Hershkovits Ayelet Sarah, Gelley Sivan, Hanna Rawad, Kleifeld Oded, Shulman Avidor, Fishman Ayelet
Department of Biotechnology and Food Engineering Technion-Israel Institute of Technology, Haifa, Israel.
The Interdisciplinary Program for Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel.
Front Aging Neurosci. 2023 May 17;15:1171123. doi: 10.3389/fnagi.2023.1171123. eCollection 2023.
Accumulation of amyloid β in the brain is regarded as a key initiator of Alzheimer's disease pathology. Processing of the amyloid precursor protein (APP) in the amyloidogenic pathway yields neurotoxic amyloid β species. In the non-amyloidogenic pathway, APP is processed by membrane-bound ADAM10, the main α-secretase in the nervous system. Here we present a new enzymatic approach for the potential treatment of Alzheimer's disease using a soluble form of ADAM10.
The ability of the soluble ADAM10 to shed overexpressed and endogenous APP was determined with an ADAM10 knockout cell line and a human neuroblastoma cell line, respectively. We further examined its effect on amyloid β aggregation by thioflavin T fluorescence, HPLC, and confocal microscopy. Using N-terminal and C-terminal enrichment proteomic approaches, we identified soluble ADAM10 substrates. Finally, a truncated soluble ADAM10, based on the catalytic domain, was expressed in for the first time, and its activity was evaluated.
The soluble enzyme hydrolyzes APP and releases the neuroprotective soluble APPα when exogenously added to cell cultures. The soluble ADAM10 inhibits the formation and aggregation of characteristic amyloid β extracellular neuronal aggregates. The proteomic investigation identified new and verified known substrates, such as VGF and N-cadherin, respectively. The truncated variant also exhibited α-secretase capacity as shown with a specific ADAM10 fluorescent substrate in addition to shedding overexpressed and endogenous APP.
Our study demonstrates that exogenous treatment with a soluble variant of ADAM10 would shift the balance toward the non-amyloidogenic pathway, thus utilizing its natural neuroprotective effect and inhibiting the main neurotoxic amyloid β species. The potential of such a treatment for Alzheimer's disease needs to be further evaluated .
大脑中β淀粉样蛋白的积累被认为是阿尔茨海默病病理的关键启动因素。淀粉样前体蛋白（APP）在淀粉样生成途径中的加工会产生神经毒性β淀粉样蛋白。在非淀粉样生成途径中，APP由膜结合的ADAM10加工，ADAM10是神经系统中的主要α分泌酶。在此，我们提出一种使用可溶性形式的ADAM10潜在治疗阿尔茨海默病的新酶促方法。
分别使用ADAM10基因敲除细胞系和人神经母细胞瘤细胞系，测定可溶性ADAM10切割过表达和内源性APP的能力。我们进一步通过硫黄素T荧光、高效液相色谱和共聚焦显微镜检查其对β淀粉样蛋白聚集的影响。使用N端和C端富集蛋白质组学方法，我们鉴定了可溶性ADAM10的底物。最后，首次在大肠杆菌中表达了基于催化结构域的截短可溶性ADAM10，并评估了其活性。
当外源添加到细胞培养物中时，可溶性酶水解APP并释放出具有神经保护作用的可溶性APPα。可溶性ADAM10抑制特征性β淀粉样蛋白细胞外神经元聚集体的形成和聚集。蛋白质组学研究分别鉴定了新的和已验证的已知底物，如VGF和N-钙黏蛋白。截短变体除了能切割过表达和内源性APP外，还表现出α分泌酶活性，如使用特定的ADAM10荧光底物所示。
我们的研究表明，用ADAM10的可溶性变体进行外源治疗将使平衡转向非淀粉样生成途径，从而利用其天然的神经保护作用并抑制主要的神经毒性β淀粉样蛋白。这种治疗方法对阿尔茨海默病的潜力需要进一步评估。
