Presenilin-1 Mutations Increase Levels of Ryanodine Receptors and Calcium Release in PC12 Cells and Cortical Neurons*

Many cases of early-onset inherited Alzheimer’s disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. PS1 mutations may perturb cellular Ca 2 1 homeostasis and thereby render neurons vulnerable to excitotoxicity and apoptosis. We now report that PC12 cells expressing PS1 mutations and primary hippocampal neurons from PS1 mutant knockin mice exhibit greatly increased levels of ryanodine receptors (RyR) and enhanced Ca 2 1 release following stimulation with caffeine. Double-labeling immunostaining and co-immu-noprecipitation analyses indicate that PS1 and RyR are colocalized and interact physically. Caffeine treatment sensitizes neurons expressing mutant PS1 to apoptosis induced by amyloid b -peptide, a neurotic peptide linked to the pathogenesis of AD. When taken together with recent evidence for alterations in RyR in brains of AD patients, our data suggest that PS1 mutations may promote neuronal degeneration in AD by increasing tran-scription and translation of RyR and altering functional properties of ryanodine-sensitive Ca 2 1 pools.

Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. PS1 mutations may perturb cellular Ca 2؉ homeostasis and thereby render neurons vulnerable to excitotoxicity and apoptosis. We now report that PC12 cells expressing PS1 mutations and primary hippocampal neurons from PS1 mutant knockin mice exhibit greatly increased levels of ryanodine receptors (RyR) and enhanced Ca 2؉ release following stimulation with caffeine. Double-labeling immunostaining and co-immunoprecipitation analyses indicate that PS1 and RyR are colocalized and interact physically. Caffeine treatment sensitizes neurons expressing mutant PS1 to apoptosis induced by amyloid ␤-peptide, a neurotic peptide linked to the pathogenesis of AD. When taken together with recent evidence for alterations in RyR in brains of AD patients, our data suggest that PS1 mutations may promote neuronal degeneration in AD by increasing transcription and translation of RyR and altering functional properties of ryanodine-sensitive Ca 2؉ pools.
Alzheimer's disease (AD) 1 is an age-related neurodegenerative disorder that is a leading cause of death and disability (1). Although the mechanisms of neuronal degeneration in AD are not clear, they appear to involve increased oxidative stress and disruption of cellular calcium homeostasis (2). Whereas most cases of AD are not caused by a specific genetic defect and have a late age of onset, some cases are characterized by an early age of onset and a dominant inheritance pattern. Mutations in the gene encoding presenilin-1 (PS1) on chromosome 14 are responsible for many such cases of inherited AD (3,4). PS1 is an integral membrane protein that is expressed in neurons throughout the brain wherein it is localized primarily in the endoplasmic reticulum (ER). Two pathogenic mechanisms for PS1 mutations have been proposed. One mechanism involves altered proteolytic processing of the amyloid precursor protein, resulting in increased production of neurotoxic forms of amy-loid ␤-peptide (A) and decreased levels of the neuroprotective secreted form of amyloid precursor protein (5)(6)(7)(8)(9)(10). A second mechanism involves perturbed Ca 2ϩ regulation, which results in enhanced elevations of intracellular Ca 2ϩ levels under conditions of oxidative and excitotoxic stress (11)(12)(13)(14). Hippocampal neurons from PS1 mutant knockin mice exhibit increased vulnerability to excitotoxicity, which is associated with enhanced elevations of intracellular Ca 2ϩ levels (13). Moreover, Ca 2ϩ imaging analyses (15) and electrophysiological studies (16) reveal excessive synaptic Ca 2ϩ responses in transgenic mice expressing mutant PS1.
