Alzheimer's disease-associated peptide Aβ₄₂ mobilizes ER Ca²⁺ via InsP₃R-dependent and -independent mechanisms

Introduction

Alzheimer's disease (AD) is a progressive and irreversible brain disorder, which results in severe memory loss, behavioral as well as personality changes and a decline in cognitive abilities. While the most common type of AD remains idiopathic in origin, with age the most significant risk factor for disease onset (sporadic AD, sAD), ~5% of cases show a Mendelian pattern of inheritance (familial AD, fAD). The amyloid β-peptide (Aβ) is hypothesized to be central to the pathogenesis of both sporadic and familial AD (Hardy and Selkoe, 2002). Aβ is a small, hydrophobic polypeptide, consisting of 39–42 amino acid residues, which occurs principally as a 40 or 42 amino acid peptide, Aβ₄₀ and Aβ₄₂, respectively (Zhang et al., 2011). An imbalance between the production and clearance of Aβ, as occurs in fAD and sAD, respectively, leads to the accumulation of Aβ and, in turn, to its aggregation. This aggregation process represents a critical step in the pathogenesis of AD because the neurotoxic properties of Aβ are associated only with aggregated forms of the peptide (Kuperstein et al., 2010). Protein aggregation is highly dynamic and involves a wide range of intermediate structures such as oligomers, comprising dimers, trimers, dodecamers, and higher-molecular weight complexes, before aggregating into protofibrils and finally into mature amyloid fibrils (Dobson, 2003).

A mounting body of evidence now suggests that soluble oligomeric forms of Aβ constitute the primary neurotoxic species rather than monomers or fibrils (Lambert et al., 1998; Chromy et al., 2003; Gong et al., 2003; Demuro et al., 2005; Klyubin et al., 2005). Soluble oligomers have proved toxic when applied to cultured cells and primary neuronal cultures in vitro (Lambert et al., 1998; Bucciantini et al., 2002; Dahlgren et al., 2002; Kayed et al., 2003; Whalen et al., 2005). In addition, they are capable of inducing cognitive deficits when administered in vivo (Cleary et al., 2005; Rowan et al., 2007) and adversely affect hippocampal LTP in vivo (Walsh et al., 2002; Cleary et al., 2005; Klyubin et al., 2009, 2011).

Dysregulation of intracellular Ca²⁺ homeostasis is associated with cell exposure to Aβ and likely underlies its neurotoxic effects (Bezprozvanny and Mattson, 2008; Green and Laferla, 2008; Berridge, 2010; Demuro et al., 2010). A number of mechanisms by which Aβ elicits its effects on intracellular Ca²⁺ homeostasis have been put forward. These include direct effects on the plasma membrane, where it has been proposed to destabilize its structure (Mueller et al., 1995; Mason et al., 1996), to induce a generalized increase in membrane permeability (Bucciantini et al., 2002; Kayed et al., 2003) or to insert into the membrane forming cation-conducting pores (Arispe et al., 1993; Mueller et al., 1995; Mason et al., 1996; Bucciantini et al., 2002; Kayed et al., 2003; Kawahara, 2004; Simakova and Arispe, 2006; Arispe et al., 2007; Demuro et al., 2011). Aβ has also been reported to activate plasma membrane receptors, including N-methyl-d-aspartate (NMDA) receptors coupled to Ca²⁺ influx (Guo et al., 1996; Dobson, 2003; Blanchard et al., 2004; De Felice et al., 2007), to alter neuronal excitability which, in turn, influences the extent of Ca²⁺ influx (Good et al., 1996) and to induce dysregulation of endoplasmic reticulum (ER) Ca²⁺ homeostasis (Ferreiro et al., 2004, 2006; Resende et al., 2008). In addition to acting from the extracellular space, where it accumulates in the diseased brain, Aβ also has an intracellular site of action (Wirths et al., 2004). Indeed, as a result of uptake from the extracellular space or via its intracellular synthesis and processing, Aβ has been reported to accumulate within the cell (Pierrot et al., 2004; Bayer and Wirths, 2011; Kaminski Schierle et al., 2011). This intracellular Aβ is also neurotoxic and has been shown to target the ER and the mitochondria, inducing a stress response and causing permeability transition, respectively (Yao et al., 2009; Umeda et al., 2011).

In this study, we investigated (1) the contribution of Ca²⁺ mobilization from the ER to the increase in intracellular Ca²⁺ induced by oligomeric Aβ₄₂, (2) the mechanism (s) by which Aβ₄₂ elicited this effect, (3) the capacity for Aβ₄₂ to mobilize Ca²⁺ directly from the ER. To allow isolation of effects on the ER from other plasma membrane targets of Aβ₄₂, model cells systems were used that allowed fundamental aspects of ER Ca²⁺ regulation to be studied. We determined that Ca²⁺ release from the intracellular ER substantially contributed to the increase in intracellular Ca²⁺ concentration induced by oligomeric Aβ₄₂. The Aβ₄₂-induced Ca²⁺ elevation comprised InsP₃ dependent and independent components. Using DT40 cells deficient in the three InsP₃R isoforms that were permeabilized to allow direct access of Aβ₄₂ to the ER, we also demonstrated that it had the capacity to release Ca²⁺ from the ER independent of InsP₃Rs. Together, these data place the ER and Ca²⁺ released from it as central to the actions of both extracellular Aβ and Aβ that has reached an intracellular location.

Materials and Methods

Materials

Peptides were purchased from The American Peptide Company and rPeptide. Cell culture reagents and chemicals were from Invitrogen or Sigma, unless otherwise stated.

