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J. Biol. Chem., Vol. 275, Issue 24, 18195-18200, June 16, 2000

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

Sic L. Chan^§¶, Michael Mayne^¶, Clark P. Holden^¶, Jonathan D. Geiger^¶, and Mark P. Mattson^§**

From the  Sanders-Brown Research Center on Aging, University of Kentucky, Lexington, Kentucky 40536, the ^§ Laboratory of Neurosciences, NIA, National Institutes of Health, Baltimore, Maryland 21224, and the  Department of Pharmacology and Therapeutics, University of Manitoba Faculty of Medicine, Winnipeg, Manitoba, Canada

Received for publication, January 3, 2000, and in revised form, April 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ pools.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 amyloid -peptide (A) and decreased levels of the neuroprotective secreted form of amyloid precursor protein (5-10). A second mechanism involves perturbed Ca²⁺ regulation, which results in enhanced elevations of intracellular Ca²⁺ levels under conditions of oxidative and excitotoxic stress (11-14). Hippocampal neurons from PS1 mutant knockin mice exhibit increased vulnerability to excitotoxicity, which is associated with enhanced elevations of intracellular Ca²⁺ levels (13). Moreover, Ca²⁺ imaging analyses (15) and electrophysiological studies (16) reveal excessive synaptic Ca²⁺ responses in transgenic mice expressing mutant PS1.

ER contains two main types of Ca²⁺ release channels, the inositol 1,4,5-trisphosphate receptors (IP₃R, ~300 kDa) and the ryanodine receptors (RyR,~565 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. IP₃Rs (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 Ca²⁺-induced Ca²⁺ release mechanism following Ca²⁺ entry across the plasma membrane through voltage-operated Ca²⁺ 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 IP₃R 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). IP₃Rs and RyRs have been extensively studied for their roles in the generation of intracellular Ca²⁺ oscillations and Ca²⁺ waves that convey information required for many vital cellular functions (20). Recent reports implicate IP₃R- and RyR-mediated Ca²⁺ release from ER in apoptotic signaling and induction in lymphocytes (21-23). Ca²⁺ release from ER can promote neuronal excitotoxicity and apoptosis, as indicated by the ability of blockers of ER Ca²⁺ 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 Ca²⁺ responses to caffeine and increased neuronal vulnerability to excitotoxicity and apoptosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 × 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 A1-42 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 Ca²⁺- and Mg²⁺-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 L-glutamine, 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% CO₂ 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 × 10⁶ 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 reverse-transcribed into cDNA according to the procedures outlined by the manufacturer (First Strand Synthesis Kit; PharMingen, Mississauga, Ontario, Canada). Hot-start PCR amplification of 2 µl of cDNA was performed using hot wax beads (Invitrogen) and specific primers for the RyR3 receptor (forward 5'-GGC GCT GCG GAA GAC CTG CAC-3' and reverse 5'-GCC GGG CCG AAG CAC TC-3') 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 [³²P]dCTP nested RyR3 PCR product generated from the RyR3 PCR product described above (forward 5'-AAT CCG CTC CCT CCT CAG TGT CAG-3' and reverse 5'-AAC GGC AGC AGC TAG CAA CCA TC-3' 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 µg 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 µg/ml leupeptin, 5 µg/ml aprotinin, 2 µg/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 peroxidase-conjugated secondary antibody and a chemiluminescence detection kit (Amersham Pharmacia Biotech). The PS1 antibody was an affinity-purified 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 60× 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 ([Ca²⁺]_i) were quantified by fluorescence ratio imaging of the Ca²⁺ 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 µM fura-2) and imaged using a Zeiss AttoFluor system with a 40× oil objective. The average [Ca²⁺]_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 Ca²⁺ or a saturating level of Ca²⁺ (1 mM) using the formula: [Ca²⁺]_i = K_d[(R  R_min)/(R_max  R)](F_o/F_s).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 Ca²⁺ 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 Ca²⁺ 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).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Levels of RyR mRNA and protein are increased in PC12 cells and primary neurons expressing mutant PS1. A, Southern blot analysis showing levels of RyR3 RT-PCR products from PC12 cells overexpressing L286V and M146V mutant PS1 (PS1-MT), wild-type (PS1-WT), and untransfected and vector-transfected PC12 cells. Relative RyR3 RT-PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase RT-PCR levels. Values are the mean and S.E. of determinations made in samples from four to six cultures. ***, p < 0.001 compared with values for untransfected cells, vector-transfected cells, and cells overexpressing wild-type PS1 (ANOVA with Student-Neuman-Kuels post hoc tests). B, RyR3 mRNA levels in hippocampus and cerebellum of PS1M146V knockin and wild-type mice. Values are the mean and S.E. of determinations made in samples from six mice. ***, p < 0.001 compared with values for wild-type mice (ANOVA with Student-Neuman-Kuels post hoc tests). C, quantitative immunoblot analysis showing RyR protein levels in whole brain homogenates and isolated brain microsomes of wild-type (WT, lane 1 and 3) and mutant PS1 knockin mice (PS1M146V, lanes 2 and 4), and in crude lysates of vector-transfected PC-12 cells (lane 5) and several PC-12 clones overexpressing wild-type PS1 (lanes 6 and 7) or mutant PS1 (lanes 8-10). Similar results were obtained in a separate experiment.

