Amyloid β oligomers increase ER-mitochondria Ca2+ cross talk in young hippocampal neurons and exacerbate aging-induced intracellular Ca2+ remodeling

ORIGINAL RESEARCH published: 08 February 2019 doi: 10.3389/fncel.2019.00022 Edited by: Francesco Moccia, University of Pavia, Italy Reviewed by: Cesar Cardenas, Universidad Mayor, Chile Cláudia Pereira, Universidade de Coimbra, Portugal *Correspondence: Maria Calvo-Rodriguez [email protected] †Present address: Maria Calvo-Rodriguez, Alzheimer Research Unit, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, United States Received: 20 August 2018 Accepted: 17 January 2019 Published: 08 February 2019 Citation: Calvo-Rodriguez M, Hernando-Perez E, Nuñez L and Villalobos C (2019) Amyloid β Oligomers Increase ER-Mitochondria Ca2+ Cross Talk in Young Hippocampal Neurons and Exacerbate Aging-Induced Intracellular Ca2+ Remodeling. Front. Cell. Neurosci. 13:22. doi: 10.3389/fncel.2019.00022 Amyloid βOligomers Increase ER-Mitochondria Ca2+ Cross Talk in Young Hippocampal Neurons and Exacerbate Aging-Induced Intracellular Ca2+ Remodeling Maria Calvo-Rodriguez1*†,Elena Hernando-Perez1,Lucia Nuñez1,2 and Carlos Villalobos1 1Instituto de Biología y Genética Molecular (IBGM), Consejo Superior de Investigaciones Científicas (CSIC) and Universidad de Valladolid, Valladolid, Spain, 2Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid, Valladolid, Spain Alzheimer’s disease (AD) is the most common neurodegenerative disorder and strongly associated to aging. AD has been related to excess of neurotoxic oligomers of amyloid β peptide (Aβo), loss of intracellular Ca2+homeostasis and mitochondrial damage. However, the intimate mechanisms underlying the pathology remain obscure. We have reported recently that long-term cultures of rat hippocampal neurons resembling aging neurons are prone to damage induced by Aβoligomers (Aβo) while short-term cultured cells resembling young neurons are not. In addition, we have also shown that aging neurons display critical changes in intracellular Ca2+homeostasis including increased Ca2+store content and Ca2+transfer from the endoplasmic reticulum (ER) to mitochondria. Aging also promotes the partial loss of store-operated Ca2+entry (SOCE), a Ca2+entry pathway involved in memory storage. Here, we have addressed whether Aβo treatment influences differentially intracellular Ca2+homeostasis in young and aged neurons. We found that Aβo exacerbate the remodeling of intracellular Ca2+induced by aging. Specifically, Aβo exacerbate the loss of SOCE observed in aged neurons. Aβo also exacerbate the increased resting cytosolic Ca2+concentration, Ca2+store content and Ca2+release as well as increased expression of the mitochondrial Ca2+ uniporter (MCU) observed in aging neurons. In contrast, Aβo elicit none of these effects in young neurons. Surprisingly, we found that Aβo increased the Ca2+transfer from ER to mitochondria in young neurons without having detrimental effects. Consistently, Aβo increased also colocalization of ER and mitochondria in both young and aged neurons. However, in aged neurons, Aβo suppressed Ca2+transfer from ER to mitochondria, decreased mitochondrial potential, enhanced reactive oxygen species (ROS) generation and promoted apoptosis. These results suggest that modulation of ER—mitochondria coupling in hippocampal neurons may be a novel physiological role of Aβo. However, excess of Aβo in the face of the remodeling of intracellular Ca2+homeostasis associated to aging may lead to loss of ER—mitochondrial coupling and AD. Keywords: calcium, Alzheimer’s disease, aging, endoplasmic reticulum, mitochondria, store-operated calcium entry, hippocampal neurons Frontiers in Cellular Neuroscience | www.frontiersin.org 1February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta INTRODUCTION Alzheimer’s disease (AD) is the most common form of dementia, accounting for 60%–80% of all dementia cases. Despite of all the efforts, the mechanisms underlying the pathology are still unknown. The two main hallmarks of AD are extracellular plaques composed of amyloid β(Aβ) peptide and intracellular neurofibrillary tangles composed of hyperphosphorylated and misfolded tau. Most AD cases are sporadic (late-onset AD) and affect elderly people, but some cases (around 5%) are inherited in an autosomal dominant fashion, caused by mutations in the genes that encode presenilin1 (PS1), PS2 and amyloid precursor protein (APP; early-onset AD or familial AD). One of the most accepted hypothesis of AD is the ‘‘amyloid cascade hypothesis,’’ which postulates that the deposition of the Aβis a central event to the pathology of AD (Hardy and Selkoe, 2002). This hypothesis considers that the pathology of AD is caused by an abnormal processing of the protein Aβ, occasioned by a reduction in its clearance or by an increase in its production. Most AD cases are sporadic, being aging the most important risk factor for the disease. During aging and, particularly, in neurodegenerative diseases, the systems that regulate intracellular Ca2+homeostasis are affected, leading to synaptic dysfunction, plasticity detriment and neuronal degeneration. Consequently, a wide amount of studies has shown that the disturbance of Ca2+homeostasis is associated with the neurotoxicity that occurs in AD (Mattson, 2007; Bezprozvanny, 2009; Berridge, 2010). The ‘‘Ca2+ hypothesis of AD’’ (Khachaturian, 1989) postulates that the activation of the amyloidogenic pathway induces a remodeling of the neuronal Ca2+signaling, disturbing the normal Ca2+ homeostasis and therefore, the mechanisms responsible for learning and memory. In addition, oxidative stress, alterations in mitochondrial energetic metabolism and protein aggregation, such as Aβin AD, affect negatively the intracellular Ca2+ homeostasis. Recent studies have also shown that impaired function of intracellular organelles such as the endoplasmic reticulum (ER) and mitochondria plays an important role in the regulation of Ca2+during aging and AD (Toescu and Verkhratsky, 2003). In this manner, PSs, located in the membrane of the ER, have