Cell Culture

Human neuroblastoma SH-SY5Y cells were cultured in F-12 Nutrient Mixture (Ham) containing FBS (10%), penicillin (100 units/ml), streptomycin (100 μg/ml), non-essential amino acids (0.1 mM), and L-glutamine (2 mM). Prior to all experiments, SH-SY5Y cells were cultured overnight in Opti-MEM Reduced Serum Medium, containing FBS (1.5%), penicillin (100 units/ml), streptomycin (1.0 μg/ml), non-essential amino acids (0.1 mM), and L-glutamine (2 mM). For live-cell Ca²⁺ imaging experiments, cells were plated onto poly-L-lysine-coated coverslips at a density of 3.2 × 10⁴ cell/cm². For the MTT reduction assay, cells were plated at a density of 9 × 10³ cells/cm². To overexpress GFP-tagged type 1 InsP₃ 5′-Phosphatase (GFP-5′P) or GFP (Peppiatt et al., 2004; Higazi et al., 2009), cells were infected with adenovirus for 8 h prior to overnight culture. Culture of DT40 cells and DT40 cells deficient in the three InsP₃R isoforms (DT40 TKO) was performed as previously described (Tovey et al., 2006).

Preparation of Aβ₄₂ Oligomers

Wild type and scrambled Aβ₄₂ were obtained at a purity of >95%. Peptide mass was verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptides from the same batch were used throughout. Samples of synthetic Aβ₄₂ oligomers were prepared as previously described (Demuro et al., 2005) and remained stable for at least 3 weeks. Samples of Aβ _1–42 scrambled peptide (KVKGLIDGAHIGDLVYEFMDSNSAIFREGVGAGHVHVAQVEF) were prepared in the same way as Aβ₄₂ oligomers. All Aβ samples were stored at 4°C and were used within 10–15 days of preparation. Toxicity of Aβ₄₂ preparations was confirmed by MTT assay before use in Ca²⁺ imaging experiments (Figure S1A). The oligomeric nature of the Aβ₄₂ preparation was established by surface plasmon resonance (SPR) spectroscopy using an antibody specific to oligomeric Aβ₄₂ (Figure S1B). All Aβ₄₂ concentrations stated are based on the molar mass of the peptide.

Live Cell Ca²⁺ Imaging

Methods for single cell analysis of intracellular Ca²⁺ concentration were as previously described (Peppiatt et al., 2003). Cells were loaded at 37°C with 2 μM of the acetoxymethyl (AM) ester form of fura-2 for 30 min followed by an equivalent period in dye free media to allow de-esterification of the indicator. Imaging experiments were performed using either Ca²⁺-containing (121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 6 mM NaHCO₃, 25 mM HEPES, 5.5 mM glucose, pH 7.3) or Ca²⁺ free (as for Ca²⁺ containing with 1.8 mM CaCl₂ replaced with 1 mM EGTA) buffer as indicated. Fura-2 imaging was carried out using an imaging system configured around a Nikon TE300 inverted epi-fluorescence microscope equipped with a 20× 0.75 NA multi-immersion objective. Samples were illuminated by alternate excitation at 340 and 380 nm using a Sutter filter changer (340HT15 and 380HT15; Sutter Industries) and emitted light was filtered at >460 nm (1 ratio pair per 2 s). Images were captured using a Hamamatsu ORCA ER CCD camera. The imaging system was controlled with Ultraview software (PerkinElmer Life Sciences Ltd., UK). Acquired images were processed with Ultraview software and analyzed in MATLAB. Background subtracted fura-2 ratios were calibrated according to standard procedures (Grynkiewicz et al., 1985), using the maximum and minimum ratio values obtained through exposing cells sequentially to Ca²⁺ free and Ca²⁺ containing imaging buffer to which 2 μM ionomycin had been added. Parameters analyzed from the Ca²⁺ responses included the peak amplitude, the time to peak and the integral of the response (the area under the curve) and the percentage of responding cells.

InsP₃-induced Ca²⁺ release (IICR) from permeabilized wild type and InsP₃R null DT40 cells (three InsP₃R isoforms deleted by homologous recombination; DT40 TKO) (Sugawara et al., 1997) was performed as previously described (Tovey et al., 2006). Briefly, the ER of cells was loaded with the low-affinity Ca²⁺ indicator mag-fluo-4 and Aβ-induced Ca²⁺ release was measured from the saponin-permeabilized cells using a fluorescence plate reader (FlexStation 3, Molecular Devices).

MTT Reduction Assay

The Cell Titer 96 Non-Radioactive Cell Proliferation Assay (Promega) was used to validate the cytotoxic effect of Aβ₄₂ on SH-SY5Y cells and was performed according to manufacturer's instructions. Briefly, cells were incubated with Aβ₄₂ (n = 4) for 24 h prior to the addition of the MTT dye solution and a further 4 h incubation at 37°C, 5% CO₂. Thereafter, the solubilization/stop solution was added and incubated overnight at room temperature. Absorbances were read at 570 nm with a reference wavelength of 650 nm using a fluorescence plate reader (Synergy HT, BIO-TEK). The data is expressed as the percentage of MTT reduction relative to both live- and dead-cell controls and thus represents the percentage of viable cells. Aβ₄₂ samples were considered to be toxic if 25–40% of cells remained metabolically healthy at an Aβ₄₂ concentration of 1 μ M and if more than 50% remained metabolically healthy at a concentration of 100 nM.

Statistical Analysis

Data is presented as the mean value of the combined datasets ± SEM. Statistical significance was determined by Student's t-test (two-tailed). Data was accepted as significant when p < 0.05 and is denoted by *p < 0.05, **p < 0.01, or ***p < 0.001.

