Internalized Amyloid-β (1-42) Peptide Inhibits the Store-Operated Calcium Entry in HT-22 Cells

Abstract

Dysregulation in calcium signaling pathways plays a major role in the initiation of Alzheimer’s disease (AD) pathogenesis. Accumulative experimental evidence obtained with cellular and animal models, as well as with AD brain samples, points out the high cytotoxicity of soluble small oligomeric forms of amyloid-β peptides (Aβ) in AD. In recent works, we have proposed that Aβ-calmodulin (CaM) complexation may play a major role in neuronal Ca2+ signaling, mediated by CaM-binding proteins (CaMBPs). STIM1, a recognized CaMBP, plays a key role in store-operated calcium entry (SOCE), and it has been shown that the SOCE function is diminished in AD, resulting in the instability of dendric spines and enhanced amyloidogenesis. In this work, we show that 2 and 5 h of incubation with 2 μM Aβ(1-42) oligomers of the immortalized mouse hippocampal cell line HT-22 leads to the internalization of 62 ± 11 nM and 135 ± 15 nM of Aβ(1-42), respectively. Internalized Aβ(1-42) oligomers colocalize with the endoplasmic reticulum (ER) and co-immunoprecipitated with STIM1, unveiling that this protein is a novel target of Aβ. Fluorescence resonance energy transfer measurements between STIM1 tagged with a green fluorescent protein (GFP) and Aβ(1-42)-HiLyte™-Fluor555 show that STIM1 can bind nanomolar concentrations of Aβ(1-42) oligomers at a site located close to the CaM-binding site in STIM1. Internalized Aβ(1-42) produced dysregulation of the SOCE in the HT-22 cells before a sustained alteration of cytosolic Ca2+ homeostasis can be detected, and is elicited by only 2 h of incubation with 2 μM Aβ(1-42) oligomers. We conclude that Aβ(1-42)-induced SOCE dysregulation in HT-22 cells is caused by the inhibitory modulation of STIM1, and the partial activation of ER Ca2+-leak channels.

Store-Operated Calcium Entry Inhibition and Plasma Membrane Calcium Pump Upregulation Contribute to the Maintenance of Resting Cytosolic Calcium Concentration in A1-like Astrocytes

“…Moreover, several ER proteins have been shown to be involved in Aβ(1–42) production, and also in the Aβ(1–42) dysregulation of intracellular calcium, reviewed in [ 21 ]. In a recent work, we reported that nanomolar concentrations of the Aβ(1–42) peptide can inhibit SOCE [ 30 ]. Therefore, we experimentally measured the SOCE response in U251 astrocytes treated with cytokines and in untreated Control U251 astrocytes ( Figure 4 ).…”

“…In this work, we found that the treatment of U251 cells with the cytokines TNF-α (30 ng/mL), IL-1α (3 ng/mL) and C1q (400 ng/mL) for 24 h produced a large depletion of ER calcium content, and also a large attenuation of SOCE. The latter result can be seen as a direct effect of Aβ(1–42) generation in these A1-like astrocytes, because we previously demonstrated that only nanomolar concentrations of intracellular Aβ(1–42) are needed to inhibit SOCE activity [ 30 ]. In addition, the enhanced release of calcium from the ER can generate a feed-forward cycle, leading to potentiation of the production of neurotoxic Aβ peptides in A1-like reactive astrocytes, since it has been demonstrated that Aβ-induced BACE1 upregulation can be blocked by preventing calcium influx through treatment with 2-aminoethoxydiphenyl borate, an inhibitor of inositol trisphosphate-dependent calcium release from the ER, and U73122, an inhibitor of phospholipase C [ 54 ].…”

“…Also, attenuated Ca 2+ entry through store-operated calcium entry (SOCE) is consistently observed in sporadic AD and in skin fibroblasts from familial AD patients [ 29 ]. Recently, we showed that the Ca 2+ entry through SOCE is highly sensitive to Aβ, because intracellular nanomolar concentrations of the Aβ oligomer can bind to stromal interaction molecule 1 (STIM1) and produce significant inhibition of Ca 2+ entry [ 30 ]. On the other hand, the overexpression of plasma membrane calcium pumps (PMCAs) in striatal astrocytes has been shown to inhibit intracellular Ca 2+ signals [ 31 ].…”

Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research

“…The dysregulation of calcium homeostasis observed in Alzheimer’s disease was attributed to the inhibition of LTCC by CaM–Aβ complexes. In a subsequent study, using the same fluorescence-labeled Aβ and Alexa488-labeled secondary antibodies for primary anti-PDI and anti-CaM or stromal interaction molecule 1 (STIM1) labeled with green fluorescent protein (STIM1-GFP), Gutiérrez-Merino and colleagues demonstrated that prior to the internal dysregulation of calcium homeostasis, a perturbation of store-operated calcium entry occurs through the internalization of Aβ and its inhibition of STIM1, along with partial activation of the ER Ca 2+ -leak channels [ 102 ]. For these measurements, variations in donor fluorescence were analyzed, and the R 0 and the spectral overlap integral (J) values were obtained for the Aβ*555–SPM1-GFP donor–acceptor pair [ 102 ].…”

Brain Hydrophobic Peptides Antagonists of Neurotoxic Amyloid β Peptide Monomers/Oligomers–Protein Interactions

“…Particularly relevant among intracellular targets of Aβ(1–42) oligomers are neuronal non-amyloidogenic proteins showing high affinity for nanomolar concentrations of Aβ peptides, because critical concentration values in the sub micromolar range have been reported for the induction of Aβ(1–42) fibrillization [ 37 , 38 ], and concentrations of non-fibrillar Aβ peptides within the nanomolar range have been reported in the brain [ 39 , 40 , 41 ]. Among these proteins, the dissociation constant of Aβ(1–42) has been reported to be around 1 nM only for tau [ 42 ], cellular prion protein (PrP C ) [ 43 ], glycogen synthase kinase 3α (GSK3α) [ 44 ], calmodulin (CaM) [ 45 ], and likely stromal interaction molecule 1 (STIM1) [ 46 ]. Human PrP C is a glycoprotein that largely localizes to cholesterol-rich lipid rafts on the outer surface of the cell membrane, thereby acting as a high-affinity receptor for extracellular Aβ oligomers in concert with the low-density lipoprotein receptor-related protein-1 [ 47 ].…”