been associated to familial AD. They are integral membrane proteins that form the catalytic core of the γ-secretase complex that cleaves APP. Mutations in PSs increase the production of Aβ1–42 form or reduce the production of Aβ1–40 (Borchelt et al., 1996; Lemere et al., 1996). In addition, PSs are believed to form low conductivity channels, being involved in the formation of the leak channels of the ER, releasing Ca2+in a passive manner from the ER to the cytoplasm (Tu et al., 2006). Mutations in PSs result in an increased release of Ca2+from the ER (Nelson et al., 2007), probably due to a loss of function of the leak channel. The other key player of intracellular Ca2+homeostasis, mitochondria, are dynamic organelles that generate ATP and contribute to many cellular functions. They play a main role in apoptotic signaling, lipid synthesis and buffering of intracellular Ca2+. Many reports have also proposed mitochondrial dysfunction in AD. In this manner, dysfunction of mitochondrial bioenergetics (Atamna and Frey, 2007; Yao et al., 2009), increased fission and decreased fusion (Wang et al., 2009; Santos et al., 2010), morphological changes (Hirai et al., 2001; DuBoff et al., 2013; Xie et al., 2013) and redistribution of mitochondria (Kopeikina et al., 2011) have been extensively reported. ER and mitochondria are connected through the mitochondria-associated ER membranes (MAMs). They play a central role in the processes that occur between ER and mitochondria, such as communication between the two organelles, which includes Ca2+transport. When the Ca2+ from the ER is released to the cytoplasm, part of this Ca2+ is taken up by mitochondria, acting as a buffer of Ca2+ (Szabadkai et al., 2003). If normal flux is affected, the amount of Ca2+taken up by mitochondria could be increased. If there is mitochondrial Ca2+overload, the mitochondrial permeability transition pore (PTPm) will open, mitochondrial membrane potential will collapse and proapoptotic factors such as cytochrome c will be released, activating the caspase pathway and triggering apoptosis. In addition, an excessive Ca2+increase will enhance the production of reactive oxygen species (ROS), that will also contribute to the opening of the PTPm (Berridge, 2010). Alteration of the correct MAM function has been previously shown in AD (Müller et al., 2018). In this manner, increased connectivity ER-mitochondria was found in human fibroblasts from patients with familiar AD mutations as well as in fibroblasts from patients with sporadic AD (Area-Gomez et al., 2012; Area-Gomez and Schon, 2017). Our group has also previously reported that Aβoligomers (Aβo), unlike the fibrils, promote the entry of Ca2+into the cell, causing mitochondrial Ca2+ overload and cell death by apoptosis (Sanz-Blasco et al., 2008; Calvo-Rodríguez et al., 2016a). Also, exposure of primary hippocampal neurons to conditioned media containing Aβo increased the contact points ER-mitochondria. In addition, these media also increased ER-mitochondria Ca2+transfer in neuroblastoma cells (Hedskog et al., 2013). However, the effects of oligomers in the context of aging has not been addressed. Moreover, this study has never been performed in primary neurons. We have previously described an intraorganellar Ca2+ remodeling with aging, involving ER-mitochondria cross-talk and loss of store-operated Ca2+entry (SOCE) in rat hippocampal neurons (Calvo-Rodríguez et al., 2016b). Most studies on mechanisms underlying the pathology of AD use transgenic animal models that develop the pathology at early ages (i.e., around 6 months of age), cells from AD patients that are therefore already diagnosed, or cell lines exposed to Aβ peptides. In consequence, the alterations in the intraorganellar Ca2+remodeling in AD in the context of aging have not been described, and the full spectrum of mechanisms through which Ca2+homeostasis is altered in AD in aging is still missing. Based on this, we decided to study whether the soluble oligomers of the most toxic peptide involved in AD, Aβ1–42 (Aβo), are able to alter: (1) resting levels of cytosolic free Ca2+concentration ([Ca2+]cyt); (2) SOCE; (3) the content and release of Ca2+from the internal stores, as well as; (4) the Frontiers in Cellular Neuroscience | www.frontiersin.org 2February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta Ca2+transfer from ER-to mitochondria; (5) the interaction ER-mitochondria; and (6) the expression of the mitochondrial Ca2+uniporter (MCU) and inositol trisphosphate receptors (IP3Rs) in young and aged hippocampal neurons. In order to model aging, we have employed an in vitro culture of hippocampal rat neurons maintained for several days in vitro (DIV). Primary rat hippocampal neurons in long-term culture have been previously used as a model of aging by our group and others (Porter et al., 1997; Brewer et al., 2007; Calvo-Rodríguez et al., 2016b, 2017). Importantly, some of the changes that occur in aging are mimicked by this in vitro model of aging, such as accumulation of ROS, lipofuscin granules, heterochromatic foci, activation of the Jun N-terminal protein kinase (pJNK) and p53/p21 pathways and changes in NMDA receptor expression (Sodero et al., 2011; Calvo et al., 2015a). By using fluorescence Ca2+imaging of fura-2 loaded cells, we have evaluated the change in the Ca2+content in the ER, SOCE, the Ca2+depletion from ER induced by different physiological agonists and the coupling ER-mitochondria, in young (4–8 DIV, short-term) and aged (15–21 DIV, long term) hippocampal neurons cultured in vitro and exposed to Aβo as a model of AD and in vitro aging. MATERIALS AND METHODS Animals and Reagents Wistar rat pups (newborn P0–1) were obtained from the Valladolid University animal facility. All animals were handled according to the ethical standard of Valladolid University under protocols approved by the animal housing facility and in accordance with the European Convention 123/Council of Europe and Directive 86/609/EEC. Fura2/AM and Rhod-5N are from Invitrogen (Barcelona, Spain). Fetal bovine serum (FBS) is from Lonza (Barcelona, Spain). Horse