Single organelle fret-based analysis of intracellular calcium: different effects of presinilis carrying Familial Alzheimer's Disease mutations

Calcium (Ca2+) is one of the major intracellular messengers that impacts nearly every aspect of cell life. In particular, it plays essential roles in neuronal development, synaptic transmission and plasticity, as well as in the regulation of metabolic pathways and cell fate decisions. The first goal of my work was to study more in details the Golgi apparatus (GA), as an intracellular Ca2+ store. The Golgi complex may store up to 5% of the total cellular Ca2+ at higher concentrations and contains several different luminal Ca2+-binding proteins and Ca2+-release channels. The use of a specific Cameleon Ca2+ sensor targeted to the trans-Golgi, allowed us to directly demonstrate the functional GA heterogeneity by showing the distinct behaviour of this sub-compartment: it takes up Ca2+ almost exclusively via SPCA1 (and not by SERCA); it does not release Ca2+ in response to IP3 generation, but rather accumulates the cation as a consequence of the cytoplasmic Ca2+ rise. Using a novel GFP-based Ca2+ probe selectively localized to the medial-Golgi (mGo-D1cpV), we have demonstrated that this sub-compartment behaves in a unique way, releasing Ca2+ upon cell stimulation not only with IP3-generating agonists, but also through Ryanodin receptors (RyRs) and relying, for its Ca2+ uptake, on both SERCA and SPCA1 pumps. The GA represents, thus, an intracellular Ca2+ store with an high complexity level. In a space of a few microns and despite its continuous inter-mixing dynamics, the Ca2+ toolkit undergoes major changes and distinct, separate functional sub-regions, with different Ca2+ features, contribute to its Ca2+ handling. The second aim of my work was to study, at the single cell level by employing specifically targeted cameleon Ca2+ probes, the effect of Familial Alzheimer's Disease (FAD) presenilin 1 and 2 (PS1, PS2) mutants on Ca2+ homeostasis in different sub-cellular compartments: the endoplasmic reticulum (ER), the medial and trans-Golgi. Ca2+ dyshomeostasis has been demonstrated in AD, in particular in the rare genetically inherited FAD cases (mostly due to mutations in PS1 and PS2 genes). In our lab has been recently shown, by employing an ER-targeted aequorin Ca2+ sensor at the cell population level, that FAD-PS2 mutants reduce the Ca2+ content of the major intracellular Ca2+ store, the ER, mostly by inhibiting SERCA activity. In order to deeper investigate the effect of the expression of FAD-PS mutants on ER Ca2+ handling in single cell FRET-experiments, we created a novel ER-targeted cameleon Ca2+ probe (ER-D4) with optimized Ca2+ affinity and dynamic range. In addition, the new cameleon Ca2+ probe targeted to the medial-Golgi, above described, was also used for a similar purpose. Ca2+ handling by the distinct intracellular compartments, at single cell level, was evaluated in SHSY5Y cell line over-expressing PS2 wild type (wt), FAD-PS2-T122R, PS1 wt or FAD-PS1-A246E and also in human fibroblasts from a FAD-PS2-N141I and a FAD-PS1-A246E patient (and corresponding controls from individuals of the same age and sex). Ca2+ concentration ([Ca2+]) of the ER and the medial-Golgi, in resting cells, is strongly reduced by PS2, but not PS1, mutants expression, while that of the trans-Golgi compartment results unaffected by both mutants. The Ca2+ uptake rate in these stores is reduced by PS2 mutants expression and, only within the medial-Golgi, also the Ca2+ leak rate is potentiated. These findings strength our previous data showing the inhibitory effect of FAD-PS2 on SERCA activity. They are also consistent with the fact that, within the Golgi, only the medial-compartment is endowed with IP3 receptors and SERCA pumps, whereas the trans-Golgi is devoid of both. In conclusion, the data highlight the molecular heterogeneity of different intracellular Ca2+ stores (and in distinct sub-compartments of the same organelle) and provide new insights for the comprehension of the mechanisms through which PSs influences cellular Ca2+ homeostasis and FAD pathogenesis. In the future, these cameleon probes will be applied to study Ca2+ alterations induced by FAD-PS2 mutations and, eventually, in vivo by means of viral vectors.