Results

Intracellular ca²⁺ is elevated in cells exposed to oligomeric Aβ₄₂

Experiments were first performed to validate the Ca²⁺ mobilizing properties of oligomeric Aβ₄₂ over the concentration range of its toxicity. Application of Aβ₄₂ spanning its cytotoxic range (1, 5 and 10 μM) caused an elevation in intracellular Ca²⁺ (Figure 1A). The increase in cytosolic Ca²⁺ concentration immediately followed the addition of Aβ₄₂, developed to a peak within minutes of application and subsequently returned to baseline, despite the continued presence of the peptide. No Ca²⁺ responses were detected when Aβ₄₂ below 1 μM was applied (data not shown). Between 1 μM and 10 μM Aβ, the number of responding cells, the peak amplitude and the integral of the Ca²⁺ responses increased in a concentration-dependent manner. The number of responding cells reached 100% at 5 μM Aβ₄₂ (Figures 1Bi,iii,v). To test cell viability as well as to determine whether metabotropic Ca²⁺ signaling was affected by Aβ, carbachol (CCH) was applied subsequent to Aβ. CCH elicited Ca²⁺ responses in 100% of cells pre-exposed to 1 or 5 μM oligomeric Aβ₄₂ or to a vehicle control (10%) (Figures 1Bii,iv,vi). At 10 μM Aβ, however, the number of cells responding to CCH was significantly reduced (Figure 1Bii). The peak amplitude and integral of the Ca²⁺ responses to CCH subsequently applied were inversely related to the magnitude of the Ca²⁺ responses elicited by oligomeric Aβ₄₂ (Figures 1Biv,vi). This observation suggested that exposure to Aβ₄₂ oligomers was depleting the intracellular CCH-sensitive ER Ca²⁺ store. These Ca²⁺ mobilizing effects of oligomeric Aβ₄₂ were significantly greater than observed in cells exposed to Aβ₄₂ that had been prepared in a manner to yield a monomeric form of the peptide (Figure S2, S1B). From these results, due to its potency in mobilizing Ca²⁺ whilst preserving agonist responses, a concentration of 5 μM oligomeric Aβ₄₂ was selected for use in subsequent experiments.

Figure 1. Aβ₄₂ oligomers induce Ca²⁺ transients in a concentration-dependent manner. (A) Example fura-2 Ca²⁺ traces of SH-SY5Y cells exposed to a concentration range of Aβ₄₂ oligomers followed by 100 μM CCH. A trace taken from cells in which Aβ₄₂ oligomers were substituted with double-distilled water (dd H₂O; vehicle) is also shown (for each group, n > 744 cells). (B) Quantitative analysis of the Ca²⁺ responses illustrated in A. The magnitude of Ca²⁺ responses elicited by Aβ₄₂ oligomers, dd H₂O and CCH is presented as (Bi,ii) percentage of responding cells, (Biii,iv) peak amplitude and (Bv,vi) integral of the response. Aβ₄₂ oligomer-induced Ca²⁺ transients were normalized to the responses induced with the highest concentration (10 μM) of the respective Aβ₄₂ preparation. CCH-induced Ca²⁺ responses were normalized to control experiments conducted on the same experimental day. Bar graphs are mean ± SEM from at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Aβ₄₂ Oligomer-Induced Ca²⁺ transients are peptide sequence specific

As a control for the application of peptide, experiments were also performed using a scrambled Aβ sequence, which had been prepared in the same manner as the wild type Aβ₄₂. Although significantly less toxic than the wild type sequence (Figure S1A), scrambled Aβ peptide also evoked Ca²⁺ responses in all cells (Figure 2Ai). However, consistent with its lower toxicity, both the amplitude and the integral of the Ca²⁺ transients elicited by scrambled Aβ were significantly lower than those induced by oligomeric Aβ₄₂ and, in addition, they required a significantly longer time to reach peak (Figures 2Bi,Ci,Di). Furthermore, concordant with the less potent effect of scrambled Aβ in mobilizing intracellular Ca²⁺, the amplitude and integral of CCH-induced Ca²⁺ transients elicited following prior exposure to scrambled Aβ were significantly greater than those stimulated following prior exposure to oligomeric Aβ₄₂ (Figures 2Bii,Cii,Dii).

Figure 2. Aβ₄₂ oligomer-induced Ca²⁺ transients are sequence specific. Bar charts illustrating the magnitude of Ca²⁺ responses elicited by SH-SY5Y cells following the application of 5 μM Aβ₄₂ oligomers or Aβ scramble and 100 μM CCH (n > 370 cells). Data is presented as (A) percentage of responding cells, (B) peak amplitude, (C) integral of the response, (D) time to peak. Bar graphs are mean ± SEM from at least three independent experiments. ***p < 0.001.

Taken together, the comparison of the effects of Aβ scramble and oligomeric Aβ₄₂ demonstrates that the amino acid sequence of Aβ₄₂ has potent Ca²⁺ mobilizing properties, which are distinct from the action of Aβ scramble.

Aβ₄₂ Oligomers Mobilize Ca²⁺ from Intracellular Stores

The reduced magnitude of CCH-induced Ca²⁺ signals observed in cells previously exposed to oligomeric Aβ₄₂ suggested that this form of Aβ₄₂ was exerting an effect on intracellular Ca²⁺ stores. Therefore, we tested the relative contributions of Ca²⁺ influx from the extracellular space and its release from intracellular stores to Aβ₄₂-induced Ca²⁺ transients.

To determine the contribution of extracellular Ca²⁺ and Ca²⁺ influx to Aβ₄₂ oligomer-induced Ca²⁺ transients, we performed experiments using Ca²⁺-free imaging buffer. Under these conditions, Aβ₄₂ oligomers retained their ability to induce Ca²⁺ responses, with 100% of cells responding (Figure 3Ai). While no significant difference was observed in the peak amplitude (Figure 3Aiii) of Aβ₄₂ oligomer-induced Ca²⁺ transients, the integral of the response was significantly decreased in the absence of extracellular Ca²⁺ (Figure 3Av).