serum, neurobasal medium, HBSS medium, B27, L-glutamine and gentamycin are from Gibco (Barcelona, Spain). Papain solution is from Worthington (Lakewood, NJ, USA). The poly-D-lysine and annexin V are from BD (Madrid, Spain). DNase I and antibody against the MCU are from Sigma (Madrid, Spain). IP3R1 and IP3R2 primary antibodies are from Santa Cruz Biotechnology (Dallas, TX, USA). IP3R3 primary antibody is from BD Transduction Laboratories (Madrid, Spain). ER tracker, mitotracker, tetramethylrhodamine, methyl ester (TMRM) and CM-H2DCFDA are from ThermoFisher Scientific. Aβ1–42 peptide is from Bachem AG (Bubenforf, Switzerland). Other reagents and chemicals were obtained either from Sigma or Merck. Primary Hippocampal Neuron Culture Hippocampal neurons were prepared from Wistar rat pups under sterile conditions as reported by Brewer et al. (1993) with further modifications by Pérez-Otaño et al. (2006). Briefly, newborn rat pups were decapitated and, after brain removal, meninges were discarded and hippocampi were separated from cortex. Hippocampal tissue was cut in small pieces, transferred to papain solution (20 u./ml) and incubated at 37◦C for 30 min. After 15 min, DNase I (50 µl/ml) was added. Tissue pieces were washed with Neurobasal medium and cell suspension was obtained using a fire-polished pipette in Neurobasal medium supplemented with 10% FBS. Cells were centrifuged at 160 gfor 5 min and pellet was suspended in Neurobasal medium. Hippocampal cells were plated onto poly-D-lysinecoated, 12 mm diameter glass coverslips at 30 ×103cells/dish (plating density, 169 cells/mm2), and grown in Neurobasal media supplemented with L-glutamine (2 mM), gentamicin (1 µg/ml), 2% B27 and 10% FBS, maintained in a humidified 37◦C incubator with 5% CO2without further media exchange. Cells were cultured for 4–8 DIV for young cultures, and 15–21 DIV for aged cultures. Other details have been reported in detail elsewhere (Calvo et al., 2015a; Calvo-Rodríguez et al., 2015). Aβ1–42 Oligomers Preparation Aβ1–42 oligomers (Aβo) were prepared as previously reported by a new procedure (Caballero et al., 2016). Briefly, Aβ1–42 peptide was initially solved at 1 mM concentration in iced cold hexafluoroisopropanol (HFIP), and separated into aliquots in sterile microcentrifuge tubes. The solution was then incubated for 2 h at room temperature (RT) to allow monomerization of the peptide. HFIP was removed under vacuum in a speed vacuum (800 g×10 min at RT), and the peptide film was stored desiccated at −20◦C. For aggregation, the peptide was first suspended in dry DMSO at 5 mM concentration. For complete suspension of the peptide, it was subjected to ultrasounds for 10 min, aliquoted in propylene non-siliconized tubes, and stored at −20◦C until use. MEM (Gibco) medium supplemented with 0.5 mg/ml Fe2+, 0.5 mg/ml Cu2+and 0.5 mg/ml Zn2+was added to bring the peptide to a concentration of 80 µM. Finally, it was incubated at 37◦C for 24 h. For experiments, Aβ1–42 was solved in the culturing media to 1 µM final concentration. Composition of these oligomers has been characterized previously (Caballero et al., 2016; Calvo-Rodríguez et al., 2016b). Effects of Aβo on Apoptosis in Young and Aged Neurons For apoptosis, hippocampal neurons were exposed to either vehicle or Aβo 1 µM for 24 h. Cells were then washed with phosphate buffered saline (PBS) and apoptosis was evaluated using annexin V (1:20, 10 min) in annexin binding buffer 1×(in mM) NaCl, 140; CaCl2, 2.5; Hepes, 10 (pH 7.4) and assessed by fluorescence microscopy using a Nikon Eclipse TS100 microscope (objective 40×). Fluorescence Imaging of Cytosolic Free Ca2+ Concentration Hippocampal cells were cultured for 4–8 DIV (short-term) or 15–21 DIV (long-term) and exposed to either vehicle (supplemented MEM) or 1 µM Aβo for 24 h before the experiment. The day of the experiment, hippocampal cells were washed in standard external medium (SEM) containing (in mM) NaCl 145, KCl 5, CaCl21, MgCl21, glucose 10 and Hepes/NaOH 10 (pH 7.4). Then cells were incubated in the Frontiers in Cellular Neuroscience | www.frontiersin.org 3February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta same medium containing fura-2 (4 µM) for 60 min at RT in the dark. Then coverslips were placed on the perfusion chamber of a Zeiss Axiovert 100 TV, perfused continuously with the same pre-warmed medium at 37◦C and epi-illuminated alternately at 340 and 380 nm using a filter wheel. Light emitted at 520 nm was recorded every 1–5 s with a Hamamatsu ER camera (Hamamatsu Photonics France). Pixel by pixel ratios of consecutive frames were captured and cytosolic Ca2+concentration ([Ca2+]cyt) values from regions of interest (ROIs) corresponding to individual neurons were averaged and expressed as the ratio of fluorescence emission following excitation at 340 and 380 nm as reported in detail previously (Sanz-Blasco et al., 2008; Calvo et al., 2015b). For estimation of Ca2+increase, responses (calculated as area under curve, A.U.C.) were averaged from responsive neurons (selected by their morphology different from glial cells in the brightfield). Fraction was calculated by dividing responsive cells for the total cell number in the field, considering responsive cells the ones showing a change in the slope of the Ca2+recording after application of the stimulus. For measurements of SOCE, fura-2-loaded cells were treated with the sarcoplasmic and ER Ca2+ATPase (SERCA) pump blocker thapsigargin (1 µM) for 15 min in the same SEM devoid of extracellular Ca2+before imaging as reported elsewhere (Calvo-Rodríguez et al., 2016b). Then cells were subjected to fluorescence imaging and stimulated with 5 mM Ca2+to monitor the SOCE-dependent rise in [Ca2+]cyt. Recordings were made in the presence of TTX to prevent activation of voltage-gated Ca2+channels by connected neurons. For estimation of Ca2+store content, the rise in [Ca2+]cyt induced by low concentrations of the Ca2+ionophore ionomycin (400 nM) added in the absence of extracellular Ca2+was monitored as reported previously (Villalobos and García-Sancho, 1995). Fluorescence