Abstract
Alzheimer’s Disease (AD) is an age-associated neurodegenerative disease that is the most common form of dementia in NZ. An emerging hypothesis is that the dysregulation of calcium (Ca2+) signaling driving synaptic transmission can lead to AD symptoms. Ca2+ plays an important role in the pre- and post-synaptic cell to initiate presynaptic neurotransmitter release and postsynaptic signal amplification. A protein critical in intracellular Ca2+ release on both sides of the synapse is the ryanodine receptor (RyR). Inappropriate release, or ‘leak’ of Ca2+ through RyR shows association with AD, however why this occurs is unknown. Ca2+ leak in cardiomyocytes has been attributed to the inappropriate ultrastructural placement and movement of RyR within the cell. Whether inappropriate trafficking of RyR occurs at the synapse in AD neurons is not known. Using Fluorescence Recovery After Photobleaching (FRAP) we have analysed the diffusion characteristics of RyR2 in HEK293 cells, giving a much-needed baseline for continued work in neurons. FRAP analysis showed RyR2 tagged with a green fluorescent protein had a significantly longer half-life compared to a freely soluble cytosolic protein (soluble GFP) (87.63 s ± 8.817 vs 7.99 s ± 3.88, respectively. p < 0.0001). RyR2-GFP half-life was also significantly longer than an Endoplasmic Reticulum (ER) entrapped soluble protein (CEPIA) (87.63 s ± 8.817 vs 9.63 s ± 1.13, respectively. p < 0.0001). Subsequent analysis of the diffusion coefficients of each protein showed that RyR2-GFP had a significantly lower diffusion coefficient than both CEPIA (0.017 µm2s-1 ± 0.001 vs 0.15 µm2s-1 ± 0.02, respectively. p = 0.016) and soluble GFP (0.017 µm2s-1 ± 0.001 vs 0.6 µm2s-1 ± 0.2, respectively. p = 0.0035). Significance was tested using a Kruskal-Wallis non-parametric test with a Dunn’s post hoc test. Taken together these results suggest that membrane bound RyR2 movement in the ER is significantly slower than two independent soluble proteins. Diffusion coefficients obtained in this experiment will be valuable for future work to determine if RyR2 movement in neurons is a factor of simple diffusion or targeted transport. We have also gathered preliminary data using the phenomena of Total Internal Reflection Fluorescence (TIRF) allowing a qualitative look at single RyR2-GFP protein movement. Future work will include tracking single labelled RyR2 in cultured neurons in an amyloid-beta treatment model of AD. This work will be the first to determine how RyR is trafficked within a cell and whether this is altered in an AD phenotype.