Abstract
Alzheimer’s disease (AD), characterized by memory and cognitive deficits, is a progressive, irreversible neurodegenerative disease. A rising theory addressing AD development and progression is the Ca2+ hypothesis, suggesting that Ca2+ dyshomeostasis can result in neuropathological lesions observed in AD patients. Previous studies have shown that ryanodine type II receptors (RyR2), predominantly expressed in the heart, have a high expression in hippocampal neurons, and that Ca2+ leak through RyR2 leads to AD-like symptoms. FKBP12.6, a regulatory protein that forms a complex with RyR2, modulates Ca2+ release by manipulating RyR2 ultrastructural organization. Current treatment for AD is limited, with treatment having minimal effects in alleviating symptoms. S107, a RyR2-specific Rycal, has shown to prevent FKBP12.6 depletion, therefore reducing Ca2+ leak by stabilizing the RyR2-FKBP12.6 interaction. We propose to determine how S107 alters Ca2+ leak, and ultrastructural RyR2 and FKBP12.6 arrangement in HEK293 cells by performing real-time Ca2+ imaging, confocal microscopy, and super-resolution microscopy. If S107 acts in a similar manner as it does in the heart, this may provide a novel treatment for preventing Ca2+ leak in AD. HEK293-RyR2WT-expressing cells were either mock transfected or transfected with FKBP12.6 cDNA. Cells were incubated in either 10 µM DMSO or S107. Our functional Ca2+ assay using a ratiometric dye, Fura-2AM, showed that when exposed to increasing Ca2+ concentrations (0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 1 mM), FKBP12.6 significantly reduced Ca2+ leak, and that S107 further reduced Ca2+ leak significantly only in the presence of FKBP12.6 (p<0.05). The amount of Ca2+ released by RyR2 was also significantly reduced (p<0.05) when FKBP12.6 and S107 were present (85.54% +/-1.26% vs. 72.66% +/- 3.88% respectively; p<0.001). Our structural assay was able to determine that (1) FKBP12.6 was present in RyR2 HEK293 cells transfected with FKBP12.6 and (2) that there is no significant difference (p>0.05) between RyR2-only cells and RyR2 cells with a combination of FKBP12.6 and S107 in RyR2 count, spread, and density. Future research can explore these aspects in cultured AD neurons, as well as looking at other forms of imaging, such as tomography and DNA-PAINT, to observe cluster arrangement and organization.