Imaging of Mitochondrial Free Ca2+ Concentration Hippocampal cells cultured for 4–8 DIV (short-term) or 15–21 DIV (long-term) were exposed to either vehicle or 1µM Aβo for 24 h before the experiment. The day of the experiment, hippocampal cells were washed in SEM and incubated in the same medium containing Rhod-5N AM (1 µM) for 30 min at RT in the dark and washed in SEM for additional 30 min. Then coverslips were placed on the perfusion chamber of a Zeiss Axiovert 100 TV, perfused continuously with the same pre-warmed medium at 37◦C and epi-illuminated in the red channel (551 nm). Light emitted at 576 nm was recorded every 5 s with a Hamamatsu ER camera (Hamamatsu Photonics France). Pixel by pixel ratios of consecutive frames were captured and mitochondrial Ca2+ concentration ([Ca2+]mit) values from ROIs corresponding to individual neurons were averaged and expressed as normalized fluorescence emission (F/F0). For estimation of Ca2+increase, responses (calculated as amplitude of the peak) were averaged from responsive neurons (selected by their morphology different from glial cells in the brightfield). Fraction was calculated by dividing responsive cells for the total cell number in the field, considering responsive cells the ones showing a change in the slope of the Ca2+recording after application of the stimulus. Immunofluorescence of the Mitochondrial Calcium Uniporter (MCU) and IP3 Receptors Hippocampal cells at different culture periods and exposed to either vehicle or 1 µM Aβo for 24 h were washed with phosphate buffered saline (PBS), fixed with p-formaldehyde 4% and incubated with antibodies against MCU (1:200) and IP3 receptors IP3R1 (1:50), IP3R2 (1:50) and IP3R3 (1:50) at 4◦C overnight. Immunopositive cells were revealed using Alexafluor 488-tagged antibodies (1:300). Optical density was measured in selected ROIs corresponding to individual neurons using ImageJ software. Further details have been reported elsewhere (CalvoRodríguez et al., 2016b). ER-Mitochondria Colocalization Cells in every condition were stained for 10 min with 200 nM MitoTracker green and ERTracker red and imaged directly using a confocal microscope. High resolution images of cells were recorded by using a Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). The Mitotracker and ER-tracker were excited with 488 and 543 laser lines, respectively, and emission was acquired with a charged CCD camera. The images were analyzed in LAS AF Lite software (Leica Microsystems, Mannheim, Germany). Background was subtracted from all images. Colocalization analysis for the two markers was performed on Z-stacks acquired with steps of 0.8 µm. For colocalization analysis, green and red channel images were acquired independently. Analysis were carried out on single-plane images using ImageJ plug-ins. Manders2 and Pearson’s coefficients were calculated applying the ImageJ Colocalization analysis plug-in. Mitochondrial Membrane Potential For mitochondrial membrane potential measurements, hippocampal neurons were exposed to either vehicle or Aβo 1 µM for 24 h. Then, cells were washed in SEM and loaded with the mitochondrial potential probe (TMRM, 10 nM) for 30 min at RT in the dark. Then coverslips containing cells were placed on a Zeiss Axiovert 100 TV inverted microscope and subjected to fluorescence imaging. Fluorescence images were captured with the rhodamine filter set with a Hamamatsu ER-Orca fluorescence camera as reported previously (Calvo-Rodríguez et al., 2016b). Generation of Reactive Oxygen Species (ROS) For ROS generation measurements, hippocampal neurons were exposed to either vehicle or Aβo 1 µM for 24 h. Then, cells were washed in SEM and loaded with the ROS probe 5-(and-6)- chloromethyl-20,70-dichlorodihydrofluorescein diacetate (CMH2DCFDA 2 µM) for 40 min at RT in the dark. Then coverslips containing cells were placed on a Zeiss Axiovert 100 TV inverted Frontiers in Cellular Neuroscience | www.frontiersin.org 4February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta microscope and subjected to fluorescence imaging. Fluorescence images were captured with the FITC filter set with a Hamamatsu ER-Orca fluorescence camera. Statistics Changes in fluorescence ratio are expressed as area under the curve (AUC) and maximum increase in ratio (∆ratio). Calculation of AUC and ∆ratios were performed using Origin Lab 7.0. Data are presented as mean ±SEM. When only two means were compared, Student’s t-test was used. For more than two groups, statistical significance of the data was assessed by one-way or multifactorial analysis of variance (ANOVA), depending on the number of factors considered. Differences were considered significant at p<0.05, where 0.05 is the signification level. RESULTS AβOligomers Induce Apoptosis Only in Aged Neurons We tested the effects of Aβ1–42 oligomers (Aβo) on the rate of apoptosis in rat hippocampal neurons cultured for 4–8 DIV representing young neurons, or in neurons cultured for 15–21 DIV that represent many characteristics of aged neurons as reported previously (Calvo-Rodríguez et al., 2016a). Short-term (4–8 DIV) and long-term (15–21 DIV) cultured hippocampal neurons were exposed for 24 h to 1 µM Aβo or to vehicle (supplemented MEM, see ‘‘Materials and Methods’’ section). This latter condition was taken as control group. FIGURE 1 | Amyloid βpeptide oligomers (Aβo) induce apoptosis only in aged neurons. Short-term (young) and long-term (aged) hippocampal cultures were exposed for 24 h Aβo (1 µM) or vehicle. Then, the relative abundance (percent, %) of apoptotic neurons was assessed by incubation with annexin V. Pictures show representative bright field cultures of short-term (A) and long-term cultures (B) of rat hippocampal neurons. Bars represent mean ±standard external medium (SEM) percentage of apoptotic cells per condition. n= 339 and 330 cells from three independent experiments for young neurons (A) and 452 and 417 cells from four independent experiments, respectively for aged neurons (B).