Figure 3. Aβ₄₂ oligomer-induced Ca²⁺ transients arise through release from the ER Ca²⁺ store. Bar charts illustrating the magnitude of Ca²⁺ responses elicited following the manipulation of (A) extracellular (n > 218 cells) and (B) ER Ca²⁺ concentrations (n > 512 cells). The magnitude of Ca²⁺ responses elicited by 5 μM Aβ₄₂ oligomers and 100 μM CCH is presented as (i,ii) percentage of responding cells, (iii,iv) peak amplitude and (v,vi) integral of the response. Bar graphs are mean ± SEM from at least three independent experiments. ***P < 0.001.

In contrast to the Aβ₄₂ oligomer-induced Ca²⁺ response, the peak amplitude and the integral of the Ca²⁺ responses to CCH applied following Aβ₄₂ oligomer exposure were significantly decreased by removal of extracellular Ca²⁺ from the imaging buffer (CCH, after Aβ₄₂; Figures 3Aiv,vi). This effect on the CCH-induced Ca²⁺ responses is likely due to lack of store-operated Ca²⁺ entry, which would replenish the Ca²⁺ released from stores by Aβ₄₂. Indeed, the peak amplitude and the integral of CCH-induced Ca²⁺ responses elicited in Ca²⁺ free buffer were significantly greater in naïve cells (CCH, no Aβ₄₂) than when Aβ₄₂ oligomers were previously applied (Figures 3Aiv,vi). Since Aβ₄₂ oligomer-induced Ca²⁺ transients were not significantly affected by removal of extracellular Ca²⁺, these results suggest that oligomeric Aβ₄₂ and CCH mobilize Ca²⁺ from a common intracellular Ca²⁺ pool.

The requirement of Ca²⁺ release from the ER Ca²⁺ store for the Ca²⁺ transients elicited by Aβ-induced was next investigated. To this end, ER Ca²⁺ stores were depleted by exposure of cells the SERCA pump inhibitor thapsigargin (Tg; 2 μM, 15 min) prior to the application of Aβ₄₂. In the absence of replete ER Ca²⁺ stores, Aβ₄₂-induced Ca²⁺ transients were completely abrogated (Figures 3Bi,iii,v). Similarly, CCH-induced Ca²⁺ responses were eliminated in Tg-treated cells (Figures 3Bii,Biv,Bvi), confirming the effect of Tg. Taken together, these experiments establish that Aβ₄₂ oligomers mobilize Ca²⁺ from the ER.

Aβ₄₂-Induced Ca²⁺ Release Occurs in Part through InsP₃Rs

Having determined that Aβ₄₂ oligomers mobilize Ca²⁺ from the intracellular ER Ca²⁺ store, we aimed to identify the mechanism by which Ca²⁺ release occurs. We therefore tested whether Aβ₄₂ was causing Ca²⁺ release from the ER through activation of InsP₃R or ryanodine receptor (RyR) Ca²⁺ release channels localized to this organelle.

Although SH-SY5Y cells have been reported to express functional RyRs, application of caffeine (10 mM), an agonist of the three RyR isoforms (10 mM) did not elicit a Ca²⁺ response in the SH-SY5Y cells used in this study (Figure S2A). Furthermore, the neuronally-expressed type 2 RyR could not be detected by immunoblot analysis (Figure S2B). Based on these observations, a role for RyR2 in Aβ₄₂ oligomer-mediated Ca²⁺ release was ruled out.

SH-SY5Y cells express InsP₃Rs and elicit robust Ca²⁺ responses to InsP₃-generating agonists including CCH (Tovey et al., 2001) (Figures 1–3). Therefore, we focused our investigation on the contribution of InsP₃Rs to Aβ₄₂-induced Ca²⁺ transients. To abrogate InsP₃-mediated Ca²⁺ responses, InsP₃ signaling was inhibited pharmacologically with 10 mM caffeine (Parker and Ivorra, 1991; Bezprozvanny et al., 1994) or was prevented by adenoviral-mediated overexpression of GFP-5′P, which metabolizes the second messenger InsP₃ to inactive InsP₂ (Higazi et al., 2009). To exclude the contribution of Ca²⁺ influx to the Aβ₄₂ oligomer-induced Ca²⁺ transients, these experiments were performed in the absence of extracellular Ca²⁺.

Caffeine application did not affect the number of cells exhibiting Ca²⁺ responses following Aβ₄₂ oligomer application, with 100% of cells responding (Figure 4B). However, caffeine significantly decreased the peak amplitude and the integral of the Aβ₄₂ oligomer-induced Ca²⁺ transients (Figure 4B). In contrast, Aβ scramble-induced Ca²⁺ transients were unaffected by caffeine application (Figure 4C). Ca²⁺ responses to 0.5 μM CCH were abolished by caffeine, demonstrating its inhibitory effect upon IICR (Figures 4A–C).

Figure 4. Aβ₄₂ oligomer-induced Ca²⁺ release is sensitive to caffeine. (A) Imaging protocol employed to investigate the involvement of InsP₃Rs in Aβ₄₂ oligomer-mediated Ca²⁺ release from the ER. InsP₃Rs were inhibited by co-administration of caffeine. (B,C) Bar charts illustrating the magnitude of Ca²⁺ responses elicited by SH-SY5Y cells following the application of 5 μM Aβ₄₂ oligomers (n > 780 cells) (B) or Aβ scramble (n > 144 cells) (C) and 0.5 μM CCH (n = 512 cells) in the presence or absence of 10 mM caffeine. Data is presented as percentage of responding cells, peak amplitude and integral of the response. Bar graphs are mean ± SEM from at least three independent experiments. ***P < 0.001.

