Aging Enables Ca 2+ Overload and Apoptosis Induced by Amyloid-β Oligomers in Rat Hippocampal Neurons: Neuroprotection by Non-Steroidal Anti-Inflammatory Drugs and R-Flurbiprofen in Aging Neurons

Abstract

The most important risk factor for Alzheimer’s disease (AD) is aging. Neurotoxicity in AD has been linked to dyshomeostasis of intracellular Ca²⁺ induced by small aggregates of the amyloid-β peptide 1-42 (Aβ₄₂ oligomers). However, how aging influences susceptibility to neurotoxicity induced by Aβ₄₂ oligomers is unknown. In this study, we used long-term cultures of rat hippocampal neurons, a model of neuronal in vitro aging, to investigate the contribution of aging to Ca²⁺ dishomeostasis and neuron cell death induced by Aβ₄₂ oligomers. In addition, we tested whether non-steroidal anti-inflammatory drugs (NSAIDs) and R-flurbiprofen prevent apoptosis acting on subcellular Ca²⁺ in aged neurons. We found that Aβ₄₂ oligomers have no effect on young hippocampal neurons cultured for 2 days in vitro (2 DIV). However, they promoted apoptosis modestly in mature neurons (8 DIV) and these effects increased dramatically after 13 DIV, when neurons display many hallmarks of in vivo aging. Consistently, cytosolic and mitochondrial Ca²⁺ responses induced by Aβ₄₂ oligomers increased dramatically with culture age. At low concentrations, NSAIDs and the enantiomer R-flurbiprofen lacking anti-inflammatory activity prevent Ca²⁺ overload and neuron cell death induced by Aβ₄₂ oligomers in aged neurons. However, at high concentrations R-flurbiprofen induces apoptosis. Thus, Aβ₄₂ oligomers promote Ca²⁺ overload and neuron cell death only in aged rat hippocampal neurons. These effects are prevented by low concentrations of NSAIDs and R-flurbiprofen acting on mitochondrial Ca²⁺ overload.

Keywords

aging Alzheimer’s disease calcium hippocampal neurons mitochondria non-steroidal anti-inflammatory drugs NSAIDs R-flurbiprofen

INTRODUCTION

Aging is the most important risk factor for Alzheimer’s disease (AD). The etiology of AD and the role of aging remain largely unknown. Unfortunately, efficient therapies are lacking and concern is rising on the failure of costly clinical trials with promising compounds. A critical example is Tarenflurbil^® (R-flurbiprofen), an enantiomer of flurbiprofen without anti-inflammatory activity selected for a clinical trial because of its ability to modulate γ-secretase [1]. The reasons for failure are controversial, but researchers claim that protection efficiency may vary according to stage of the disease and/or age [2].

Increasing evidence indicates that small soluble aggregates or oligomers of Aβ_42, rather than monomers or fibrils, are the most likely neurotoxin in AD [3]. We have shown that Aβ₄₂ oligomers, but not fibrils, induce Ca²⁺ entry, mitochondrial Ca²⁺ overload and apoptosis in cerebellar granule cells [4]. In addition, we reported that several NSAIDs and R-flurbiprofen protected cerebellar neurons against apoptosis by depolarizing mitochondria partially and preventing mitochondrial Ca²⁺ overload [4, 5]. These results, together with results from other labs using mouse models of familial AD, have contributed to put forward the so-called Ca²⁺ hypothesis of AD [6 –9]. The death target and mechanism of Ca²⁺ entry induced by Aβ₄₂ oligomers remain controversial [10, 11]. In addition, the role of aging in the susceptibility of neurons to Aβ₄₂ oligomers neurotoxicity and the effects on intracellular Ca²⁺ are unclear at present.

Multiple sources of evidence suggest that long-term cultures of rat hippocampal neurons display many of the hallmarks of aging in vivo, including accumulation of reactive oxygen species, lipofuscin granules, heterochromatic foci, activation of the Jun N-terminal protein kinase and p53/p21 pathways, gradual loss of cholesterol, and changes in Ca²⁺ channel density and NMDA receptor expression [12 –17]. Therefore, long-term cultures of hippocampal neurons may provide a suitable model for investigating Ca²⁺ remodeling in aged hippocampal neurons.

Here we have used rat hippocampal neurons cultured for different days in vitro (DIV) to study the effects of in vitro aging on Ca²⁺ overload and susceptibility to neuron cell death induced by Aβ₄₂ oligomers. In addition, we have tested the effects of very low concentrations of NSAIDs and R-flurbiprofen on Ca²⁺ overload and neuron cell death induced by Aβ₄₂ oligomers in aged neurons. Our results show that Aβ₄₂ oligomers fail to increase cytosolic and mitochondrial Ca²⁺ concentrations in young neurons. Consistently, they also fail to induce cytochrome c release and apoptosis in young neurons. However, sensitivity to Aβ₄₂ oligomers is acquired during in vitro aging when Aβ₄₂ oligomers promote massive Ca²⁺ influx and mitochondrial Ca²⁺ overload leading to cytochrome c release and apoptosis. We conclude that aging enables Ca²⁺ overload and neuron cell death induced by Aβ₄₂ oligomers in hippocampal neurons. In addition, we also found that NSAIDs and R-flurbiprofen prevent mitochondrial Ca²⁺ overload and neuron cell death in aged neurons only at low concentrations while at larger concentrations they promote apoptosis.