∗p<0.05 compared to control group. Figure 1 shows that, in resting conditions, only less than 3% of the young neurons display apoptosis. Aβo do not increase the rate of apoptosis in these cultured cells. About 5% of aged neurons display apoptosis in basal conditions. In contrast, Aβo increase the percent of apoptotic cells by nearly four-fold. These results confirm our previous results reported elsewhere (Caballero et al., 2016; Calvo-Rodríguez et al., 2016a) and indicate that Aβo toxicity depends on the time in culture of rat hippocampal neurons. In our hands, cultured rat hippocampal cells display a mix of neurons and glial cells and their relative abundance changes with aging in culture mostly because glial cells proliferate while neuron cells do not. The fraction of neurons amounts 60% at 5 DIV, decreases to 23% at 14 DIV and decreases further to only 15% at 20 DIV. Therefore, for monitoring of effects of Aβo in neurons, it is convenient to carry out measurements at the single cell level in identified neurons. Fortunately, hippocampal neurons can be easily identified and distinguished from astrocytes or glial cells according to morphometric characteristics for the expert eye. Next, we addressed whether treatment of rat hippocampal neurons with Aβo promote differential changes in intracellular Ca2+homeostasis in young and aged neurons. AβOligomers Increase Resting [Ca2+]cyt and Synchronous Ca2+ Oscillations in Aged Neurons We have tested the effects of chronic treatment of Aβo on resting [Ca2+]cyt, a parameter previously reported to vary as hippocampal neurons age in culture (Calvo et al., 2015a). Short-term (4–8 DIV) and long-term (15–21 DIV) cultured hippocampal neurons were exposed for 24 h to 1 µM Aβo or to vehicle (supplemented MEM). The effects of treatments were assessed by calcium imaging of fura-2 loaded neurons. Neurons were clearly distinguished from surrounding glial cells by their morphometric characteristics as previously reported (CalvoRodríguez et al., 2016b). Figure 2 shows that short-term cultured, rat hippocampal neurons display spontaneous, synchronous [Ca2+]cyt oscillations. Notice that the average recording runs in parallel to the recordings of individual cells, indicating that oscillations are synchronous. These synchronous oscillations reflect synchronous synaptic activity and the formation of neural networks in vitro (Nunez et al., 1996; Calvo-Rodríguez et al., 2016b). Long-term cultured rat hippocampal neurons exposed to Aβo (1 µM, 24 h) display increased [Ca2+]cyt levels (resting level) when compared to the control neurons (Figure 2C). Moreover, treatment of long-term cultured hippocampal neurons with 1µM Aβo for 24 h increases significantly the synchronous and spontaneous [Ca2+]cyt oscillations compared to the control group measured as oscillations index (OI), a parameter representing both the frequency and amplitude of [Ca2+] oscillations (Figure 2D;Nunez et al., 1996). In contrast, exposure of short-term cultured neurons to 1 µM Aβo had a little or no effect on both parameters, resting [Ca2+]cyt and OI. These results suggest that prolonged exposure of hippocampal neurons in vitro to Aβo increases the excitability of the neuronal circuit, at the Frontiers in Cellular Neuroscience | www.frontiersin.org 5February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta FIGURE 2 | The exposure of aged hippocampal neurons to Aβo increases basal Ca2+and synchronous and spontaneous Ca2+oscillations. Hippocampal neurons cultured for different days in vitro (DIV), were exposed to vehicle or Aβo (1 µM, 24 h), and [Ca2+]cyt was monitored by fluorescence imaging using fura-2. (A,B) Traces show individual cells (gray lines) and mean ±SEM (black line) of the increase in [Ca2+]cyt prior to any stimulus in young (A) and aged (B) cultured hippocampal neurons exposed to either Aβo (1 µM) or vehicle. (C,D) Bars represent mean ±SEM of basal Ca2+(ratio F340/F380; C) and the oscillation index (D) for young and aged neurons, treated in absence (ctrl) of presence of the Aβo (Aβ1–42, hatched bars). n= 113, 97, 131 and 169 neurons from four to six independent experiments, respectively. ∗p<0.05 compared to control group. #p<0.05 compared to the young ones. level of basal Ca2+and spontaneous oscillations. This effect is statistically significant only in aged neurons and Aβo has no effect in this regard in young neurons. AβOligomers Dampen Further the Decreased Store Operated Ca2+ Entry (SOCE) in Aged Neurons We have previously shown that SOCE, the Ca2+entry pathway activated after depletion of intracellular Ca2+stores, decreases with in vitro aging (Calvo-Rodríguez et al., 2016b). This process has been associated to a downregulation of stromal interaction molecule 1 (Stim1) and Orai1, molecular players involved in SOCE in most cell types. Here, we assessed the effect of Aβo on SOCE in our model of in vitro aging. Short-term and long-term cultured hippocampal neurons were exposed to either vehicle (Control) or Aβo (1 µM, 24 h), and SOCE was monitored by fluorescence imaging of [Ca2+]cyt. For this end, cultured neurons were loaded with fura-2 and treated with Tg (1 µM) for 15 min in medium devoid of extracellular FIGURE 3 | Store operated Ca2+entry (SOCE) decreases in long-term cultured hippocampal neurons exposed to Aβo. Shortand long-term cultured hippocampal neurons were either exposed to vehicle (ctrl) or Aβo (1 µM, 24 h), and SOCE was monitored by fluorescence imaging of Ca2+. Neurons were incubated with fura-2 and treated with Tg (1 µM) for 15 min in medium devoid of Ca2+(0 Ca). Subsequently, Ca2+5 mM in presence of TTX (500 nM) was added to elicit an increase in the [Ca2+]cyt. La3+(100 µM) was added to block SOCE. (A–D) Pictures show typical fluorescence images of transmission (transm) and [Ca2+]cyt coded in pseudocolor of thapsigargin (Thap) and after addition of 5 Ca. Pseudocolor scale is shown at right. Bars represent 10 µm. Traces are representative recordings of fluorescence ratios of individual short-term (young, 4–8, A,C) and long-term (aged, 15–21, B,D) cultured neurons. (E) Bars correspond to the averaged SOCE (mean ±SEM). n= 65, 84, 35 and 45 individual cells studied in three and three