17通过CD40释放 结扎术。此外，ADAM10和ADAM17不是APP专一性的，可以切割各种底物，并且已经 与肿瘤发生和肿瘤进展有关。 我们最近发现，人脐血血清显著增强APPα- 切割，从而增加表达野生型人APP的CHO细胞中SAPPα的产生。此外, Cbs选择性介导神经元特异性、人重组全长APP695的α-分泌酶切割 (FAPP695)在无细胞系统中，独立于TACE和ADAM10。我们部分纯化了这种α分泌酶 利用DEAE、制备级尺寸排除和进一步的离子交换层析及目前 纯化组分(αCBS Frac.)剂量依赖地切割fAPP695，也不依赖于TACE和ADAM10。 重要的是，α哥伦比亚广播公司。不切割前体致炎细胞因子，包括IL-6和肿瘤坏死因子α。 αCBS Frac对3xTg-AD小鼠的治疗作用。注射显著增加脑部 SAPPα水平、脑Aβ生成显著减少和tauThr231异常 磷酸化。这项提案的最终目标将是进一步描述α哥伦比亚广播公司对 3xTg-AD小鼠的AD病理和行为损害，表现为Aβ和tau病理，以及 最终鉴定和表征这种新颖的和APP特有的α分泌酶。

Project Abstract We have recently shown that sAPPα directly inhibits β-secretase (BACE1) proteolysis of APP, thereby reducing Aβ production. In addition, our most recent studies show that sAPPα reduces Alzheimer’s disease (AD)-related tau-hyper-phosphorylation by inhibition of GSK3β. Moreover, while multiple low dose infusions of human umbilical cord blood-derived monocytes (CB-M) reduce AD pathology and behavioral impairment in PSAPP mice, in part via phagocytosis of Aβ, aged monocytes show impaired Aβ phagocytosis, which can be reversed by sAPPα. These results suggest that CB-M have their own α-secretase or activate endogenous α- secretase in the PSAPP mice. Thus, restoration of sAPPα levels in the brain by shifting the amyloidogenic towards the non-amyloidogenic (α-secretase) pathway could ameliorate AD-related amyloid and tau pathology, neuronal loss and cognitive impairment. An increase in α-secretase activity, therefore, is an attractive strategy for treatment of AD. Several years after descriptive reports on α-secretase, the responsible enzymes have been identified as members of the family of A Disintegrin And Metalloproteinase (ADAM). Three of these membrane-anchored zinc-dependent metalloproteinases, ADAM10, ADAM17 and ADAM9, possess α- secretase activity. However, during the past years, numerous other substrates have been linked to all three sheddases, some of which could promote AD pathogenesis via activation of cytokines or chemokines. Indeed ADAM17 is also known as the TNFα converting enzyme (TACE), which releases TNFα, a proinflammatory cytokine. It has also been reported that CD40L is released from T-cells via ADAM10 and 17 upon CD40 ligation. Furthermore, ADAM10 and ADAM17 are not APP-specific, cleave various substrates and have been associated with tumorigenesis and tumor progression. We have most recently found that human umbilical cord blood serum (CBS) markedly enhance APP α- cleavage and thereby increase sAPPα production in CHO cells expressing wild-type human APP. In addition, CBS selectively mediated the α-secretase cleavage of neuron-specific, human recombinant full-length APP695 (fAPP695) in a cell-free system, independent from TACE and ADAM10. We partially purified this α-secretase using DEAE, preparative-grade size exclusion and further ion-exchange chromatography and the present purified fraction (αCBS Frac.) dose-dependently cleaves fAPP695, also independent from TACE and ADAM10. Importantly, αCBS Frac. does not cleave precursor pro-inflammatory cytokines, including IL-6 and TNFα. Furthermore, treatment of 3XTg-AD mice with αCBS Frac by i.c.v. injection markedly increases cerebral sAPPα levels together with significant decreases in cerebral Aβ production and abnormal tauThr231 phosphorylation. The ultimate goal of this proposal will be to further characterize the effects of αCBS Frac on AD pathology and behavioral impairment in 3XTg-AD mice, which display both Aβ and tau pathology, and ultimately identify and characterize this novel and APP specific α-secretase.