MATERIALS AND METHODS

Reagents

Wistar rat pups (newborn P0 - 1) are from the Valladolid University animal facility. Fura2/AM, cytochrome c antibody (MA5-11283), wt coelenterazine and lipofectamine^® 2000 are from Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) is from Lonza (Barcelona, Spain). Horse serum, Neurobasal medium, HBSS medium, MEM medium, B27 and L-glutamine are from Gibco (Barcelona, Spain). Papain solution is from Worthington (Lakewood, NJ, USA). Coelenterazine n is from Biotium (Hayward, California, USA). NSAIDs and R-Flurbiprofen are from Cayman Chemical Company (Madrid, Spain). Aβ₄₂ peptides are from Bachem AG (Bubendorf, Switzerland). Poly-D-lysine and Annexin V are from BD (Madrid, Spain). DNase I is from Sigma (Madrid, Spain). The mitGAmut plasmid was kindly donated by P. Brulet (CNRS, Gif-sur-Yvette, France). Other reagents and chemicals are either from Sigma or Merck.

Primary hippocampal neuron culture

Hippocampal neurons are obtained from Wistar rat pups under sterile conditions as reported by Brewer et al. [18] with further modifications introduced by Pérez-Otaño et al. [19]. Briefly, rat pups are decapitated and, after brain removal, meninges are discarded and the hippocampus is separated from cortex. Hippocampal tissue is then cut in small pieces, transferred to papain solution (20μg/ml) and incubated at 37°C for 30 min with occasional gentle shaking. After 15 min, DNase I (50μg/ml) and tissue pieces are washed with Neurobasal Medium. A cell suspension is obtained using a fire-polished pipette in Neurobasal supplemented with 10% FBS. Cell suspension is then centrifuged at 160 g for 5 min and the cell pellet finally suspended in Neurobasal medium. Hippocampal cells are plated onto poly-D-lysine-coated, 12 mm diameter glass coverslips at 30×10³ cells/dish, and cultured in the same medium supplemented with L-glutamine (2 mM), gentamicin (1μg/ml), 2% B27, and 10% FBS. Cells are maintained in a humidified 37°C incubator with 5% CO₂ without further exchange of the media. Cells are cultured for 2, 8, or >13 DIV before experiments. This procedure has been described in detail elsewhere [10].

Preparation of Aβ₄₂ oligomers

Aβ₄₂ oligomers are prepared as reported recently by a new procedure [10]. Briefly, Aβ₄₂ is initially solved at 1 mM in ice cold hexafluoroisopropanol (HFIP), and separated into aliquots in sterile microcentrifuge tubes. The solution is then incubated for 2 h at room temperature (RT) to allow monomerization. HFIP is removed under vacuum in a speed vac. (800 g×10 min at RT), and the peptide film is stored desiccated at –20°C. For aggregation, the peptide is first suspended in dry dimethyl sulfoxide to a concentration of 5 mM. For complete suspension of the peptide, it is subjected to ultrasounds for 10 min, distributed in propylene non-siliconized tubes, and stored at –20°C. Medium (MEM) supplemented with 0.5 mg/ml Fe²⁺, 0.5 mg/ml Cu²⁺, and 0.5 mg/ml Zn²⁺ is added to bring the peptide to a concentration of 80μM and is incubated at 37°C for 24 h. For experiments, Aβ₄₂ is solved in medium to a final concentration of 2μM.

Fluorescence imaging of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and in situ immunofluorescence

Coverslips containing cultured hippocampal neurons for different DIV are incubated in standard external medium (SEM) containing (in mM) NaCl 145, KCl 5, CaCl₂ 1, MgCl₂ 1, glucose 10, and Hepes 10 (pH 7.42). Then, cells are incubated with fura2/AM dye (4μM) for 60 min at RT in the dark. Coverslips are placed on the perfusion chamber of a Zeiss Axiovert 100 TV microscope and perfused continuously with the same pre-warmed (37°C) SEM. For imaging, cells are epi-illuminated alternately at 340 and 380 nm lights with a filter wheel, and light emitted at 520 nm is recorded with a Hamamatsu ER camera (Hamamatsu Photonics, France) every 5 s. Pixel by pixel ratios of consecutive frames are captured, and [Ca²⁺]_cyt of regions of interest (ROIs) corresponding to individual neurons are expressed as the ratio of fluorescence emission at 520 nm following excitation at 340 and 380 nm (Ratio F340/F380), as reported in detail previously [13]. For calculations, responses were averaged from responsive neurons easily selected by their morphology different from glial cells. Responsive cells were considered all those showing a change in the slope of the Ca²⁺ recording after stimulation. In some experiments, cells were identified assessing the single cell content of β-tubulin III and glial fibrillary acidic protein (GFAP) by indirect immunofluorescence in the same cells used for calcium imaging as reported previously [4]. Briefly, after calcium imaging, cells were fixed with p-formaldehyde and incubated with anti-β-tubulin III (1:400) and anti GFAP (1:200) for 1 h at 37°C. Then, cells were washed and incubated with 1:100 labeled anti IgG antibodies. Nuclei were stained by incubation with DAPI 0.2 mg/ml for 5 min.