Although caffeine inhibits InsP₃Rs (Bezprozvanny et al., 1994), it also acts on targets other than the InsP₃R such as cyclic nucleotide phosphodiesterases and phospholipase C (PLC) (Toescu et al., 1992; Taylor and Broad, 1998). Therefore, to investigate further the role of InsP₃ signaling in the generation of Aβ₄₂ oligomer-induced Ca²⁺ transients, InsP₃ signaling was inhibited by GFP-5′P overexpression. Using this strategy, InsP₃-mediated Ca²⁺ signals induced by CCH were prevented, validating this approach for suppression of InsP₃ signaling (Figure 5A). As observed for caffeine, however, GFP-5′P overexpression did not prevent Aβ₄₂ oligomer-induced Ca²⁺ transients, with 100% of cells responding (Figure 5B). However, the peak amplitude and the integral of Aβ₄₂ oligomer-induced Ca²⁺ transients were significantly decreased by overexpression of GFP-5′P (Figure 5B) when compared to the magnitude of Ca²⁺ transients in control cells, expressing GFP alone. Significantly, Aβ scramble-induced Ca²⁺ transients were not affected by GFP-5′P overexpression with no significant impact of its expression upon the peak amplitude or the integral of Aβ scramble-induced Ca²⁺ transients (Figure 5C). Taken together, these results demonstrate that Ca²⁺ transients elicited by Aβ₄₂ oligomers arise as a result of release from the ER intracellular Ca²⁺ store and that activation of InsP₃Rs contributes to this effect.

Figure 5. Aβ₄₂ oligomer-induced Ca²⁺ release occurs is reduced by InsP₃ 5′P expression. (A) Imaging protocol employed to investigate the involvement of InsP₃Rs in Aβ₄₂ oligomer-mediated Ca²⁺ release from the ER. InsP₃ was metabolized by overexpression of InsP₃ 5′P. (B,C) Bar charts illustrating the magnitude of Ca²⁺ responses elicited by SH-SY5Y cells infected with InsP₃ 5′P or GFP alone following the application of 5 μM Aβ₄₂ oligomers (n > 207 cells) (B) or Aβ scramble (n > 115 cells) (C) and 0.5 μM CCH (n > 55 cells). Data is presented as percentage of responding cells, peak amplitude and integral of the response. Bar graphs are mean ± SEM from at least three independent experiments. *P < 0.05.

Aβ₄₂ Oligomer-Induced Ca²⁺ Leak from the ER

The data presented above indicates that externally applied Aβ₄₂ rapidly induces an increase on cytosolic Ca²⁺ that involves InsP₃-dependent and -independent Ca²⁺ release from the ER. Since Aβ₄₂ has also been shown to elicit some of its cytotoxic effects as a result of intracellular accumulation (Wirths et al., 2004), we investigated whether it mobilized Ca²⁺ from the ER when directly applied. We also tested whether InsP₃Rs were required for its intracellular action.

To this end, an established permeabilized cell high-throughput functional assay of ER Ca²⁺ release was used (Tovey et al., 2006). This model uses as substrate for specific analysis of ER Ca²⁺ release, a plasma membrane-permeabilized preparation of the DT40 chicken B-lymphocyte cell line. A derivative of this cell line in which the 3 InsP₃R isoforms have been deleted by homologous recombination (DT40 TKO), allows the requirement for InsP₃Rs for Ca²⁺ release to be tested (Sugawara et al., 1997). Cell permeabilization and substantial dilution in intracellular buffer rules out the contribution of endogenously generated InsP₃ to signaling in this assay. Using this assay, a significantly greater InsP₃ independent ER Ca²⁺ leak was observed in both wild-type (p = 0.002) and DT40 TKO cells (p = 0.0195) exposed to Aβ₄₂ oligomers compared to the passive Ca²⁺ leak detected in each cell type (Figures 6A,B). The maximal Ca²⁺ leak rate induced by Aβ₄₂ oligomers was not significantly different between wild-type and DT40 TKO cells (p = 0.2606, Figure 6C), suggesting that InsP₃Rs were not required for Aβ₄₂ oligomers to trigger Ca²⁺ release.

Figure 6. Aβ₄₂ oligomers stimulate Ca²⁺ release from the ER of permeabilized cells. (A) Representative traces of mag-fluo-4 fluorescence (relative fluorescence units, RFU) in permeabilized DT40 cells and DT40 TKO cells, illustrating changes in ER luminal Ca²⁺ levels as a function of time in response to Aβ₄₂ oligomers, Aβ scramble and buffer alone. Data represents the mean of three measurements. (B,C) Initial quantitative analysis of the maximum Ca²⁺ leak rates from the ER of permeabilized DT40 cells and DT40 TKO cells triggered by Aβ₄₂ oligomers, Aβ scramble and buffer, respectively. The maximal Ca²⁺ leak rate was calculated by taking the maximal value of the first derivative of the fluorescence values to the time. Data were calculated as −dF/dt and represents the mean Ca²⁺ leak rate obtained from three measurements in RFU over time (RFU/s). **p < 0.01.

Aβ scramble did not increase the rate of the Ca²⁺ leak in DT40 cells (p = 0.2528) or in DT40 TKO cells (p = 0.0993) compared to the passive Ca²⁺ leak observed in each cell type (Figure 6B), and thus there was no significant difference in the maximal Ca²⁺ leak rate following Aβ scramble application between these two cell types (p = 0.2522, Figure 6C). Importantly, a significant difference between the Ca²⁺ leak rates triggered by exposure to Aβ₄₂ oligomers and Aβ scramble in wild-type DT40 cells (p = 0.0056) and DT40 TKO cells (p = 0.0045) was observed, indicating that Aβ-induced Ca²⁺ leak from the ER is dependent and specific to the amino acid sequence of Aβ₄₂. Taken together, these results suggest that Aβ₄₂ oligomers trigger a Ca²⁺ leak from the ER, which does not depend upon a direct interaction with InsP₃Rs.

