Live cell imaging of β-secretase activity in SH-SY5Y cells

Abstract

Although the amyloid hypothesis states that the transmembrane protein Amyloid Precursor Protein (APP) is endoproteolytically processed by secretases to produce amyloid-β peptides (Aβ), where in a cell's membranous network such cleavages occur is not clear. The inability to directly measure constitutive secretase activities during production of the various forms of Aβ in live cell environments has resulted in an incomplete understanding of the basic pathogenesis of Alzheimer's disease (AD) including possible association between the sequelae of APP processing and cell death. Cell permeable fluorogenic substrates have been synthesized to detect secretase activities in live cell environments. Utilizing confocal microscopy, high resolution imaging has localized β-secretase activity inside and on the surfaces of live cells. Furthermore, the methods developed here allow imaging of dynamic redistribution of enzymatic activities. For example, simultaneous imaging with substrates for both secretase and caspase activities over time has enabled investigation of the possible association between β-secretase activity and apoptosis induction. β-secretase activity has been observed inside cytoplasmic vesicles of live single SH-SY5Y cells. By time-lapse imaging, vesicles containing proteolytically active β-secretase have been observed to migrate from the cytoplasm to the plasma membrane from which blebs are released into the extracellular environment. No ring staining of the plasma membrane was observed. The additional presence of cell permeable complimentary fluorogenic probes with specificity for caspase activities indicates β-secretase and caspase activities do not coexist within the same intracellular vesicles or extracellular blebs. High resolution imaging of proteolysis by β-secretase shows for the first time the subcellular localization of the latter in viable cells. The presence of β-secretase activity inside cytoplasmic vesicles which then bleb from a cell's plasma membrane suggests a revision of the model of APP processing in which β-secretase is shown to cleave APP on the extracellular side of the plasma membrane. The capability of imaging proteolytic activities believed to lead to the formation of extracellular amyloid plaques and intracellular neurofibrillary tangles provides a new tool for unraveling the dynamic processing of APP which may enable improved precision in targeting biochemical activities essential for or associated with the pathogenesis of AD.