Bioluminescence imaging of mitochondrial Ca²⁺ concentration ([Ca²⁺]_mit)

Hippocampal neurons cultured for different DIV are transfected with the mitGAmut plasmid using lipofectamine^® 2000. The mitGAmut plasmid contains a mutated, low affinity aequorin targeted to mitochondria and a GFP sequence to help selecting transfected neurons [20]. After 24 h, cells are incubated for 2 h with 4μM n or wt coelenterazine at RT in the dark, washed with SEM, and placed into a perfusion chamber thermostated to 37°C under a Zeiss Axiovert S100 TV microscope. Then cells are perfused continuously (5–10 ml/min) with test solutions based on the SEM described above prewarmed at 37°C. Bioluminescence images are taken with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor. Photonic emissions are integrated for 10 s periods. At the end of each experiment, cells are permeabilized with SEM containing 0.1 mM digitonin and 10 mM CaCl₂, added here to release all the residual aequorin photonic emissions [21]. Photons were quantified using the Hamamatsu Aquacosmos software and converted into mitochondria free Ca²⁺ concentration ([Ca²⁺]_mit) values as reported previously [22, 23]. In experiments with permeabilized cells, perfusion was performed in a standard internal medium (SIM) containing (in mM) 130 KCl, 5 NaCl, 2 MgCl₂, 5 Succinate, 2 KH₂PO₄, 1 ATP, 20 HEPES/KOH, pH 7.0. Cells were permeabilized by perfusing them with SIM containing 50μM digitonin for 1 min. Then, cells were incubated with SIM containing 200 nM Ca²⁺ that resembles resting intracellular Ca²⁺ concentration (buffered with EGTA), in the presence or absence of NSAIDs for 5–7 min. After that, perfusion is switched to SIM containing 10μM Ca²⁺, in the presence or absence of the corresponding NSAID, to induce mitochondrial Ca²⁺ uptake. Media with low concentrations of Ca²⁺ were prepared with different concentrations of CaCl₂ and the Ca²⁺ buffers H-EDTA and tris-EGTA are prepared according to the computer program MaxQuelator (Chris Patton, Stanford University). Further details have been reported previously [23].

Apoptosis measurements

Hippocampal neurons cultured for different DIV are incubated for 1 h with vehicle or 2μM Aβ₄₂ oligomers in the same SEM described above and in the presence or the absence of different NSAIDs. After Aβ₄₂ treatment, cells are washed once and returned to the original Neurobasal medium for an additional 24 h period. After that, cells are washed with phosphate buffered saline (PBS) once and apoptosis is evaluated using Annexin V (1:20, 10 min) using annexing binding buffer 1x containing (in mM) NaCl 140; CaCl₂ 2.5; Hepes 10 (pH 7.4) and assessed by fluorescence microscopy using a Nikon Eclipse TS100 microscope (objective 40x) as reported previously [12].

Measurements of cytochrome c release

Cytochrome c release from mitochondria is tested by immunofluorescence as reported previously [4, 12]. Hippocampal neurons cultured for several DIV are treated for 1 h with vehicle or Aβ₄₂ oligomers, washed and cultured for 24 h. Then, cells are fixed with p-formaldehyde and cytochrome c was tested by indirect immunofluorescence using a specific antibody against cytochrome c (1:300; Mouse anti-cytochrome c antibody MA5-11283 purchased from Invitrogen, Barcelona, Spain). Immunopositive cells are revealed using Alexafluor 488-tagged antibodies (1:300). Nuclei are identified by DAPI staining. Optical density in individual neurons is carried out to quantify cytochrome c release using Image J software (National Institute of Mental Health, Bethesda, MA, USA).

Statistics

Changes in fluorescence ratio are expressed as Δ[Ca²⁺]_cyt (ΔRatio F340/F380) using Origin Lab 7.0. Data are presented as mean±SEM. When only two means are compared, Student’s t test is used. For more than two groups, statistical significance of the data is assessed by one-way or two-way ANOVA and compared using Bonferroni’s multiple comparison tests using Origin Lab 7.0 software. Differences are considered significant at p < 0.05. Power analysis was carried out using GraphPad StatMate 2 software.

RESULTS

Apoptosis induced by Aβ₄₂ oligomers increases dramatically with culture age

The effects of Aβ₄₂ oligomers on apoptosis were investigated in hippocampal neurons cultured for 2, 8, and >13 DIV. Cultures were treated with 2μM Aβ₄₂ oligomers for 1 h and then cells were incubated in Neurobasal Medium for 24 h before measuring apoptosis. Although this oligomer concentration is higher than the physiological concentration, it has been widely used in the literature for reporting quick effects of oligomers. Apoptosis was estimated by monitoring everted phosphatidylserine using Annexin V staining. Representative bright field (transmission) and immunofluorescence images (Anx V) are shown in Fig. 1A. The relative abundance (percent) of apoptotic neurons in cell cultures at different DIV treated or not treated with Aβ₄₂ oligomers are shown in Fig. 1B. Aβ₄₂ oligomers did not induce apoptosis in young neurons (2 DIV). However, in mature neurons (8 DIV), Aβ₄₂ oligomers modestly promoted apoptosis, and this effect increased rather dramatically in aged neurons (>13 DIV). Therefore, neuronal apoptosis induced by Aβ₄₂ oligomers depends critically on the age of the cultures.

