Targeting the alpha secretase ADAM10 for the treatment of Alzheimer's disease

摘要/摘要 阿尔茨海默病(AD)是最常见的退行性疾病之一，其特点是 由淀粉样蛋白b(Ab)纤维组成的老年斑的患病率。在淀粉样蛋白生成途径中， B和g分泌酶对b-淀粉样前体蛋白(APP)的顺序蛋白分解产生抗体(40- 42个氨基酸)。AB多聚体形成以突触为靶点的神经毒性低聚物，导致 长时程增强中断、细胞死亡和记忆丧失。或者，在非淀粉样变性患者中 途径，APP被a和g分泌酶顺序切割，产生SappA，展示 神经保护特性，并阻止抗体的形成。研究发现，ADAM10是一种 锌金属蛋白水解素家族的一员，作为神经元中的结构性α-分泌酶。 目前正在评估激活ADAM10作为一种治疗干预手段。不过，这个 该策略有严重的缺点：ADAM10已知可以裂解多种底物，并被上调 在一系列的恶性肿瘤中。非特异性(非选择性)上调/增强ADAM10 作为AD的一种治疗干预手段，可能会有有害的副作用。它的重要性 ADAM10的非催化(去整合素和富含半胱氨酸)结构域在底物识别中的作用 如前面所示。识别活性构象的构象特异性单抗8C7 发现肿瘤细胞上存在ADAM10。重要的是，虽然8C7选择性地增强了应用 在基于细胞的分析中，它抑制临床前的Notch-Delta和Eph-Ephin致癌信号 模型，在小鼠身上没有任何明显的毒性效应，从而排除了有害的副作用 可能是因为它可能被用作治疗AD的药物。拟议的研究将描述 ADAM10-APP在体外的相互作用以更好地了解ADAM10α-分泌酶如何与 裂解应用程序。8C7单抗促进这种切割的分子机制也将 被调查。一种人类版本的抗ADAM10单抗8C7，命名为1H5，可增强切割 ADAM10的底物结合域是最近产生的。 与8C7类似，该人源单抗(1H5)及其衍生的单链可变区(ScFv)， 现在将在基于细胞的分析中进行评估，以确定它们在促进APP方面的有效性 乳沟。APP的脱落将在大鼠神经母细胞瘤和人脑胶质母细胞瘤细胞中进一步检测 在mAbs(8C7和1H5)和ScFv存在下的表达。这项建议，以加强非 抗ADAM10单抗的淀粉样变性通路有助于开发新的AD治疗方法， 作为治疗潜力原理的证明。

Summary/Abstract Alzheimer’s disease (AD), one of the most prevalent degenerative diseases, is characterized by the prevalence of senile plaques that consist of fibrils of amyloid b (Ab). In the amyloidogenic pathway, sequential proteolysis of the b-amyloid precursor protein (APP) by b and g secretases generate Ab (40- 42 amino acid) peptides. Ab multimerizes into neurotoxic oligomers that target the synapse, leading to disruption of long-term potentiation, cell death, and memory loss. Alternatively, in the non-amyloidogenic pathway, APP is cleaved sequentially by a and g secretase to generate sAPPa that exhibits neuroprotective properties and precludes the formation of Ab. Studies have identified ADAM10, a member of the adamlysin family of zinc metalloproteinases, as the constitutive a-secretase in neurons. Activation of ADAM10 is currently being evaluated as a means of therapeutic intervention. However, this strategy has severe disadvantages: ADAM10 is known to cleave multiple substrates and is upregulated in a wide range of malignancies. A non-specific (non-selective) up-regulation/enhancement of ADAM10 as a means of therapeutic intervention in AD would likely have deleterious side effects. The importance of the non-catalytic (disintegrin and cysteine rich) domains of ADAM10 in substrate recognition was shown previously. A conformation specific mAb, 8C7, which recognizes an active conformation of ADAM10 found on tumor cells was developed. Importantly, while 8C7 selectively enhances APP cleavage in cell-based assay, it inhibits Notch-Delta and Eph-ephrin oncogenic signaling in preclinical models, without any discernible toxicity effects in mice, thereby ruling out deleterious side effects that might result from its potential use as AD therapeutic. The proposed studies will characterize the ADAM10-APP interactions in vitro to better understand how the ADAM10 a-secretase interacts with and cleaves APP. The molecular mechanism of action of the 8C7 mAb in promoting this cleavage will also be investigated. A human version of the anti-ADAM10 mAb 8C7, named 1H5, that augments cleavage of peptide substrates and binds to the substrate binding domain of ADAM10, was generated recently. Similar to 8C7, this human mAb (1H5), as well as a single-chain variable fragment (scFv) derived from it, will now be evaluated in cell-based assays in order to determine their efficacies in promoting APP cleavage. The shedding of APP will further be examined in rat neuronal and human glioblastoma cell lines in the presence of the mAbs (8C7 and 1H5) and the scFv. This proposal, to enhance the non- amyloidogenic pathway with anti-ADAM10 mAbs towards the development of novel AD treatments, serves as a proof of principle of therapeutic potential.