We describe a spectral FRET based method to quantitatively measure CaN activation dynamics at a subcellular level in cultured neurons. The spectral FRET based assay defines two localization states of CaN activation in response to soluble Aβ oligomer stimulation: first, a robust CaN activation is initiated in spines after a brief Aβ exposure; second, a delayed but sustained CaN activation is observed in the cytoplasm and the nucleus by prolonged Aβ exposure. Acute activation of CaN in spines caused by Aβ containing conditioned media is sufficient to induce spine structural changes and affect levels of postsynaptic proteins including F-actin and GluR1-containing AMPA receptors. Notably, the disease associated Aβ species, which are isolated by SEC from human AD brain, induces a rapid CaN activation specifically and locally in spines with consequent alteration in spine morphology and reduction in postsynaptic proteins. Our results provide the first demonstration that the temporal and spatial precision of CaN activation in response to Aβ stimulation involves compartmentalized modulation; the disease associated Aβ oligomers initiate rapid, local posttranslational responses downstream of CaN in spines.
FRET based assays have been extensively used as a method to image molecular processes in the natural environment such as protein-protein interaction of several signaling pathways, primarily taking advantage of overexpressed tagged proteins (Stryer, 1978
; Selvin, 2000
; Jares-Erijman and Jovin, 2003
). We have extended this technology to CaN and have used different approaches to detect CaN activation in vitro
for live cell imaging using GFP-RFP tags and endogenous proteins after immunostaining. CaN activation is widely studied in the immune system and in numerous disease processes, including congestive heart failure (Clipstone and Crabtree, 1992
; Crabtree and Schreiber, 2009
). In the central nervous system, activation of CaN has been implicated in the context of normal learning and memory, as well as diseases including glaucoma, spinal cord trauma, kainate injury and ischemia (Morioka et al., 1999
; Springer et al., 2000
; Wu et al., 2004
; Huang et al., 2005
). In the context of Alzheimer’s disease, CaN was shown to be abnormally activated in neurons but also in glia, thus participating to neuroinflammatory processes that are tighly associated with the progression of the disease (Abdul et al., 2009
). The tools we describe here may therefore prove useful in monitoring CaN's role in different cell types, with improved spatial and temporal resolution.
The FRET based tools described here allow us to specifically address questions such as the kinetics of CaNA/CaM and CaNA/CaNB interactions following stimulation in neuronal cells, and provides a tool for evaluating the kinetics of spine or nuclear activation of calcineurin after physiologic or pathologic stimuli. A previous attempt to monitor CaN activation using a phosphatase activity-dependent molecular switch was based on the N-terminal regulatory domain of the NFAT1 that were sandwiched between the enhanced cyan fluorescent protein (ECFP) and a circularly-permuted version of the yellow fluorecent protein, Venus, as a specific substrate of CaN (Newman and Zhang, 2008
). However, the overexpressed ECFP-venus FRET pair has a limited response, is indirect in that it is restricted to report only CaN-mediated NFAT activation, is sensitive to NFAT phosphorylation events, and does not provide spatial information about CaN activation. By contrast, the conformational changes induced by the interaction of CaNA-CaM or CaNA-CaNB directly report CaN activation toward all its substrates. This assay can also be adapted for transfected or endogenous protein assays and provides outstanding spatial and temporal resolution. As demonstrated by Stemmer and Klee in an earlier work (Stemmer and Klee, 1994
), the Kd of the CaNB Ca2+ binding sites is very high (10nM), so that CaNA and CaNB may be associated even at baseline. Recent pull down/mass spectroscopy data suggest that the CaNA-CaNB interaction increases significantly with cellular stress (Kozubowski et al., 2011
). From our data, we can not distinguish if CaNA and CaNB interact at baseline and then change their conformation when activated by calcium, or if the association increases with an influx of calcium, or both; regardless, activation of calcineurin leads to a change in the conformation so that the fluorophores come close enough to one another to support FRET.
Our results indicate that CaN activity is significantly increased in spines over a period of 0–60 min after Aβ oligomer application. Although overt spine loss does not occur acutely, substrates located in the spine compartment are immediately targeted by CaN and synaptic function could be substantially affected during the early phase of CaN activation. For example, the early phase of CaN activation alters spine morphology and affects levels of surface GluR1-containing receptors in spines, confirming a strong local cellular consequence of CaN activation caused by acute Aβ oligomer stimulation (Hsieh et al., 2006
). On the other hand, neurons have a late phase CaN activation in soma that starts 6 hours after Aβ oligomer application, lasts for at least 24 hours, and induces NFAT nuclear translocation (Wu et al., 2010
). Neuron function could be potentially affected by both CaN-mediated post-translational and transcriptional processes. For example, spine loss occurs in 24 hours, and can be blocked by VIVIT which prevents NFAT transcriptional activation (Wu et al., 2010
), whereas changes in spine morphology, which is associated with loss of postsynaptic proteins including F-actin and GluR1 subunit of AMPA receptors, occurs within the first hour and is not affected by VIVIT. Moreover, the FRET ratios of both CaNA-CaM and CaNA-CaNB obtained from all three compartments in neurons exposed to Aβ oligomers for 60 min followed by washing off and then returned to original conditioned media for 24 hours showed only basal level CaN activation (). This result indicates that the effect of the 60 min exposure to Aβ oligomers on CaN activation is temperate, reversible, and is a completely local event, which is not sufficient to extend its effect to the soma and initiate the late phase CaN activation. This also suggests that prolonged Aβ oligomer exposure is required for CaN activation in cytoplasm and nuclei. As altering CaN phosphatase activity is considered to be critical for physiological or pathological neuronal plasticity, the subcellular temporal properties of CaN activation in response to Aβ oligomer stimulation we observed here may be important to understand synaptic alterations during AD pathogenesis.