Cytosolic Ca²⁺ responses to Aβ₄₂ oligomers are increased in aged neurons

The effects of Aβ₄₂ oligomers on [Ca²⁺]_cyt were studied in young, mature, and aged neurons in vitro. Hippocampal neurons cultured for different time periods (2, 8, and >13 DIV) were incubated with fura2/AM and subjected to fluorescence Ca²⁺ imaging before and after stimulation with Aβ₄₂ oligomers and N-methyl D-aspartate (NMDA). Representative Ca²⁺ images coded in pseudocolor and recordings are shown in Fig. 2A. We found that [Ca²⁺]_cyt rises induced by Aβ₄₂ oligomers depended strongly on culture time. At 2 DIV, only less than 40% of the neurons responded to oligomers (Fig. 2B). In addition, the size of the [Ca²⁺]_cyt rises in responsive cells was very small. The same cells responded to NMDA 100μM. In contrast, nearly all mature (8 DIV) and aged (>13 DIV) neurons responded clearly to Aβ₄₂ oligomers (Fig. 2B). In addition, [Ca²⁺]_cyt rises induced by Aβ₄₂ oligomers were much larger in mature and aged neurons than in young cells. Figure 2C compares the average responses to Aβ₄₂ oligomers at different culture periods. Rises in [Ca²⁺]_cyt increased significantly in mature (8 DIV) relative to young (2 DIV) neurons, and effects increased further in aged neurons (>13 DIV). Thus, Ca²⁺ responses to Aβ₄₂ oligomers depend strongly on the age of the cultures. Consistent with previous reports [12], Ca²⁺ responses induced by NMDA increased with culture time as well (Fig. 2A). Finally, it is also noteworthy that, as reported previously [12], resting [Ca²⁺]_cyt levels were larger in aged neurons compared with young neurons.

To confirm that the effects of Aβ₄₂ oligomers were restricted to neurons, two-fold immunofluorescence was carried out in the same cells used for calcium imaging. Figure 3 shows that identified neurons displayed large changes in cytosolic Ca²⁺ while identified glial cells in the same microscopic fields did not respond to oligomers.

Mitochondrial Ca²⁺ responses to Aβ₄₂ oligomers are increased in aged neurons

We tested next the effects of Aβ₄₂ oligomers on mitochondrial Ca²⁺ ([Ca²⁺]_mit) in rat hippocampal neurons cultured for different DIV. For this end, we carried out bioluminescence imaging of neurons transfected with a plasmid expressing a low-affinity aequorin targeted to mitochondria. This probe also contains GFP for easy selection of transfected neurons for bioluminescence imaging (mitGAmut) [20]. Figure 4A shows typical GFP fluorescence (GFP, top) and AEQ bioluminescence (aequorin, bottom) images of transfected neurons stimulated with 2μM Aβ₄₂ oligomers. It also shows the acute effects of 2μM Aβ₄₂ oligomers on [Ca²⁺]_mit in neurons at different DIV. Aβ₄₂ oligomers failed to increase [Ca²⁺]_mit in young neurons (2 DIV). However, Aβ₄₂ oligomers raised [Ca²⁺]_mit in mature neurons (8 DIV) and these effects increased further in aged (>13 DIV) neurons (Fig. 4A). Average data shows that Aβ₄₂ oligomers have no effect on [Ca²⁺]_mit in 2 DIV neurons while promoting mitochondrial Ca²⁺ uptake in mature and aged cultures, since the effects are significantly larger in aged neurons (Fig. 4B). Therefore, effects of Aβ₄₂ oligomers on mitochondrial Ca²⁺ uptake in intact cells also strongly depends on the age of the cultures.

Mitochondrial Ca²⁺ uptake contributes to apoptosis induced by Aβ₄₂ oligomers in aged neurons

To evaluate the contribution of mitochondrial Ca²⁺ overload to the neurotoxicity induced by Aβ₄₂ oligomers, we studied whether inhibition of mitochondrial Ca²⁺ uptake affects apoptosis in aged neurons. To this end, we tested whether mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) prevented specifically mitochondrial Ca²⁺ uptake. Permeabilized neurons were perfused with intracellular medium containing 10μM Ca²⁺ in the absence and the presence of FCCP (see Materials and Methods). Figure 5A illustrates a typical recording of [Ca²⁺]_mit in permeabilized neurons transfected with mitochondria-targeted aequorin and stimulated with 10μM Ca²⁺. FCCP prevented the rise in [Ca²⁺]_mit induced by 10μM Ca²⁺. This effect is specific for mitochondria as it does not affect the rise in [Ca²⁺]_cyt induced by Aβ₄₂ oligomers (Fig. 5B).

Once we established a method to inhibit specifically mitochondrial Ca²⁺ uptake, we studied the effects of FCCP on Aβ₄₂-induced apoptosis in aged neurons. Figure 5C shows that FCCP significantly inhibits apoptosis induced by Aβ₄₂ oligomers in aged neurons (>13 DIV). To confirm the role played by mitochondria in apoptosis induced by Aβ₄₂ oligomers in aged neurons, the effects of Aβ₄₂ oligomers on cytochrome c release were tested at different DIV. We found that Aβ₄₂ oligomers promoted release of cytochrome c in aged cultures (>13 DIV) but not in the young neurons as shown by indirect immunofluorescence against cytochrome c (Fig. 6). Thus, inhibition of mitochondrial Ca²⁺ overload prevents cytochrome c release and apoptosis induced by Aβ₄₂ oligomers in aged neurons.