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 wild-type 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 PS1-positive. However, PS1 immunoreactivity was not limited to RyR-containing ER, as considerable PS1 immunoreactivity was present elsewhere in the cells.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   PS1 and RyR proteins are colocalized and interact. A, confocal laser scanning micrograph showing a merged image (anaglyph) of PC12 cells double-labeled with PS1 and RyR antibodies. Red, PS1 immunoreactivity; green, RyR immunoreactivity; yellow, sites of colocalization. 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), and homogenates from hippocampi of wild-type (lane 4) and PS1 mutant (lane 5) mice. For size reference, lysates from PS1-transfected cells were immunoprecipitated with the PS1 antibody and immune complexes were probed with the same antibody (lanes 6-8). Similar results were obtained in two additional experiments. FL, full-length PS1; NTF, N-terminal fragment; CTF, C-terminal fragment.

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 (>500 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 Ca²⁺ levels ([Ca²⁺]_i) following exposure to caffeine, an agent that induces Ca²⁺ release from ryanodine-sensitive stores, in PC12 cells and primary hippocampal neurons expressing mutant or wild-type PS1. The elevation of [Ca²⁺] 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 Ca²⁺ release from caffeine-sensitive stores in primary neurons, we established primary cortical cultures from embryonic PS1 mutant knockin mice and wild-type mice. The Ca²⁺ 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 Ca²⁺ release are a consequence of PS1 mutations under physiological conditions.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Calcium responses to caffeine are significantly enhanced in PC-12 cells and primary neurons expressing mutant PS1. A, representative recordings showing changes of [Ca2+]i after addition of 20 mM caffeine (indicated by an arrow) in untransfected and vector-transfected PC12 cells, and in PC-12 cells stably overexpressing either wild-type PS1 (PS1-WT) or mutant PS1 (PS1-MT L286V and PS1-MT M146V). B, values for peak [Ca2+]i after stimulation with caffeine in the indicated PC12 cell clones. Values are the mean and S.E. of determinations made in 12 separate cultures (measurements made in at least 40 cells/culture). *, p < 0.01 compared with values for untransfected and vector-transfected cells, and p < 0.05 compared with values for cells expressing mutant PS1. ***, p < 0.001 compared with values for untransfected and vector-transfected cells. C, representative recordings showing changes of [Ca2+]i after addition of 20 mM caffeine (indicated by an arrow) in neurons from wild-type and homozygous PS1 mutant knockin mice. Each trace is the mean of 12-16 neurons. D, values for basal [Ca2+]i and peak [Ca2+]i after stimulation with caffeine in neurons from wild-type and PS1 mutant mice. Values are the mean and S.E. of determinations made in six to eight separate cultures (measurements made in at least 10 neurons/culture). *, p < 0.01 compared with base-line value and to peak value for neurons from wild-type mice (ANOVA with Scheffe post hoc tests).