independent experiments, respectively. ∗p<0.05 compared to control group. #p<0.05 compared to the young ones. Ca2+(0 Ca) in order to fully deplete the intracellular Ca2+ stores. Subsequently, medium containing Ca2+5 mM and TTX (500 nM) was added for 5 min to activate SOCE. TTX was added to block the electrical connectivity of cultured neurons. La3+(100 µM) was added later to block this pathway. Pictures in Figure 3 show typical bright field images (transm) and fluorescence images coded in pseudocolor representing [Ca2+]cyt before (basal) and after addition of 5 mM Ca2+(5 Ca). Representative recordings of fluorescence ratios of individual short-term (young, 4–8 DIV, Figures 3A,C) and long-term (aged, 15–21 DIV, Figures 3B,D) cultured neurons are also Frontiers in Cellular Neuroscience | www.frontiersin.org 6February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta shown. Figure 3E shows bars corresponding to the averaged SOCE calculated as ∆RatioF340/F380 (mean ±SEM). Data confirm our previous results showing that SOCE is reduced significantly in in vitro aged neurons relative to young neurons. In addition, they show that AβO decrease SOCE further but this effect is only statistically significant in aged neurons. AβOligomers Increases Further the Enhanced Ca2+ Store Content in Aged Neurons We have reported previously that in vitro aging increases Ca2+ store content in rat hippocampal neurons (Calvo-Rodríguez et al., 2016b). Ca2+store content was assessed after chronic exposure to Aβo in young and aged neurons. For this end, Ca2+ store content was tested using the Ca2+ionophore ionomycin added in medium devoid of external Ca2+. This procedure has been reported previously to estimate Ca2+store content in living cells (Villalobos and García-Sancho, 1995;CalvoRodríguez et al., 2016b). Ca2+store content was monitored by fluorescence imaging in short-term and long-term cultured hippocampal neurons, exposed to Aβo (1 µM, 24 h), and compared to the controls (exposed to the vehicle for dilution of Aβo, MEM). These experiments were always carried out in medium devoid of extracellular Ca2+(0 Ca), in order to avoid the entry of Ca2+from the external medium. The stimulation of hippocampal neurons with 400 nM ionomycin in Ca2+free medium (0 Ca; Figure 4) increased [Ca2+]cyt in all neurons studied (measured as AUC). Exposure of the short-term cultured cells to Aβo did not modify the increase in [Ca2+]cyt induced by ionomycin (Figures 4A,C). However, in the long-term cultured cells, chronic exposure to Aβo for 24 h increased significantly the content of the stores, as shown by the enhanced increase in the [Ca2+]cyt induced by ionomycin in 0 Ca medium (Figures 4B,D). These results suggest that the chronic treatment of cultured hippocampal neurons with Aβo increases further Ca2+store content exclusively in the long-term cultured neurons, an effect that could be related to the increase in the basal Ca2+levels that were appreciated in the aged neurons exposed to the Aβo, but that does not occur in the young cultured ones. AβOligomers Increase Further the Enhanced Release of Ca2+ Induced by Acetylcholine in Aged Neurons In order to understand the functional consequences of the increase in the levels of Ca2+in the intracellular stores, we then analyzed the release of Ca2+from the ER that is mobilized by different agonists. The release of Ca2+mediated by the IP3receptor was first evaluated using the neurotransmitter acetylcholine (ACh). In order to cancel the contribution of the entry of Ca2+and just focus on the release from the stores, all the experiments were carried out in the absence of free Ca2+in the extracellular medium (0 Ca). Application of ACh to cultured hippocampal neurons in 0 Ca produced an increase in the [Ca2+]cyt, as described previously (Calvo-Rodríguez et al., 2016b). Figure 5 shows representative recordings in short-term FIGURE 4 | The exposure of aged hippocampal neurons to Aβo increases Ca2+store content. Hippocampal neurons exposed to either Aβo (1 µM) or vehicle and loaded with fura-2 were stimulated with ionomycin (400 nm) in external medium devoid of Ca2+(0 Ca) and subjected to fluorescence imaging to estimate the content of Ca2+deposits. (A–D) Pictures show transmission (transm) and pseudocolor images of fluorescence ratios in basal conditions (basal) and after ionomycin (Ionom) stimulation in short-term (A,C, Young 4–8 DIV) and long-term (B,D, Aged 15–21 DIV) cultured hippocampal neurons not exposed (A,B, control) or exposed (C,D, Aβ1–42) to Aβo (1 µM, 24 h). Pseudocolor bars are shown at right. Bar represent 10 µm. Traces are representative recordings of the Ca2+release induced by ionomycin (400 nM) in medium devoid of Ca2+(0 Ca) in individual neurons subjected to fluorescence imaging. (E) Bars represent the average (mean ±SEM) in the increase of [Ca2+]cyt in response to ionomycin (measured as area under the curve, AUC) in short-term (young) and long-term (aged) hippocampal neurons exposed to either Aβo (1 µM, 24 h) or vehicle. n= 55, 42, 81 and 114 individual cells from three to six independent cultures, respectively. ∗p<0.05 compared to control group. #p<0.05 compared to the young ones. and long-term cultured hippocampal neurons exposed to either Aβo (1 µM, 24 h) or vehicle, and subjected to fluorescence Ca2+ imaging. It can be observed that the chronic treatment with Aβo does not affect the increase in the [Ca2+]cyt induced by ACh in short-term cultured hippocampal neurons (Figures 5A,C). In contrast, chronic exposure of long-term cultured hippocampal neurons to Aβo promoted a larger [Ca2+]cyt response to ACh Frontiers in Cellular Neuroscience | www.frontiersin.org 7February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta FIGURE 5 | The exposure of aged hippocampal neurons to Aβo increases Ca2+responses to acetylcholine (ACh) in rat hippocampal neurons. Hippocampal neurons incubated with vehicle or Aβo (1 µM, 24 h) and loaded with fura-2 were subjected to fluorescence imaging for monitoring the Ca2+ released by ACh in short-term and long-term cultured neurons. Cells were stimulated with ACh in Ca2+free medium. (A–D) Pictures show transmission images (transm) and pseudocolor images of fluorescence ratios taken before (Basal) and after ACh stimulation. Pseudocolor scale is shown at right. Bars correspond to 10 µm. Traces represent significant fluorescence recordings in single neurons of the Ca2+release induced by ACh (100 µM) in young and aged cultures not exposed (A,B, control) or exposed (C,D, Aβ1–42) to Aβo (1 µM, 24 h). (E–G) Bars represent the summary results for 102, 94, 39 and 59 cells from four independent cultures, respectively (mean ±SEM, AUC) of responsive cells (E), the fraction of responsive cells (F), and the product of both parameters (G).