Disintegrin-like metalloproteases (ADAMs) comprise the major family of ectodomain sheddases. In inflammatory settings, ADAMs mediate signaling of cytokines, including TNFα and IL-6, and control leukocyte recruitment by the cleavage of cell adhesion molecules. ADAM17 and ADAM10 have been implicated in diverse diseases associated with inflammation, including psoriasis, asthma, arthritis, atherosclerosis, inflammation associated tumors, diabetes and Alzheimer’s disease (AD) [1-3]. With regard to the latter, it is assumed that decreased α-secretase (ADAM10 and ADAM17) activity and increased β-secretase (BACE1) activity results in abnormal processing of the amyloid precursor protein (APP). This leads to elevated levels of neurotoxic amyloid β, a key pathognomonic feature of AD. Due to the important role of ADAMs and BACE in amyloid β production, altering the ADAM10/17 and/or BACE function are being considered as a promising therapeutic approach to AD .
Although ADAM10 and ADAM17 were identified over a decade ago, the regulation of their proteolytic activity is still poorly understood. In the last funding period, we learned that even striking up- or downregulation of ADAM RNA expression levels does not correlate with changes in functionality. Instead, we found that environmental factors, in particular nutrition, significantly modulated the amount of substrate release.
AD is an inflammatory disease, which is well known to be influenced by environmental factors such as caloric restriction, hypoxia, oxidative stress, cholesterol metabolism or uptake of excitotoxins. We would now like to extend our current investigations into the role of environmental factors on the regulation of ADAMs and related transmembrane proteases (e.g. BACE1) in the context of AD, using cell culture studies and an AD mouse model.
We want to:
- to learn more about how environmental factors might influence ADAM-mediated substrate release
- to gain insights into the dietary regulation of ADAMs and related transmembrane proteases (beta-secretase) in a mouse model of AD
- to investigate the effects of environmental factors on events relevant to AD pathology in cell culture
APP/PS transgenic mice will be fed different diets and cognitive function of the animals will be studied with our cooperation partners in Hamburg, who have longstanding expertise in this field. The mice will be sacrificed and serum samples will be harvested for quantification of released ADAM substrates. Organ tissues will be frozen for further RT-PCR and immunoblot analysis of protease expression and substrate processing. Brain sections will be subjected to immunohistochemical analyses of plaque formation and neuronal degeneration. In parallel, we will investigate the effect of environmental factors on neurons in cell culture models. Cell lines overexpressing either wildtype APP or swedish-mutant APP will be analyzed under conditions mimicking environmental factors that affect AD development such as caloric restriction (resveratrol, rapamycin), enhanced glucose metabolism (Insulin-like growth factor), excitototoxins (glutamate) and membrane/cholesterol modulating agents. APP processing will be analyzed by immunoblot and ELISA. Immunocytochemical analyses and live cell imaging will be performed to learn more about protease/substrate trafficking. The balance between ROS generation and the cellular antioxidant defense system, which essentially influences ADAM function, will be analyzed using the cell permeable fluorescent probe DCFH-DA. Phosphatidylserine exposure, which plays a critical role for ADAM-mediated shedding but also for vesicle trafficking in the cell, will be visualized using Annexin-V staining. Protease activity will be determined using peptide-based activity assays and substrate processing analysis of APP and additional substrates important for cognitive functions (e.g. N-cadherin, L1CAM, NrCAM). Functional consequences of ADAM-mediated substrate cleavage will be additionally monitored by real time electrical impedance measurements with the xCelligence system.
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