NSAIDs and R-flurbiprofen inhibit mitochondrial Ca²⁺ uptake induced by Aβ₄₂ oligomers in aged hippocampal neurons

Once we established the importance of mitochondrial Ca²⁺ uptake for apoptosis induced by Aβ₄₂ oligomers in aged neurons, we investigated whether low concentrations of salicylate and other NSAIDs, including R-flurbiprofen that lacks anti-inflammatory activity, may prevent mitochondrial Ca²⁺ overload and apoptosis induced by Aβ₄₂ oligomers in aged hippocampal neurons. These compounds are considered mild mitochondrial uncouplers that dissipate partially the mitochondrial potential (Δψ), the driving force for mitochondrial Ca²⁺ uptake as reported previously [4]. Figure 7 shows typical single-cell recordings of [Ca²⁺]_mit in permeabilized neurons challenged with 10μM Ca²⁺ in the absence and the presence of low concentrations of NSAIDs and R-flurbiprofen. We found that 10μM Ca²⁺ increased [Ca²⁺]_mit in permeabilized hippocampal neurons (Fig. 7A). This effect was inhibited by 100μM salicylate, 1μM sulindac sulphide, and 1μM R-flurbiprofen as shown by representative recordings (Fig. 7B–D). All three compounds tested significantly inhibited mitochondrial Ca²⁺ uptake in permeabilized neurons (Fig. 7E).

Effects of NSAIDs and R-flurbiprofen are specific for mitochondria since none of the above compounds decreased the rise in [Ca²⁺]_cyt induced by Aβ₄₂ oligomers (Fig. 8). However, a deepened, more sophisticated quantification of calcium imaging results could provide new insights on cytosolic Ca²⁺ responses in cultured hippocampal neurons. Thus, at fairly low concentrations, NSAIDs inhibit mitochondrial Ca²⁺ uptake without affecting the rise in cytosolic Ca²⁺. Thus, NSAIDs inhibit specifically mitochondrial Ca²⁺ uptake at very low concentrations.

NSAIDs and R-flurbiprofen prevent apoptosis induced by Aβ₄₂ oligomers in aged hippocampal neurons

The effects of NSAIDs and R-flurbiprofen on apoptosis induced by Aβ₄₂ oligomers were tested in aged neurons (>13 DIV). NSAIDs were added 30 min prior, during, and 30 additional min after treatment with Aβ₄₂ oligomers. Figure 9A shows representative bright-field images of hippocampal neurons treated with vehicle (Control) and Aβ₄₂ oligomers in the presence and the absence of 100μM salicylate. Apoptosis was estimated in similar cultures using annexin V staining. All compounds tested including 100μM salicylate (Fig. 9B), 0.5μM sulindac sulphide (Fig. 9C), and 0.5μM R-flurbiprofen (Fig. 9D) significantly prevented apoptosis induced by Aβ₄₂ oligomers. We noticed that neuroprotection afforded by R-flurbiprofen decreased as we increased its concentration. Accordingly, we tested the dose-dependent effects of R-flurbiprofen alone on apoptosis and compared them with the effects of Aβ₄₂ oligomers. Figure 9E shows that R-flurbiprofen has no effect on apoptosis at 0.1μM. However, at 1μM R-flurbiprofen, apoptosis becomes apparent, and at larger concentrations (>10μM), R-flurbiprofen induced apoptosis significantly and to an extent quite similar to the effects of Aβ₄₂ oligomers. Thus, R-flurbiprofen protects neurons against Aβ₄₂ oligomers at low concentrations while promoting apoptosis at large concentration.

DISCUSSION

Aging is the most important risk factor for AD. Aβ is the main component of amyloid plaques and small aggregates of Aβ₄₂ are involved critically in AD. However, how aging influences susceptibility to neurotoxicity induced by Aβ₄₂ oligomers is largely unknown. In this study we have investigated the acute effects of Aβ₄₂ oligomers on cell death and subcellular Ca²⁺ in an in vitro model of neuronal aging: the long-term culture of rat hippocampal neurons. We found that Aβ₄₂ oligomers at μM concentrations do not induce apoptosis in young neurons. In contrast, Aβ₄₂ oligomers promote apoptosis in mature neurons, and this effect increases further in long-term cultures of rat hippocampal neurons. These results open the question as to how aged cells acquire increased sensitivity to Aβ₄₂ oligomers. Consistently, we show that cytosolic and mitochondrial Ca²⁺ responses to Aβ₄₂ oligomers are missing in young neurons (2 DIV), becoming significant in mature neurons (8 DIV) and increasing dramatically in aged neurons (>13 DIV). These results strongly suggest that enhanced cytosolic and mitochondrial Ca²⁺ responses of aging neurons contribute to explain the increased susceptibility to neuron cell damage induced by Aβ₄₂ oligomers in aged neurons.