ER contains two main types of Ca 2ϩ release channels, the inositol 1,4,5-trisphosphate receptors (IP 3 R, ϳ300 kDa) and the ryanodine receptors (RyR,ϳ565 kDa), each represented by three different isoforms with similar structural properties (17). These tetrameric channels display distinct but overlapping tissue distribution and may co-exist in the same cell in which they are modulated by different second messengers. IP 3 Rs (types I, II, and III) are activated upon binding of inositol 1,4,5-trisphosphate, generated by phospholipase C-mediated polyphosphoinostide breakdown following cell surface receptor activation. In neuronal cells, RyR are activated via the classical Ca 2ϩ -induced Ca 2ϩ release mechanism following Ca 2ϩ entry across the plasma membrane through voltage-operated Ca 2ϩ channels. Three RyR subtypes, encoded by different genes, were originally identified in skeletal muscle (type 1 RyR), heart (type 2 RyR), and brain (type 3 RyR). Within the brain, levels of IP 3 R are highest in cerebellar Purkinje cells and CA1 hippocampal neurons, whereas RyRs are present at particularly high levels in pyramidal neurons in the hippocampus and cerebral cortex (18,19). IP 3 Rs and RyRs have been extensively studied for their roles in the generation of intracellular Ca 2ϩ oscillations and Ca 2ϩ waves that convey information required for many vital cellular functions (20). Recent reports implicate IP 3 R-and RyR-mediated Ca 2ϩ release from ER in apoptotic signaling and induction in lymphocytes (21)(22)(23). Ca 2ϩ release from ER can promote neuronal excitotoxicity and apoptosis, as indicated by the ability of blockers of ER Ca 2ϩ release to protect neurons against cell death induced by glutamate (24) and amyloid ␤-peptide (12). We therefore examined possible mechanisms by which FAD-linked mutations in PS1 disrupt cellular calcium homeostasis and endanger neurons. We show that levels of type 3 RyR mRNA and protein are increased in PC12 cell clones stably expressing mutant PS1 and in brain tissue from PS1 knockin mice expressing mutant PS1 at normal levels. The increased level of RyR is associated with enhanced Ca 2ϩ responses to caffeine and increased neuronal vulnerability to excitotoxicity and apoptosis.

PS1 Mutant Knockin Mice and Primary Neuronal Cell
Culture Methods-The targeting strategy used to generate PS1 mutant knockin mice is detailed elsewhere (13). Mice were maintained on a C57BL/6 ϫ 129/Sv background. Previous studies have characterized these mice, showing that the knockin mice express mutant PS1 at normal levels. PS1 mutant mice have no overt developmental abnormalities, but do exhibit increased levels of A␤1-42 in brain tissue and increased vulnerability of hippocampal neurons to apoptosis and excitotoxicity (13,14). Cultures of dissociated cortical cells were prepared from embryonic day 18 wild-type and homozygous PS1M146V knockin mouse pups using methods similar to those described previously (13). Briefly, cerebral cortices were removed and incubated for 15 min in Ca 2ϩ -and Mg 2ϩ -free Hanks' balanced saline solution (Life Technologies, Inc.) containing 0.2% trypsin. Cells were dissociated by trituration and plated into polyethyleneimine-coated plastic or glass-bottom culture dishes containing minimum essential medium with Earle's salts supplemented with 10% heat-inactivated fetal bovine serum, 2 mM Lglutamine, 1 mM pyruvate, 20 mM KCl, 10 mM sodium bicarbonate, and 1 mM Hepes (pH 7.2). Following cell attachment (3-6 h after plating), the culture medium was replaced with neurobasal medium with B27 supplements (Life Technologies, Inc.). Experiments were performed in 6 -8-day-old cultures; greater than 90% of the cells in the cultures were neurons, and the remaining cells were astroctyes as judged by cell morphology and immunostaining with antibodies against neurofilaments and glial fibrillary acidic protein.
PC12 Cell Clones-PC12 cell lines stably overexpressing human wild-type PS1 or mutant PS1 (L286V or M146V mutations), and a control clone transfected with empty vector were established using methods described in our previous studies (11,12). Cells were maintained at 37°C (5% CO 2 atmosphere) in RPMI medium supplemented 10% with heat-inactivated horse serum and 5% with heat-inactivated fetal bovine serum. For experiments, cells were subcultured into 35-or 60-mm polyethyleneimine-coated culture dishes.
Experimental Treatments and Quantification of Neuronal Survival-Amyloid ␤-peptide 25-35 (Bachem, Torrance, CA) was prepared as a 1 mM stock in water 2 h prior to use. Caffeine was prepared as a concentrated stock in Locke's buffer. The methods for quantification of neuron survival (apoptosis and necrosis) were described previously (25,26). For assessment of cell death by apoptosis, cells were fixed and stained with the fluorescent DNA-binding dye Hoechst 33342 (Molecular Probes, Eugene, OR). Neurons with condensed and fragmented nuclear DNA were considered apoptotic. For assessment of secondary necrosis, live cells were stained with trypan blue (Sigma); neurons that took up the dye were considered necrotic. For morphological evaluation of cell survival in primary hippocampal neurons, the same microscope fields of neurons were photographed prior to, and at designated time points following, exposure to treatments. Neurons with fragmented neurites and a crenated cell body were considered nonviable.