∗p<0.05 compared to control group. #p<0.05 compared to the young ones. when compared to control cells (Figures 5B,D). Figures 5E–G show the quantification of the response. Bars represent the mean value of the [Ca2+]cyt increase, represented as AUC (AUC ±SEM) of the Ca2+response (Figure 5E). The fraction of cells that presented an increase in the [Ca2+]cyt is also showed (Figure 5F). It can be observed that the fraction of cells that responded to the stimulation with ACh is higher in long-term cultured hippocampal neurons. In order to compare the response between young and aged neurons, a measure of global release of Ca2+involving both parameters (Ca2+released and fraction of responding cells) was calculated. For this end we multiplied the AUC of the cells that showed an increase in the [Ca2+]cyt by the fraction of cells that responded to the stimulus. Figure 5G shows the global difference in Ca2+release from the stores among the four populations. Notice that whereas aging increases the fraction of neurons responsive to ACh, treatment with Aβo enhances instead the release of Ca2+in each individual cell. Thus, aging increases the fraction of neurons responsive to ACh and Aβo enhances the rise in Ca2+elicited by ACh in aged neurons. No effect was observed in young neurons. AβOligomers Increase Further the Enhanced Release of Ca2+ Induced by Caffeine in Aged Neurons Next, we assessed the effects of Aβo on Ca2+release induced by activation of ryanodine receptors (RyRs) in short-term and long-term cultured neurons. Caffeine (Caff; 20 mM) was employed as an agonist of the RyRs. Caffeine is an agonist of the RyRs, which are sensitive to Ca2+-induced Ca2+released (CICR) processes. In this case, nominally free Ca2+(without adding EGTA) was used as external cellular medium to be able to activate CICR, while minimizing the effect of Ca2+ entry from the extracellular medium into the cytosol. To avoid high osmotic variation following the application of 20 mM Caff, 20 mM glucose was added to the extracellular medium prior to its addition, subsequently replaced by 20 mM Caff (Calvo-Rodríguez et al., 2016b). When hippocampal neurons were stimulated by caffeine in nominally free Ca2+, a rapid and transient increase in the [Ca2+]cyt could be observed (Figure 6). The bars shown in Figure 6E represent the mean ±SEM value of the increase in [Ca2+]cyt (quantified as AUC). ER Ca2+ mobilized by caffeine was similar in the young neurons (exposed or not exposed to 1 µM Aβo for 24 h) and in the non-exposed cultured-aged neurons. However, in cultured-aged neurons previously exposed to Aβo (1 µM, 24 h), the increase in [Ca2+]cyt was significantly higher than in the control group. Figure 6G shows the global increase (AUC Ca2+increase ×fraction) produced by stimulation with caffeine under these conditions. Therefore, Ca2+release induced by both IP3receptor and RyR agonists is further enhanced by Aβo treatment in aged neurons but not in young neurons. AβOligomers Enhance ER-Mitochondria Cross Talking in Young Neurons but They Promote Opposite Effects in Aged Neurons Next, we tested the effects of Aβo on ER-mitochondria crosstalk, i.e., Ca2+transfer from ER to mitochondria. For this end, we measured the increase in mitochondrial [Ca2+] ([Ca2+]mit) induced by Ca2+release elicited by ACh in medium devoid of Ca2+(0 Ca), in the same manner as for Figure 5. Short-term and long-term cultured hippocampal neurons were incubated either with vehicle (Control) or Aβo (1 µM, 24 h). Then cells were washed and preincubated with the mitochondrial Ca2+probe Rhod-5N, a fluorescent calcium probe that targets mitochondria (de la Fuente et al., 2012), and subjected to fluorescence imaging in single neurons. Neurons treated with either vehicle (control) or Aβo were perfused with the agonist ACh in medium devoid of Ca2+(0 Ca) to induce the release of Ca2+from the ER. Frontiers in Cellular Neuroscience | www.frontiersin.org 8February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta FIGURE 6 | The exposure of aged hippocampal neurons to Aβo increases Ca2+responses to caffeine in rat hippocampal neurons. Short-term (young, 4–8 DIV) and long-term (aged, 15–21 DIV) cultured hippocampal neurons were either exposed to vehicle or Aβo (1 µM, 24 h) and loaded with fura-2 and subjected to fluorescence imaging. Pictures show representative images of transmission (transm) and pseudocolor coded of basal and the maximum response of Ca2+to caffeine in young (A,C) and aged cells (B,D). Pseudocolor scale is shown at right. Bars correspond to 10 µm. Traces represent single-cell recordings of young and aged cells non-exposed (ctrl) or exposed (Aβ1–42) to Aβo (1 µM, 24 h) of the Ca2+response induced by caffeine 20 mM in medium nominal Ca2+.