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

人脐带血血清中新型 APP 特异性 α 分泌酶的鉴定

• 批准号: 9380529
• 负责人: Jun Tan
• 金额: $ 22.43万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9380529
• 关键词: Abeta synthesis; Age; Alpha Cell; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Antibodies; Brain; Cell-Free System; Cerebrum; Chinese Hamster Ovary Cell; Cleaved cell; Complex; Cues; Cytokine Activation; Dementia; Deposition; Development; Diagnosis; Disease; Disintegrins; Dose; Elderly; Enzymes; Exclusion; Family member; Frequencies; Goals; Human; Human Amyloid Precursor Protein; Impaired cognition; Impairment; Individual; Inflammatory; Infusion procedures; Injection of therapeutic agent; Interleukin-1 beta; Interleukin-6; Ion-Exchange Chromatography Procedure; Length; Ligation; Link; Mediating; Membrane; Metalloproteases; Mus; Neurocognition; Neurons; Pathogenesis; Pathology; Pathway interactions; Peptides; Phagocytosis; Phosphorylation; Preparation; Prevalence; Process; Production; Proteolysis; Recombinants; Reporting; Serum; T-Lymphocyte; TNF gene; TNFRSF5 gene; TNFSF5 gene; Therapeutic; Umbilical Cord Blood; Umbilical cord structure; Work; Zinc; aged; alpha secretase; behavior test; behavioral impairment; beta secretase; beta-site APP cleaving enzyme 1; chemokine; cognitive testing; conditioned fear; cytokine; improved; intraperitoneal; monocyte; neurogenesis; neuron loss; neuroprotection; novel; prophylactic; restoration; secretase; tau Proteins; treatment strategy; tumor progression; tumorigenesis

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.

项目摘要 我们最近发现sAPPα直接抑制APP的β-分泌酶（BACE 1）蛋白水解，从而 减少Aβ的产生。此外，我们最近的研究表明，sAPPα减少阿尔茨海默病 通过抑制GSK 3 β的AD相关tau蛋白过度磷酸化。此外，虽然多次低剂量输注 人脐带血来源单核细胞（CB-M）减少AD病理和行为障碍 在PSAPP小鼠中，部分通过吞噬Aβ，老年单核细胞显示出受损的Aβ吞噬作用，这可能是 由sAPPα逆转。这些结果提示CB-M有自身的α-分泌酶或激活内源性α-分泌酶。 PSAPP小鼠中的分泌酶。因此，通过转移淀粉样蛋白原， 向非淀粉样蛋白生成（α-分泌酶）途径的转化可以改善AD相关的淀粉样蛋白和tau病理， 神经元丧失和认知障碍。因此，增加α-分泌酶活性是一种有吸引力的策略 治疗AD。在对α-分泌酶进行描述性报道几年后， 已被鉴定为A解整合素和金属蛋白酶（ADAM）家族的成员。其中三 膜锚定的锌依赖性金属蛋白酶，ADAM 10，ADAM 17和ADAM 9，具有α- 分泌酶活性然而，在过去的几年里，许多其他基板已连接到所有三个 脱落酶，其中一些可以通过激活细胞因子或趋化因子促进AD发病。确实 ADAM 17也被称为TNFα转化酶（TACE），它释放TNFα，一种促炎因子， 细胞因子也有报道称，CD 40 L通过ADAM 10和17从T细胞释放， 结扎此外，ADAM 10和ADAM 17不是APP特异性的，切割各种底物，并且已经被证实是APP特异性的。 与肿瘤发生和肿瘤进展相关。 我们最近发现，人脐带血血清（CBS）可显著增强APP α- 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α-分泌酶切割神经元特异性人重组全长APP 695 （fAPP 695）在无细胞系统中，独立于TACE和ADAM 10。我们部分纯化了这种α-分泌酶 使用DEAE，纯化级尺寸排阻和进一步的离子交换色谱， 纯化组分（αCBS Frac.）剂量依赖性地切割fAPP 695，也独立于TACE和ADAM 10。 重要的是，αCBS Frac。不裂解前体促炎细胞因子，包括IL-6和TNFα。 此外，通过i. c. v.注射αCBS Frac治疗3XTg-AD小鼠， sAPPα水平以及脑Aβ产生的显著减少和异常tauThr 231 磷酸化本提案的最终目标是进一步表征αCBS Frac对 3XTg-AD小鼠中的AD病理学和行为障碍，其显示Aβ和tau病理学，以及 最终鉴定和表征这种新型APP特异性α-分泌酶。