Discussion

Here we show that the oligomeric form of the AD-associated peptide Aβ₄₂ has potent Ca²⁺ mobilizing properties and we identify mechanisms responsible for its action. Using both intact and permeabilized cell assays to investigate the effects of extracellular and internalized Aβ₄₂, respectively, we establish that Ca²⁺ release from the ER makes the greatest contribution to the Ca²⁺ mobilizing effects of Aβ₄₂. The InsP₃ signaling pathway also contributes to the Ca²⁺ mobilizing properties of oligomeric Aβ₄₂ in intact cells. InsP₃Rs were not required for Aβ₄₂-stimulated Ca²⁺ flux in permeabilized cells ruling out a direct regulation of InsP₃Rs by Aβ₄₂.

Central to the Ca²⁺ hypothesis of amyloid toxicity is the property of Aβ to induce Ca²⁺ elevations in its target cells. This sets in motion a cascade of events, which culminates in neuronal death. Ever since this hypothesis was put forward more than 20 years ago (Khachaturian, 1989, 1994), numerous reports have described Aβ-induced changes in intracellular Ca²⁺ in a number of cell types including primary neurons and astrocytes as well as neuroblastoma cell lines (Abramov et al., 2004b; Demuro et al., 2005). While there is general consensus that Aβ affects Ca²⁺ homeostasis, the mechanisms underlying this action of Aβ are many. Contributing to this diversity are the different experimental models used, the peptide sequence applied, the conformational state of the peptide and the method used for peptide preparation. Indeed, a number of shorter Aβ sequences have been employed in in vitro studies and depletion of ER Ca²⁺ store content reported (Ferreiro et al., 2004, 2008). Since Aβ₄₂ is considered to be more relevant to the pathology of AD, we focused on its effects on intracellular Ca²⁺ homeostasis. Not only is an accumulation of Aβ₄₂ observed in AD, this longer and more hydrophobic peptide is also more prone to self-assemble than Aβ ₄₀, the other principle length at which Aβ occurs. As a result, Aβ₄₂ exerts a greater degree of neurotoxicity (Jarrett and Lansbury, 1993). Consistent with the growing body of evidence that soluble oligomeric forms of Aβ constitute the primary neurotoxic species (Walsh et al., 2002; Gong et al., 2003; Cleary et al., 2005; Klyubin et al., 2005), this species of Aβ₄₂ potently induced Ca²⁺ fluxes and cytotoxicity in this study (Figures 1, 2 and Figure S2). Highlighting the requirement for appropriate peptide controls when studying Aβ₄₂, Ca²⁺ release and cytotoxicity was also induced by a scrambled peptide sequence of Aβ₄₂, although the magnitude of these responses was significantly lower than that induced by the wild type sequence. From these results, we concluded that the peptide sequence of Aβ₄₂ was the major contributor to the toxicity and Ca²⁺ mobilizing properties. The temporal properties of the Ca²⁺ transients we observed were reminiscent of those reported elsewhere, being relatively slow in reaching peak and returning to baseline levels after a few minutes (Demuro et al., 2005; Simakova and Arispe, 2006). The return of these Ca²⁺ signals to baseline does, however, suggest that the Ca²⁺ elevations induced by Aβ₄₂ were not immediately toxic. The Ca²⁺ mobilizing properties of the scrambled peptide, however, may reflect the previously described intrinsic properties of an oligomeric/amyloid peptide (Bucciantini et al., 2002; Yoshiike et al., 2008). For example, oligomeric forms of polyQ and insulin have been shown to induce Ca²⁺ transients (Demuro et al., 2005). The solvent HFIP used for preparation of the peptide has also previously been shown to exhibit cytotoxicity and to affect ion conductance of membranes (Capone et al., 2009).

Both Ca²⁺ influx from the extracellular space and release from ER-localized intracellular stores have been reported to be induced by Aβ and involved in its toxic action (Blanchard et al., 2004; Ferreiro et al., 2004, 2006; Kayed et al., 2004; Demuro et al., 2005, 2011; Kelly and Ferreira, 2006; Simakova and Arispe, 2006; Arispe et al., 2007; De Felice et al., 2007; Resende et al., 2008; Demuro and Parker, 2013). Although Ca²⁺ entry from the extracellular space was a component of the Ca²⁺ elevation induced by Aβ₄₂ in this study, the greatest contribution was due to release from the ER. Moreover the lack of an effect of removal of extracellular Ca²⁺ upon the initial peak of the Ca²⁺ response or the number of responding cells suggested that Ca²⁺ entry across the plasma membrane was secondary to Ca²⁺ release from the ER. Since Aβ₄₂ was acting to deplete the ER stores, the Ca²⁺ influx could arise via a store-operated Ca²⁺ entry pathway. These observations are not, however, incompatible with an additional mechanism for Ca²⁺ entry via plasma membrane pores formed by Aβ₄₂, which have been shown to require a longer period to develop (Demuro et al., 2011). Whether the Ca²⁺ fluxes associated with the formation of membrane pores, which were generally local to the pore and were of a relatively small magnitude, contribute to the global Ca²⁺ transient is not clear (Demuro et al., 2011).

Analysis of the mechanisms underlying Ca²⁺ release from the ER revealed that while InsP₃Rs contributed to Aβ₄₂-induced Ca²⁺ release from the ER in intact cells, the greater part of the Ca²⁺ elevation induced by Aβ₄₂ was due to an alternative mechanism. However, IICR did not contribute to the Ca²⁺ responses induced by scrambled peptide. From these results, we concluded that Aβ₄₂-induced Ca²⁺ release from the ER comprises an Aβ₄₂ sequence-specific component, which is InsP₃-dependent, and a second component, which is peptide sequence- and InsP₃-independent. Comparison of these Aβ₄₂ and Aβ₄₂ scrambled datasets reveals that although the InsP₃-dependent component of the total Aβ₄₂ signal is relatively minor, when considered as a fraction of the Aβ₄₂-specific Ca²⁺ signal (i.e., Aβ₄₂—Aβ₄₂ scrambled Ca²⁺ transient), its importance is increased.