Importantly, the apoptotic response of aged neurons to Aβ oligomers depends on the mitochondrial Ca²⁺ overload rather than the cytosolic one. This view is supported by the fact that specific inhibition of mitochondrial Ca²⁺ uptake with mitochondrial uncouplers (FCCP) prevents apoptosis without preventing rises in cytosolic Ca²⁺. Consistently with the key role of mitochondrial Ca²⁺ overload, we show that Aβ₄₂ oligomers promote cytochrome c release only in aged neurons. Therefore, aged neurons are much more sensitive to Aβ₄₂ oligomers than young neurons because they underlie much larger Ca²⁺ loads in response to Aβ₄₂ oligomers than their young counterparts. We have reported recently that Ca²⁺ responses to NMDA are also increased in aged neurons in vitro and this effect is mediated by changes in NMDA receptor subunit expression similar to those found in vivo [12, 16]. Consistently, Ca²⁺ responses induced by NMDA also increased along culture time. Accordingly, age-associated changes in NMDA receptor subunit expression may contribute to the enhanced sensitivity to Aβ₄₂ oligomers and increased risk of AD with aging. Nevertheless, changes in expression of other death targets related to intracellular Ca²⁺ may also be involved in the enhanced sensitivity to Aβ oligomers and increased risk of AD with aging. For example changes in the activity and/or expression of the neuron specific isoform of Na,K ATPase α3 are also consisting with our results [24]. Triggering mechanisms and pathways for Ca²⁺ entry induced by Aβ₄₂ oligomers may include activation of NMDA receptors [25], α7 nicotinic acetylcholine receptors [26], metabotropic glutamate receptor 5 [27], and the above mentioned neuron specific Na,K ATPase α3 [24]. Interestingly, changes in expression of some of these receptors could be strongly influenced by aging in vivo and in vitro [12, 24], thus contributing to age-related changes in susceptibility to Aβ₄₂ oligomers and AD.

Regardless of the Ca²⁺ entry pathway activated by Aβ₄₂ oligomers in aged neurons, our results point to mitochondrial Ca²⁺ overload as key player in neuron cell death induced by Aβ₄₂ oligomers. As stated above, this view is supported by the effects of low concentrations of FCCP that prevent mitochondrial Ca²⁺ overload without affecting the rise in [Ca²⁺]_cyt and protect largely against apoptosis induced by Aβ₄₂ oligomers. Therefore, any compound that limits mitochondrial Ca²⁺ uptake may potentially protect against Aβ₄₂ oligomers. Compelling evidence indicates that different NSAIDs may protect against AD [28, 29]. Multiple mechanisms have been proposed for neuroprotection afforded by NSAIDs including inhibition of pro-inflammatory activity of surrounding glia, modulation of γ-secretase activity involved in Aβ processing and inhibition of mitochondrial Ca²⁺ overload [4 , 29]. In fact, we showed that NSAIDs and R-flurbiprofen inhibit mitochondrial Ca²⁺ overload and neuron cell death induced by Aβ₄₂ oligomers in cerebellar granule cells [4] and by NMDA in aged rat hippocampal neurons [12]. Unfortunately, a large phase III clinical trial using Tarenflurbil (R-flurbiprofen) did not slow cognitive decline or the loss of activities of daily living in patients with mild AD [1]. It has been argued that this failure may be related to the fact that anti Aβ activity could be decreased in aged individuals with well-developed AD. We show that NSAIDs including salicylate, sulindac sulphide, and R-flurbiprofen prevent mitochondrial Ca²⁺ overload induced by Ca²⁺ in permeabilized neurons and apoptosis induced by Aβ₄₂ oligomers, strongly suggesting that NSAIDs and R-flurbiprofen may protect against AD by preventing mitochondrial Ca²⁺ overload induced by Aβ₄₂ oligomers in aging neurons.

NSAIDs and R-flurbiprofen are considered mild mitochondrial uncouplers. Thus, at low concentrations, they depolarize partially mitochondria [4]. However, at large concentrations, such as those required for anti-inflammatory activity or for modulating γ secretase, they collapse the mitochondrial potential [4], thus compromising energy supply. This may be particularly true for aged neurons, including aged neurons in vitro that show a significant loss of mitochondrial potential compared to young neurons [12, 30]. Consistently, we found that large concentrations of R-flurbiprofen that may collapse the mitochondrial potential promoted apoptosis to almost the same extent than Aβ₄₂ oligomers. It is difficult to extent these results to the in vivo situation. However, while low concentrations (<1μM) of NSAIDs and R-flurbiprofen protect efficiently against mitochondrial calcium overload acting as partial mitochondrial uncouplers, at high concentrations (>10μM) NSAIDs and R-flurbiprofen may become toxic, particularly in the context of aging, where mitochondrial potential of neurons is compromised.

In summary, we show here that young neurons are permissive to Aβ₄₂ oligomers. However, when neurons age, they become sensitive to cell damage induced by Aβ₄₂ oligomers that promote Ca²⁺ entry, mitochondrial Ca²⁺ overload, cytochrome c release and neuronal apoptosis. Changes in Ca²⁺ responses could be triggered by age-associated changes in the expression of Aβ₄₂ oligomer death targets including probably NMDA receptors and neuron specific Na,K ATPase 3α. In addition, changes in subcellular Ca²⁺ handling related to aging may also contribute to enhanced sensitivity to oligomers. For instance, it has been reported that the loss of calcium buffering may contribute to selective neuronal vulnerability in AD [31]. Further research is required to ascertain more precisely the remodeling of subcellular Ca²⁺ in aging and its contribution to enhanced sensitivity to Aβ₄₂ oligomers and AD.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants VA145U13, BIO/VA33/13, BIO103/VA45/11 from Regional Government of Castilla y León Spain BFU2012-37146 from Ministry of Economy and Competitivity of Spain. María Calvo-Rodríguez was supported by a pre-doctoral fellowship from Regional Government of Castilla y León and the European Social Fund.