Total RNA Extraction and RT-PCR-Total RNA was purified from approximately 10 ϫ 10 6 cells from transfected PC12 cell lines or approximately 50 mg (wet weight) of hippocampus or cerebellum dissected from wild type and PS1M146V knockin mice using a SNAP isolation protocol (Invitrogen, CA). One microgram of total RNA was reversetranscribed into cDNA according to the procedures outlined by the manufacturer (First Strand Synthesis Kit; PharMingen, Mississauga, Ontario, Canada). Hot-start PCR amplification of 2 l of cDNA was performed using hot wax beads (Invitrogen) and specific primers for the RyR3 receptor (forward 5Ј-GGC GCT GCG GAA GAC CTG CAC-3Ј and reverse 5Ј-GCC GGG CCG AAG CAC TC-3Ј) that yielded a 699-base pair product or glyceraldehyde-3-phosphate dehydrogenase-specific primers that yielded a 343-base pair product (27). PCR conditions were 25 cycles at 95°C for denaturation (60 s), 53°C for annealing (60 s), and 72°C for extension (60 s). Linear amplification of PCR products was determined as described previously (28). Products were separated by agarose gel electrophoresis (1.3%), transferred to a positively charged nylon membrane under alkaline conditions, and probed using a randomly labeled [ 32 P]dCTP nested RyR3 PCR product generated from the RyR3 PCR product described above (forward 5Ј-AAT CCG CTC CCT CCT CAG TGT CAG-3Ј and reverse 5Ј-AAC GGC AGC AGC TAG CAA CCA TC-3Ј that yielded a 218-base pair product using identical PCR conditions outlined above) or human glyceraldehyde-3-phosphate dehydrogenase cDNA (28). Densitometric analyses of RT-PCR products were performed using NIH Image software (version 1.60).
Immunoprecipitation and Western Blot Analysis-Aliquots of cell lysates or brain homogenates containing 300 g of protein were incubated with rabbit anti-PS1 (12) or mouse monoclonal anti-RyR (Affinity Bioreagents) antibodies in immunoprecipitation buffer (150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 5 g/ml leupeptin, 5 g/ml aprotinin, 2 g/ml pepstatin A, 0.25 mM phenylmethylsulfonyl fluoride, 50 mM Tris, pH 7.6). Antigen-antibody complexes were precipitated with immobilized protein A (for anti-PS1 antibody) or G (for anti-RyR antibody), washed three times in immunoprecipitation buffer, and solubilized by heating in Laemmli buffer containing 2-mercaptoethanol at 100°C for 4 min. The solubilized proteins were separated by electrophoresis on a 4 -12% gradient SDS-polyacrylamide gel and then transferred to a nitrocellulose sheet. After blocking with 5% milk and a 1-h incubation in the presence of primary anti-PS1 and anti-RyR antibodies, the nitrocellulose sheet was further processed using horseradish peroxidaseconjugated secondary antibody and a chemiluminescence detection kit (Amersham Pharmacia Biotech). The PS1 antibody was an affinitypurified polyclonal antibody, which was raised against a synthetic peptide corresponding to the loop region (amino acids 331-345) of human PS1. This antibody has been shown in immunoblotting analysis to recognize both the full length wild-type and mutant PS1 proteins, as well as their N-and C-terminal derivatives (PS1-NTF and -CTF) in neural cell lysates (12).
Immunocytochemistry-Cells were fixed in 4% paraformaldehyde, membranes permeabilized by exposure for 5 min to 0.2% Triton X-100 in phosphate-buffered saline, and placed in blocking serum (5% horse or goat serum in phosphate-buffered saline). Cells were then exposed to primary antibodies (1:100 dilution of rabbit polyclonal PS1 antibody and 1:1000 dilution of RyR antibody) overnight at 4°C, followed by incubation for 1 h with a mixture of Texas Red-labeled anti-rabbit and fluorescein-labeled anti-mouse secondary antibodies (Vector). Images of immunofluorescence were acquired using a confocal laser scanning microscope (dual wavelength scan) with a 60ϫ oil immersion objective.