Our demonstration of the participation of InsP₃ signaling in Aβ₄₂-induced Ca²⁺ responses provides robust evidence in support of this pathway in Aβ₄₂-mediated Ca²⁺ signals thus far. In particular, the use of InsP₃ 5′phosphatase overexpression to suppress InsP₃ signaling is a highly selective strategy, overcoming issues regarding incomplete knockdown of InsP₃Rs and contribution of the isoforms not targeted when using siRNA approaches. The inhibition of Ca²⁺ signals by caffeine is also consistent with a role for the InsP₃ signaling pathway in the Ca²⁺ mobilizing effects of Aβ (Parker and Ivorra, 1991; Bezprozvanny et al., 1994). Not only does caffeine inhibit InsP₃Rs directly (Bezprozvanny et al., 1994), by also inhibiting PLC, caffeine is a potent inhibitor of InsP₃ generation (Taylor and Broad, 1998). These findings are consistent with the reduction in the Aβ₄₂-induced Ca²⁺ transient observed following application of the PLC inhibitor U73122 (Resende et al., 2008) although U73122 has numerous non-specific effects. The mechanism by which InsP₃ signaling is engaged by Aβ₄₂ in this study remains to be established. Since the effects of inhibition of InsP₃ signaling persist in the absence of extracellular Ca²⁺, activation of PLC and InsP₃ generation by Aβ₄₂-stimulated Ca²⁺ influx can be excluded. Thus, a more likely scenario would involve Aβ₄₂ engagement of a PLC-coupled G protein coupled-receptor (GPCR). Indeed, a number of different GPCRs, including metabotropic glutamate receptors, are activated by Aβ₄₂, contributing to modulation of LTP, Aβ₄₂ synthesis and processing and cytotoxicity (Wang et al., 2004; Thathiah and De Strooper, 2011).

The internalization of Aβ from the extracellular space (Bucciantini et al., 2004; Pierrot et al., 2004; Wirths et al., 2004; Kaminski Schierle et al., 2011) raises a further possibility that Aβ acts to either directly activate/sensitize InsP₃Rs or to alter InsP₃ generation/metabolism. Since significant intracellular Aβ₄₂ accumulation would require up to 1 h (Bucciantini et al., 2004; Kaminski Schierle et al., 2011), it is unlikely that this endocytosed Aβ₄₂ contributes to the acute modulation of Ca²⁺ fluxes observed in this study and elsewhere in intact cells. Endocytosis of Aβ₄₂ may, however, contribute to the more chronic effects on Ca²⁺ homeostasis as well as cytotoxicity previously reported (Ferreiro et al., 2004, 2006; Resende et al., 2008). The possibility that Aβ₄₂ could directly affect ER Ca²⁺ homeostasis from an intracellular location was therefore also considered. Using a permeabilized cell assay to allow control of cytosolic conditions and access of Aβ₄₂ to the ER, an Aβ₄₂-stimulated Ca²⁺ efflux from the ER was observed. Unlike that observed for intact cells, the difference between Aβ₄₂ and Aβ₄₂ scrambled was dramatic, revealing a highly specific effect of Aβ₄₂ upon ER Ca²⁺ mobilization. These effects were observed in the absence of exogenous InsP₃ suggesting that the effects were InsP₃R-independent. The extensive dilution of cytosol following permeabilization of the DT40 cells would also likely preclude a contribution of Aβ₄₂-stimulated InsP₃ generation. More significantly, InsP₃Rs were not required for the Ca²⁺ mobilizing properties of Aβ₄₂, since deficiency in all three InsP₃R isoforms did not affect the Ca²⁺ mobilizing properties of Aβ₄₂. The absence of a requirement for InsP₃Rs for Aβ₄₂-stimulated Ca²⁺ flux in the permeabilized cell system does not rule out the possibility that IICR contributes to Ca²⁺ fluxes and toxicity mediated by intracellular Aβ _42. Indeed, by activating Ca²⁺-sensitive PLC and generation of InsP₃, Ca²⁺ mobilized by Aβ₄₂ could promote IICR. Consistent with this notion, microinjected Aβ₄₂ was recently shown to promote Ca²⁺ signals in Xenopus oocytes in a manner that involved InsP₃ generation (Demuro and Parker, 2013).

The depletion of the ER Ca²⁺ store by Aβ₄₂ has important implications for the mechanisms of its toxicity. Depletion of ER Ca²⁺ stores results in the accumulation of unfolded proteins and activation of the ER stress response, which via caspase 12 activation and Bap31 cleavage can subsequently induce mitochondrial apoptotic cascades (Verkhratsky, 2005; Xu et al., 2005; Mekahli et al., 2011). The engagement of InsP₃Rs during Aβ₄₂-stimulated depletion of ER Ca²⁺ may be of greater consequence. Specifically, InsP₃R-induced Ca²⁺ release from the ER and its subsequent sequestration by neighboring mitochondria could lead to mitochondrial Ca²⁺ overload, permeability transition and death (Csordas et al., 2006). These pathways also lead to increased reactive oxygen species generation, which is commonly observed in AD (Ferreiro et al., 2004, 2008; Arduino et al., 2009; Clark et al., 2010).