(E–G) Bars represent mean ±SEM of the response of [Ca2+]cyt to caffeine (expressed as AUC) of 56, 78, 94 and 105 cells from three to five independent cultures, respectively (E). Panel (F) shows the fraction of cells that responded. Panel (G) represents the overall result of the multiplication of both factors. ∗p<0.05 compared to control group. #p<0.05 compared to the young ones. Figure 7 shows bright field and Rhod-5N fluorescence images in basal conditions and after stimulation with ACh (100 µM) in short-term (4–8 DIV) and long-term (15–21 DIV) cultured hippocampal neurons. Representative recordings of [Ca2+]mit obtained in single cells before and after stimulation with ACh are also shown. Bars represent the quantification of the rises in [Ca2+]mit induced by ACh in 0 Ca (left), the fraction of responsive cells (middle) and the global response by taking into account both factors (right). As shown previously using mitochondria-targeted aequorin (Calvo-Rodríguez et al., 2016b), the rise in [Ca2+]mit induced by ACh is larger in aged neurons than in young neurons. Therefore, these results confirm our previous results and validate monitoring of [Ca2+]mit with Rhod5N. The larger rise in [Ca2+]mit in aged neurons is due to both the increase in mitochondrial Ca2+uptake in every cell (left panel) and the fraction of responsive cells (middle panel) corresponding to a large global ER-mitochondria cross talk in aged neurons relative to young neurons (right panel; Figure 7C). Interestingly, Aβo increased the rise in [Ca2+]mit induced by ACh in young neurons (left panel) and this effect was not due to changes in the fraction of responsive cells (middle panel). Therefore, Aβo increase ER-cross talk in young neurons (right panel, Figure 7C). It is noteworthy that this effect cannot be due to increased ER Ca2+content (Figure 4E) and is the only effect induced by Aβo that we have observed in young neurons. It is also remarkable that the effects of Aβo on the rise in [Ca2+]mit induced by ACh in young and aged neurons are opposite. Aβo increases Ca2+transfer from ER to mitochondria in young neurons and decreases it in aged neurons. In fact, Aβo nearly abolishes the rise in [Ca2+]mit induced by ACh in aged neurons and this effect is due to decreases in both the amount of Ca2+transferred per neuron and the fraction of neurons responsive to ACh. Taken together, these results show that Aβo significantly increases ER-mitochondria cross-talk in young neurons, but they rather abolish it in aged neurons. AβOligomers Enhance Further the Increased Expression of the Mitochondrial Ca2+ Uniporter in Aged Neurons Ca2+enters the mitochondria through the MCU, located into the inner mitochondrial membrane (Baughman et al., 2011; De Stefani et al., 2011). To address the opposite effects of Aβo on mitochondrial Ca2+uptake in young and aged neurons, we also tested the effects of Aβo on MCU expression. Short-term and long-term cultured neurons were incubated either with vehicle (ctrl) or Aβo (1 µM, 24 h). Then, immunostaining against MCU was evaluated by immunofluorescence. Figure 8 shows representative images of the fluorescence detection of MCU in short-term (young) and long-term (aged) cultured hippocampal neurons and the quantitative analysis of fluorescence intensity (optical density, arbitrary units) of the MCU. As reported previously (Calvo-Rodríguez et al., 2016b), we found that optical density of MCU immunofluorescence is enhanced in aged neurons relative to young neurons, thus confirming our previous results. In addition, we found that Aβo significantly increased the expression of MCU in the long-term cultured hippocampal neurons but not in young neurons. These results indicate that the opposite effects of Aβo on ER-mitochondria cross talking in young and aged neurons are not due to changes in expression of MCU. Aging Increases Expression of IP3 Receptors in Rat Hippocampal Neurons Ca2+release from intracellular stores takes place after activation of IP3receptors at the ER. In addition, IP3receptors are an ER component of the MAMs (Hedskog et al., 2013). Therefore, these receptors are critical to the coupling between ER and Frontiers in Cellular Neuroscience | www.frontiersin.org 9February 2019 | Volume 13 | Article 22 Calvo-Rodriguez et al. ER-Mitochondria Calcium Cross-Talk and Amyloid Beta molecular players involved in Ca2+transfer increases, thus leading to mitochondrial damage and apoptosis. It has been reported that MAM-associated proteins are up-regulated in AD brain and APP Swe/Lon mouse models (Hedskog et al., 2013), and this effect is mirrored by nanomolar concentrations of Aβ-peptide. Changes in MAM associated proteins in AD could be a rescue response to dysfunctional ER-mitochondria coupling. However, in the face of increased Ca2+store content associated to age and/or AD-related mutations, the further rise in ER-mitochondria coupling likely induced by Aβo may somehow collapse the own coupling leading to neuron damage. Further research is required to address this critical question. In addition, all the above results have been obtained in identified neurons in an in vitro model of neuronal aging. As stated above, the fraction of glial cells in hippocampal cultures increases with in vitro aging. Whether glial cell may contribute to Ca2+ remodeling in aged neurons treated with Aβo remains to be established. Taken together, our data show that intraorganellar Ca2+ remodeling is a hallmark of the aging that is exacerbated in the presence of Aβo. Aging decreases SOCE required for spine stability and increases Ca2+store content and MCU and IP3 receptors expression that favors Ca2+transfer from ER to mitochondria. Most of these effects are exacerbated by Aβo but only in aged neurons. In contrast, in young neurons Aβo increase ER-mitochondrial colocalization and Ca2+transfer from ER to mitochondria without having effects on ROS generation or apoptosis and only a minor decrease in mitochondrial membrane potential, probably related to increased use of this potential for ATP synthesis. However, the effect of Aβo may become highly detrimental in the aged neuron and the mechanism for this switch may play a fundamental role in AD and warrants further consideration. AUTHOR CONTRIBUTIONS MC-R, LN and CV designed the study and wrote the manuscript. MC-R and EH-P performed research and analyzed data. FUNDING This work was supported by grants BFU2012-37146 and BFU2015-70131R from Ministry of Economy and Competitivity (Spain) and grants VA145U13, BIO/VA33/13 and VA294P18 from Regional Government of Castilla y León (Spain). MCR was supported by a pre-doctoral fellowship from Regional Government of Castilla y León (Spain) and the European Social Fund. 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