Authors’ disclosures available online ().

References

Green

, Schneider

, Amato

, Beelen

, Wilcock

, Swabb

, Zavitz

(2009) Effect of tarenflurbil on cognitive decline and activities of daily living in patients with mild Alzheimer disease: A randomized controlled trial. JAMA 302, 2557–2564.

Imbimbo

, Solfrizzi

, Panza

(2010) Are NSAIDs useful to treat Alzheimer’s disease or mild cognitive impairment? Front Aging Neurosci 2, 19.

De Felice

, Velasco

, Lambert

, Viola

, Fernandez

, Ferreira

, Klein

(2007) Aβ oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. J Biol Chem 282, 11590–11601.

Sanz-Blasco

, Valero

, Rodriguez-Crespo

, Villalobos

, Nunez

(2008) Mitochondrial Ca²⁺ overload underlies Aβ oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs. PLoS One 3, e2718.

Calvo-Rodriguez

, Nunez

, Villalobos

(2015) Non-steroidal anti-inflammatory drugs (NSAIDs) and neuroprotection in the elderly: A view from the mitochondria. Neural Regen Res 10, 1371–1372.

Berridge

(2010) Calcium hypothesis of Alzheimer’s disease. Pflugers Arch 459, 441–449.

Walsh

, Klyubin

, Fadeeva

, Cullen

, Anwyl

, Wolfe

, Rowan

, Selkoe

(2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535–539.

Klyubin

, Walsh

, Lemere

, Cullen

, Shankar

, Betts

, Spooner

, Jiang

, Anwyl

, Selkoe

, Rowan

(2005) Amyloid beta protein immunotherapy neutralizes Abeta oligomers that disrupt synaptic plasticity in vivo. Nat Med 11, 556–561.

Kuchibhotla

, Goldman

, Lattarulo

, Wu

, Hyman

, Bacskai

(2008) Aβ plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks. Neuron 59, 214–225.

10.

Caballero

, Calvo-Rodriguez

, Gonzalo-Ruiz

, Villalobos

, Nuñez

(2016) A new procedure for amyloid beta oligomers preparation enables the unambiguous testing of their effects on cytosolic and mitochondrial Ca²⁺ entry and cell death in primary neurons. Neurosci Lett 612, 66–73.

11.

Zempel

, Thies

, Mandelkow

(2010) Aβ oligomers cause localized Ca²⁺ elevation, missorting of endogenous Tau into dendrites, Tau phosphorylation, and destruction of microtubules and spines. J Neurosci 30, 11938–11950.

12.

Calvo

, Sanz-Blasco

, Caballero

, Villalobos

, Nunez

(2015) Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non-steroidal anti-inflammatory drugs: Role of mitochondrial calcium. J Neurochem 132, 403–417.

13.

Calvo-Rodriguez

, Villalobos

, Nuñez

(2015) Fluorescence and bioluminescence imaging of subcellular Ca2+ in aged hippocampal neurons. J Vis Exp Dec 1, 106 doi: 10.3791/53330.

14.

Porter

, Thibault

, Chen

, Landfield

(1997) Calcium channel density and hippocampal cell death with age in long-term culture. J Neurosci 17, 5629–5639.

15.

Sodero

, Weissmann

, Ledesma

, Dotti

(2011) Cellular stress from excitatory neurotransmission contributes to cholesterol loss in hippocampal neurons aging in vitro. Neurobiol Aging 32, 1043–1053.

16.

Cui

, Feng

, Jacobs

, Duan

, Wang

, Cao

, Tsien

(2013) Increased NR2A:NR2B ratio compresses long-term depression range and constrains long-term memory. Sci Rep 3, 1036.

17.

Kaur

, Sharma

, Singh

(2001) Acetyl-L-carnitine enhances Na⁺,K⁺-ATPase glutathione-S-transferase and multiple unit activity and reduces lipid peroxidation and lipofuscin concentration in aged rat brain regions. Neurosci Lett 301, 1–4.

18.

Brewer

, Torricelli

, Evege

, Price

(1993) Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35, 567–576.

19.

Perez-Otaño

, Lujan

, Tavalin

, Plomann

, Modregger

, Liu

, Jones

, Heinemann

, Lo

, Ehlers

(2006) Endocytosis and synaptic removal of NR3A-containing NMDA receptors by PACSIN1/syndapin1. Nat Neurosci 9, 611–621.

20.

Rogers

, Stinnakre

, Agulhon

, Jublot

, Shorte

, Kremer

, Brulet

(2005) Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters. Eur J Neurosci 21, 597–610.

21.

Montero

, Alonso

, Carnicero

, Cuchillo-Ibanez

, Albillos

, Garcia

, Garcia-Sancho

, Alvarez

(2000) Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca²⁺ transients that modulate secretion. Nat Cell Biol 2, 57–61.

22.