The use of SH-SY5Y neuroblastoma cell line and permeabilized DT40 B-lymphocytes in this study, rather than primary neurons allowed careful dissection of the role of ER Ca²⁺ signaling to Aβ-induced Ca²⁺ signals independent from Ca²⁺ fluxes that may arise in neurons as a result of electrical or synaptic activity. Moreover, using this cell line, contributions from other Aβ targets described in neurons such as NMDA receptors are excluded. Analogous to a number of other studies in electrically non-excitable primary and cultured cells including Xenopus oocytes (Demuro and Parker, 2013) astrocytes and PC12 cells (Abramov et al., 2003, 2004a; Simakova and Arispe, 2006), our data indicates that certain of the Ca²⁺ mobilizing properties of Aβ₄₂ are neuron-independent and do not require the expression of any other of its reported targets. Fundamental aspects of the Ca²⁺ mobilizing properties of Aβ₄₂ were further revealed and exemplified by the Aβ₄₂-stimulated Ca²⁺ flux from the InsP₃R-deficient ER of permeabilized DT40 B-lymphocytes. These latter data demonstrate for the first time that Aβ₄₂ has the capacity to directly induce Ca²⁺ flux from the ER. Given the importance of the ER and InsP₃Rs in neuronal functions, future studies will be required to test whether InsP₃Rs contribute to Aβ-mediated neuronal pathology.

Author Contributions

Laura E. Jensen: substantial contributions to conception and design, acquisition, analysis and interpretation of data as well as writing of manuscript. H. Llewelyn Roderick: substantial contributions to conception and design, interpretation of data as well as writing of manuscript. Geert Bultynck and Tomas Luyten: designed, acquired, analysed and interpreted data of Figure 6. Hozeefa Amijee: designed, acquired and interpreted data of Figure S2. Martin D. Bootman: proof-read manuscript.

Conflict of Interest Statement

Some of this work was supported by a grant from Senexis, Babraham Research Campus and Hozefa Amijee was employed by Senexis at the time this work was carried out.

Acknowledgments

This study was supported by The Babraham Institute, Senexis, The Royal Society (University Research Fellowship to H. Llewelyn Roderick) and The Gates Cambridge Trust [Gates Cambridge Scholarship to Laura E. Jensen (née Allan)]. Work in the Geert Bultynck's laboratory was supported by the Research Council of the KU Leuven via the Concerted Actions program (GOA/09/012) and via an OT START (STRT1/10/044), and by the Interuniversity Attraction Poles Program (Belgian Science Policy; P6/28 and P7/13).

Supplementary Material

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fnmol.2013.00036/abstract

Figure S1. Validating the toxicity and conformation of Aβ₄₂ oligomers. (A) Assessment of toxicity of homogeneous Aβ preparations. Bar chart illustrating the cytotoxic effects of Aβ₄₂ preparations upon SH-SY5Y cells determined using the MTT assay. Data is expressed as a percentage of MTT reduced by test samples to the dead cell controls following 24-h treatment with Aβ₄₂ oligomers and scrambled Aβ at the respective concentrations. (Bi) Schematic diagram illustrating the epitopes of Aβ₄₂ recognized by the conformation dependent, anti-oligomer antibody, A11 (Kayed et al., 2003), and the sequence dependent, anti-amyloid antibody, 12F4 (Parvathy et al., 2001). (Bii,iii) Sensorgrams obtained from surface plasmon resonance spectroscopy, as described (Maezawa et al., 2008). A Biacore T-100, equipped with four flow cells on a sensor chip, was used for these real-time binding studies. Biotinylated Aβ₄₂was prepared by mixing a 1:10 molar ratio of biotinylated and unbiotinylated Aβ₄₂. In preparation for the binding studies, Aβ₄₂ was injected onto a streptavidin chip at a concentration of 10 μM to immobilize Aβ₄₂by streptavidin-biotin coupling. The streptavidin chip of flow cell (Fc) 2 was partially (50%) and of Fc-4 fully saturated (100%) with Aβ₄₂ oligomers. As a control, the surface of Fc-3 was partially saturated (50%) with Aβ₄₂monomers. Antibodies (Bii) A11 and (Biii) 12F4 were injected over the immobilized Aβ₄₂ of each flow cell at a concentration of 50 μg/ml and 10 μg/ml, respectively. The injection of the anti-oligomer antibody, A11, was followed by a regeneration step prior to injection of 12F4. The binding of injected antibodies, present in the flow phase, to the immobilized Aβ₄₂was measured by response units (RU) elicited. All values were corrected for the RU obtained from the reference cell, flow cell 1, which was saturated with biotinylated Aβ₄₂ only.

Figure S2. Comparison of Ca²⁺ responses elicited by Aβ₄₂ oligomers and monomers in SH-SY5Y cells. (A) Imaging protocol employed to assess the effects of homogeneous preparations of Aβ₄₂ on the Ca²⁺ signaling capacity of fluo-4-loaded SH-SY5Y cells. Cellular Ca²⁺ responses were recorded by wide-field epifluorescence. The magnitude of Ca²⁺ responses elicited by 5 μM Aβ₄₂ monomers and oligomers and the subsequent application of 100 μM CCH is presented as (B) percentage of responding cells, (C) peak amplitude and (D) integral of the response. Soluble Aβ monomers and Aβ oligomers were prepared as previously described (Demuro et al., 2005). This method of Aβ preparation reportedly results in homogeneous populations of Aβ monomers and oligomers (also Figure S1B). All Aβ₄₂ concentrations stated were based on the molar mass of the peptide.

Figure S3. Human neuroblastoma SH-SY5Y cells lack RyR expression. (A) Representative Ca²⁺ trace illustrating that SH-SY5Y cells do not elicit Ca²⁺ responses following the application of 10 mM caffeine, indicating that cells lack RyRs (n = 239 cells). However, SH-SY5Y cells do exhibit InsP₃-mediated Ca²⁺ responses. (B) Immunoblot analysis corroborating the lack of RyR2 expression in SH-SY5Y cells. RyR2 expression is observed in control samples of adult hippocampal tissue and primary hippocampal cultures maintained for 4, 8, 11, and 15 days in vitro (DIVs).

References