Anaglyphs showing sites of colocalization of immunoreactivities were generated using Imagespace software (Molecular Dynamics).
Measurement of Intracellular Free Calcium Levels-Intracellular free calcium levels ([Ca 2ϩ ] i ) were quantified by fluorescence ratio imaging of the Ca 2ϩ indicator dye fura-2 using methods described previously (13,25). Briefly, cells were loaded with the acetoxymethylester form of fura-2 (30-min incubation in the presence of 10 M fura-2) and imaged using a Zeiss AttoFluor system with a 40ϫ oil objective. The average [Ca 2ϩ ] i in individual neuronal cell bodies was determined from the ratio of the fluorescence emissions obtained using two different excitation wavelengths (334 and 380 nm). The system was calibrated with solutions containing either no Ca 2ϩ or a saturating level of Ca 2ϩ (1 mM) using the formula:

Levels of RyR Are Increased in PC12 Cells Overexpressing Mutant PS1 and in Brains of PS1 Mutant Knockin Mice-
Because PS1 is localized primarily in ER and ER Ca 2ϩ homeostasis is altered in PC12 cells overexpressing mutant PS1 (11), we sought to determine whether neurons expressing mutant PS1 exhibit alterations in levels of proteins that regulate ER Ca 2ϩ release. Levels of mRNA encoding the type 3 RyR, assessed using RT-PCR analysis, were increased 2-3-fold in PC12 cell clones overexpressing either the L286V mutation or the M146V mutation compared with clones overexpressing wild-type PS1 and untransfected and vector-transfected control clones (Fig. 1A). We next measured levels of RyR type 3 mRNA in tissue from hippocampus and cerebellum of PS1 mutant knockin and wild-type mice. Levels of type 3 RyR mRNA were increased 4-fold in hippocampus of PS1 mutant knockin mice compared with wild-type mice (Fig. 1B). A similar overall increase in RyR type 3 mRNA level was observed in cerebellar tissue from PS1 mutant mice, although there was considerable variability in levels of RyR3 mRNA among mice (Fig. 1B).
Western blot analysis showed that levels of RyR type 3 protein were increased 7-10-fold in PC12 cell clones overexpressing mutant PS1 compared with clones overexpressing wild-type PS1 and vector-transfected control clones (Fig. 1C). Similarly, RyR type 3 protein levels were increased approximately 5-8-fold in hippocampal tissue from PS1 mutant knockin mice compared with wild-type mice (Fig. 1C). As expected, levels of RyR were increased in microsomes from PS1 mutant knockin mice compared with microsomes from wildtype mice (Fig. 1C).
PS1 and RyR Are Colocalized and Directly Interact-Although PS1 has been localized to ER in several different cell types including neurons, it is not known whether PS1 and RyR are co-localized in the same ER pools. Double-labeling confocal analysis of PC12 cells ( Fig. 2A) and cultured cortical neurons (data not shown) using a polyclonal antibody against PS1 and a monoclonal antibody against the RyR revealed that essentially all detectable RyR-positive compartments were also PS1positive. However, PS1 immunoreactivity was not limited to RyR-containing ER, as considerable PS1 immunoreactivity was present elsewhere in the cells.
Co-immunoprecipitation studies were performed on homogenates of PC12 clones overexpressing mutant or wild-type PS1 and control clones, and on homogenates of brain tissue from wild-type and homozygous PS1 mutant mice. When immunoprecipitation was performed using a RyR antibody, three PS1 immunoreactive bands were detected on the immunoblot with molecular sizes consistent with full-length (46 kDa) and N-and C-terminal fragments of PS1 (Fig. 2B). The relative amounts of PS1 protein present in the RyR immunoprecipitates were greater in the PC12 cell clones overexpressing either wild-type or mutant PS1 compared with the control PC12 clones, and were not different in brain tissue from PS1 mutant mice and wild-type mice, suggesting that AD-linked mutations do not alter the interaction of PS1 with RyR. Immunoprecipitation of PC12 cell lysates using the PS1 antibody identified a high molecular mass protein (Ͼ500 kDa) that immunoreacted with the RyR antibody and, again, there was no obvious difference in samples from cells expressing wild-type and mutant PS1 (Fig. 2B).
