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

总结/摘要 阿尔茨海默病（AD）是最普遍的退行性疾病之一，其特征在于 由淀粉样蛋白B（Ab）纤维组成的老年斑的流行。在淀粉样蛋白生成途径中， 通过B和g分泌酶对B-淀粉样前体蛋白（APP）的连续蛋白水解产生Ab（40- 42个氨基酸）肽。抗体多聚化成神经毒性寡聚体，靶向突触，导致 长时程增强的破坏、细胞死亡和记忆丧失。或者，在非淀粉样蛋白生成的 在该途径中，APP被a和g分泌酶顺序切割以产生sAPPa，其表现出 神经保护特性并阻止Ab的形成。研究已经确定了ADAM 10， 锌金属蛋白酶的adamlysin家族的成员，作为神经元中的组成性α-分泌酶。 目前正在评估激活ADAM 10作为治疗干预的一种手段。但这 策略有严重的缺点：已知ADAM 10可以切割多种底物并上调 广泛的恶性肿瘤中。ADAM 10的非特异性（非选择性）上调/增强 作为AD的治疗干预手段可能具有有害的副作用。的重要性 在底物识别中，ADAM 10的非催化（去整合素和富含半胱氨酸）结构域 之前显示的。一种构象特异性mAb，8 C7，其识别 开发了在肿瘤细胞上发现的ADAM 10。重要的是，虽然8 C7选择性地增强APP 在基于细胞的测定中切割，其在临床前试验中抑制Notch-Delta和Eph-ephrin致癌信号传导。 模型，在小鼠中没有任何可辨别的毒性作用，从而排除了 可能是由于其作为AD治疗剂的潜在用途。拟议的研究将描述 体外ADAM 10-APP相互作用，以更好地了解ADAM 10 α-分泌酶如何与 8 C7 mAb促进这种切割的分子作用机制也将 追究抗ADAM 10 mAb 8 C7的人类版本，命名为1H 5，可增强切割 的肽底物，并结合到底物结合域的ADAM 10，最近产生。 类似于8 C7，该人mAb（1H 5）以及源自其的单链可变区片段（scFv）， 现在将在基于细胞的测定中进行评估，以确定它们在促进APP 乳沟将在大鼠神经元和人胶质母细胞瘤细胞中进一步检查APP的脱落 在mAb（8 C7和1H 5）和scFv的存在下，这一建议，以加强非- 抗ADAM 10单克隆抗体用于开发新型AD治疗的淀粉样蛋白生成途径， 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.