Villalobos

, Caballero

, Sanz-Blasco

, Nunez

(2012) Study of neurotoxic intracellular calcium signalling triggered by amyloids. Methods Mol Biol 849, 289–302.

23.

Nuñez

, Senovilla

, Sanz-Blasco

, Chamero

, Alonso

, Villalobos

, Garcia-Sancho

(2007) Bioluminescence imaging of mitochondrial Ca²⁺ dynamics in soma and neurites of individual adult mouse sympathetic neurons. J Physiol 580, 385–395.

24.

Ohnishi

, Yanazawa

, Sasahara

, Kitamura

, Hiroaki

, Fukazawa

, Kii

, Nishiyama

, Kakita

, Takeda

, Takeuchi

, Arai

, Ito

, Komura

, Hirao

, Satomura

, Inoue

, Muramatsu

, Matsui

, Tada

, Sato

, Saijo

, Shigemitsu

, Sakai

, Umetsu

, Goda

, Takino

, Takahashi

, Hagiwara

, Sawasaki

, Iwasaki

, Nakamura

, Nabeshima

, Teplow

, Hoshi

(2015) Na, K-ATPase alpha3 is a death target of Alzheimer patient amyloid-β assembly. Proc Natl Acad Sci U S A 112, E4465–E4474.

25.

Texidó

, Martín-Satué

, Alberdi

, Solsona

, Matute

(2011) Amyloid β peptide oligomers directly activate NMDA receptors. Cell Calcium 49, 184–190.

26.

, Lorke

, Yang

, Petroianu

(2013) On the interaction of β-amyloid peptides and α7-nicotinic acetylcholine receptors in Alzheimer’s disease. Curr Alzheimer Res 10, 618–630.

27.

Hamilton

, Esseltine

, DeVries

, Cregan

, Ferguson

(2014) Metabotropic glutamate receptor 5 knockout reduces cognitive impairment and pathogenesis in a mouse model of Alzheimer’s disease. Mol Brain 7, 40.

28.

Heneka

, Kummer

, Weggen

, Bulic

, Multhaup

, Münter

, Hüll

, Pflanzner

, Pietrzik

(2011) Molecular mechanisms and therapeutic application of NSAIDs and derived compounds in Alzheimer’s disease. Curr Alzheimer Res 8, 115–131.

29.

Wang

, Tan

, Wang

, Tan

, Meng

, Wang

, Tang

, Yu

(2015) Anti-inflammatory drugs and risk of Alzheimer’s disease: An updated systematic review and meta-analysis. J Alzheimers Dis 44, 385–396.

30.

Dong

, Cheng

, Huang

, Fan

, Chen

, Shi

, He

(2011) Mitochondrial dysfunction in long-term neuronal cultures mimics changes with aging. Med Sci Monit 17, BR91–BR96.

31.

Riascos

, de Leon

, Baker-Nigh

, Nicholas

, Yukhananov

, Bu

, Wu

, Geula

(2011) Age-related loss of calcium buffering and selective neuronal vulnerability in Alzheimer’s disease. Acta Neuropathol 122, 565–576.

Aging Enables Ca 2+ Overload and Apoptosis Induced by Amyloid-β Oligomers in Rat Hippocampal Neurons: Neuroprotection by Non-Steroidal Anti-Inflammatory Drugs and R-Flurbiprofen in Aging Neurons

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Reagents

Primary hippocampal neuron culture

Preparation of Aβ42 oligomers

Fluorescence imaging of cytosolic Ca2+ concentration ([Ca2+] cyt ) and in situ immunofluorescence

Bioluminescence imaging of mitochondrial Ca2+ concentration ([Ca2+] mit )

Apoptosis measurements

Measurements of cytochrome c release

Statistics

RESULTS

Apoptosis induced by Aβ42 oligomers increases dramatically with culture age

Cytosolic Ca2+ responses to Aβ42 oligomers are increased in aged neurons

Mitochondrial Ca2+ responses to Aβ42 oligomers are increased in aged neurons

Mitochondrial Ca2+ uptake contributes to apoptosis induced by Aβ42 oligomers in aged neurons

NSAIDs and R-flurbiprofen inhibit mitochondrial Ca2+ uptake induced by Aβ42 oligomers in aged hippocampal neurons

NSAIDs and R-flurbiprofen prevent apoptosis induced by Aβ42 oligomers in aged hippocampal neurons

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Preparation of Aβ₄₂ oligomers

Fluorescence imaging of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and in situ immunofluorescence

Bioluminescence imaging of mitochondrial Ca²⁺ concentration ([Ca²⁺]_mit)

Apoptosis induced by Aβ₄₂ oligomers increases dramatically with culture age

Cytosolic Ca²⁺ responses to Aβ₄₂ oligomers are increased in aged neurons

Mitochondrial Ca²⁺ responses to Aβ₄₂ oligomers are increased in aged neurons

Mitochondrial Ca²⁺ uptake contributes to apoptosis induced by Aβ₄₂ oligomers in aged neurons

NSAIDs and R-flurbiprofen inhibit mitochondrial Ca²⁺ uptake induced by Aβ₄₂ oligomers in aged hippocampal neurons

NSAIDs and R-flurbiprofen prevent apoptosis induced by Aβ₄₂ oligomers in aged hippocampal neurons