Calcium Release from Caffeine-sensitive Stores Is Increased in PC12 Cells and Primary Neurons Expressing Mutant PS1-In order to determine the functional consequences of increased levels of RyR in neurons expressing mutant PS1, we measured intracellular Ca 2ϩ levels ([Ca 2ϩ ] i ) following exposure to caffeine, an agent that induces Ca 2ϩ release from ryanodine-sensitive stores, in PC12 cells and primary hippocampal neurons expressing mutant or wild-type PS1. The elevation of [Ca 2ϩ ] following exposure to caffeine was markedly increased in PC12 cells overexpressing either the L286V or M146V mutations (Fig. 3, A and  C). Calcium responses to caffeine were also significantly increased in PC12 clones overexpressing wild-type PS1, but the magnitude of the increase was less than in clones overexpressing mutant PS1. In order to determine whether PS1 mutations had similar effects on Ca 2ϩ release from caffeine-sensitive stores in primary neurons, we established primary cortical cultures from embryonic PS1 mutant knockin mice and wild-type mice. The Ca 2ϩ response to caffeine was significantly greater in cortical neurons expressing mutant PS1 compared with neurons expressing wild-type PS1 (Fig. 3, B and D). Because PS1 protein is expressed at normal levels in the PS1 mutant knockin mice (13,14), the present findings directly demonstrate that increased levels of RyR and enhanced Ca 2ϩ release are a consequence of PS1 mutations under physiological conditions. Vulnerability to Cell Death Induced by Caffeine and Amyloid ␤-Peptide Is Increased in PC12 Cells and Primary Neurons Expressing Mutant PS1-As reported previously (11), we found that B, PS1 was co-immunoprecipitated with RyR from PC-12 cell lysates and brain homogenates. Polyclonal rabbit ␣PS1 and monoclonal mouse ␣RyR antibodies were used for immunoprecipitation (IP) of PS1 (lanes 1-8) and RyR proteins (lanes 9 -11), and detected in Western blots (WB) using their respective antibodies. Samples included lysates from vector-transfected PC12 cells (lanes 1, 6, and 9), PC12 cells overexpressing wild-type PS1 (lanes 2, 7, and 10) or mutant (L286V) PS1 (lanes 3, 8, and 11 PC12 cells overexpressing mutant PS1 were more vulnerable to apoptosis induced by A␤25-35 compared with cells overexpressing wild-type PS1 and vector-transfected cells (Fig. 4A). Exposure of vector-transfected PC12 cells to 30 mM caffeine resulted in little or no apoptosis during 12-and 24-h exposure periods (Fig.  4A). In contrast, PC12 cells overexpressing mutant PS1, and to a lesser extent cells overexpressing wild-type PS1, exhibited greatly increased sensitivity to caffeine-induced apoptosis. Moreover, caffeine cotreatment greatly exacerbated A␤25-35-induced apoptosis, which was particularly pronounced in cells expressing mutant PS1 (Fig. 4A). As another measure of cell death, we exposed cultures to caffeine and A␤25-35 alone, or in combination, and then stained cells 24 h later (a time point when many apoptotic cells have undergone secondary necrosis) with trypan blue. We found that significantly more cells were unable to exclude the dye trypan blue in cultures of cells expressing mutant PS1 compared with vector-transfected control cells and cells overexpressing wild-type PS1 (Fig. 4B). We next exposed primary cortical neurons from wild-type and PS1 mutant knockin mice to caffeine, A␤25-35, or the combination of caffeine and A␤25-35. Neuron survival in each culture, assessed by morphological criteria, was quantified 2, 4, 8, and 12 h later. Additional cultures were stained with Hoechst dye at the 4-and 8-h post-treatment time points, and neurons with apoptotic nuclei were quantified. Neurons expressing mutant PS1 were significantly more vulnerable to apoptosis induced by caffeine alone, A␤25-35 alone, and the combination of caffeine plus A␤25-35 compared with wild-type neurons (Fig. 4, C and D). DISCUSSION We found that levels of type 3 RyR are greatly increased in PC12 cells overexpressing mutant human PS1, and in brain tissue in knockin mice that express mutant PS1 at normal levels. Calcium imaging studies showed that PC12 cells and cortical neurons expressing mutant PS1 exhibit increased calcium responses to caffeine compared with cells expressing wildtype PS1. These findings suggest that one consequence of PS1 mutations is to increase levels of RyR in neurons resulting in increased Ca 2ϩ release following cell stimulation. We had previously shown that cells expressing mutant PS1 also release more Ca 2ϩ in response to thapsigargin, an agent that should deplete essentially all ER Ca 2ϩ pools (11,12), suggesting that there may also be an increase in the total pool of Ca 2ϩ available for release. The enhanced release of Ca 2ϩ from caffeine-sensitive stores in PC12 cells and cortical neurons expressing mutant PS1 was associated with greatly increased vulnerability of the cells to A␤25-35-and caffeine-induced cell death. Increased levels of RyR may therefore explain previous findings showing that neurons expressing mutant PS1 are more vulnerable to excitotoxicity (13) and apoptosis (14). The ability of dantrolene, a drug known to block RyR, to protect cells expressing mutant PS1 against apoptosis (12), is consistent with a pivotal role for Ca 2ϩ release through RyR in the pathogenic mechanism of PS1 mutations. Consistent with the latter interpretation, previous studies have shown that dantrolene can protect cultured neurons against metabolic and excitotoxic insults (24). Administration of dantrolene to neonatal brain slice immediately following an ischemia-like insult significantly enhanced cellular recovery as indicated by reduced energy depletion and suppression of poly(A)DP-ribose polymerase activation (29). Wei and Perry (30) showed that intravenous administration of dantrolene to gerbils immediately following transient global forebrain ischemia resulted in a significant decrease in loss of CA1 hippocampal neurons. In the same model Zhang et al. (31) reported a significant decrease in damage to CA1 neurons in gerbils receiving an intraventricular bolus of dantrolene 30 min following reperfusion. Release of Ca 2ϩ through RyR may play a general role in apoptosis in many different cell types including non-neuronal cells. As evidence, caffeine sensitizes Chinese hamster cells to apoptosis induced by ultraviolet irradiation (32), and potentiates apoptosis of HeLa cells induced by the DNA-damaging agent etoposide (33).
Our co-immunoprecipitation studies using PS1 and RyR antibodies suggest that PS1 and RyR protein(s) directly interact. The colocalization of PS1 and RyR, documented in our double-label confocal analysis are consistent with PS1 and RyR being present in the same ER population. Although there were no obvious differences in the abilities of wild-type and mutant PS1 to bind to RyR protein, the immunoprecipitation-Western blot analyses employed in the present study did not allow a quantitative determination as to whether the PS1 mutation affects binding to RyR. Further work will be needed to determine whether wildtype and mutant PS1 differentially modulate RyR function or whether this interaction plays a role in increasing levels of RyR.
The possibility that alterations in RyR and calcium signaling similar to those documented in neurons from PS1 mutant mice may also occur in AD patients is suggested by the recent work of Kelliher and co-workers (34). They showed that levels of radiolabeled ryanodine binding were significantly increased in subiculum and region CA1 of hippocampus in brain sections from AD patients in cases with early stage neurofibrillary pathology compared with brain sections from neurologically normal age-matched control patients. On the other hand, levels of ryanodine binding were significantly decreased in subiculum and region CA1 of hippocampus from late stage AD patients. The latter findings therefore suggest that increases in levels of RyR may precede neuronal degeneration in vulnerable neuronal populations in AD. Neurodegenerative disorders other than AD may also involve perturbed regulation of ryanodine-sensitive Ca 2ϩ stores. Gaucher disease is a glyocosphingolipid lysosomal storage disorder caused by a deficit of glucocerebrosidase and is characterized by severe loss of neurons in the central nervous system (35). It was recently reported that treatment of cultured hippocampal neurons with an inhibitor of glucocerebrosidase results in an increase in the caffeine-sensitive ER Ca 2ϩ pool and increased Ca 2ϩ responses to glutamate (36).
The molecular mechanism by which mutations in PS1 increase RyR expression remains to be determined. One possibility is that increased reactive oxygen species and calcium, which are key consequences of PS1 mutation (12)(13)(14), may promote RyR expression. Biswas et al. (37) showed that various mitochondrial metabolic inhibitors induce RyR expression. Further studies will be needed to elucidate the regulatory mechanisms involved in the expression and function of RyR calcium release channel in neurons and how these can be therapeutically modulated in order to ameliorate the adverse action of mutant PS1 on calcium homeostasis.