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 PC12 cells overexpressing mutant PS1 were more vulnerable to apoptosis induced by A25-35 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 A25-35-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 A25-35 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).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   PC12 cells and primary hippocampal neurons expressing mutant PS1 exhibit increased vulnerability to death induced by caffeine and A25-35. A, cultures of the indicated PC12 cell clones were exposed to 10 µM A25-35 alone, 30 mM caffeine alone, or a combination of 10 µM A25-35 and 30 mM caffeine for 6 or 12 h. Cells were stained with Hoechst dye, and the percentages of cells exhibiting apoptotic nuclei in each culture were quantified. Values are the mean and S.D. of determinations made in four cultures (80-100 cells analyzed per culture). *, p < 0.05, and **, p < 0.001, compared with corresponding values for vector-transfected cells and cells overexpressing wild-type PS1. B, vector-transfected PC12 cells (VT) and PC12 cells overexpressing either wild-type (WT) or mutant (MT, L286V mutation) PS1 were exposed to 10 µM A25-35 alone, 30 mM caffeine alone, or a combination of 10 µM A25-35 and 30 mM caffeine for 24 h. Cells were stained with trypan blue, and the percentages of cells stained in each culture were quantified. Values are the mean and S.D. of determinations made in four cultures. **, p < 0.001 compared with corresponding values for vector-transfected cells and cells overexpressing wild-type PS1. C and D, primary neurons from wild-type and PS1 mutant knockin mice were exposed to 10 µM A25-35 alone, 30 mM caffeine alone, or a combination of 10 µM A25-35 and 30 mM caffeine and neuronal survival was quantified at the indicated time points. Values are the mean and S.D. of determinations made in four cultures. *, p < 0.05, and **, p < 0.01, compared with corresponding value for wild-type mice (ANOVA with Scheffe post hoc tests).

We next exposed primary cortical neurons from wild-type and PS1 mutant knockin mice to caffeine, A25-35, or the combination of caffeine and A25-35. 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, A25-35 alone, and the combination of caffeine plus A25-35 compared with wild-type neurons (Fig. 4, C and D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 wild-type PS1. These findings suggest that one consequence of PS1 mutations is to increase levels of RyR in neurons resulting in increased Ca²⁺ release following cell stimulation. We had previously shown that cells expressing mutant PS1 also release more Ca²⁺ in response to thapsigargin, an agent that should deplete essentially all ER Ca²⁺ pools (11, 12), suggesting that there may also be an increase in the total pool of Ca²⁺ available for release.

The enhanced release of Ca²⁺ from caffeine-sensitive stores in PC12 cells and cortical neurons expressing mutant PS1 was associated with greatly increased vulnerability of the cells to A25-35- 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 Ca²⁺ 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 Ca²⁺ 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 wild-type 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 Ca²⁺ 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 Ca²⁺ pool and increased Ca²⁺ 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 (12-14), 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.

	ACKNOWLEDGEMENTS

We thank W. Fu and H. Zhu for technical assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health NIA Grant PO1AG10836.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ These authors contributed equally to this work.

^** To whom all correspondence should be addressed: Laboratory of Neurosciences, GRC 4F01, NIA, National Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8462; Fax: 410-558-8465; E-mail: mattsonm@grc.nia.nih.gov.

Published, JBC Papers in Press, April 6, 2000, DOI 10.1074/jbc.M000040200

	ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; PS, presenilin; ER, endoplasmic reticulum; RyR, ryanodine receptor; IP₃R, inositol 1,4,5-trisphosphate receptor; PCR, polymerase chain reaction; RT, reverse transcription; ANOVA, analysis of variance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				G. A. Kyriazis, Z. Wei, M. Vandermey, D.-G. Jo, O. Xin, M. P. Mattson, and S. L. Chan Numb Endocytic Adapter Proteins Regulate the Transport and Processing of the Amyloid Precursor Protein in an Isoform-dependent Manner: IMPLICATIONS FOR ALZHEIMER DISEASE PATHOGENESIS J. Biol. Chem., September 12, 2008; 283(37): 25492 - 25502. [Abstract] [Full Text] [PDF]

				T. Wakabayashi and B. De Strooper Presenilins: Members of the {gamma}-Secretase Quartets, But Part-Time Soloists Too Physiology, August 1, 2008; 23(4): 194 - 204. [Abstract] [Full Text] [PDF]

				V. Dror, T. B. Kalynyak, Y. Bychkivska, M. H. Z. Frey, M. Tee, K. D. Jeffrey, V. Nguyen, D. S. Luciani, and J. D. Johnson Glucose and Endoplasmic Reticulum Calcium Channels Regulate HIF-1{beta} via Presenilin in Pancreatic {beta}-Cells J. Biol. Chem., April 11, 2008; 283(15): 9909 - 9916. [Abstract] [Full Text] [PDF]

				K. De Keersmaecker, I. Lahortiga, N. Mentens, C. Folens, L. Van Neste, S. Bekaert, P. Vandenberghe, M. D. Odero, P. Marynen, and J. Cools In vitro validation of {gamma}-secretase inhibitors alone or in combination with other anti-cancer drugs for the treatment of T-cell acute lymphoblastic leukemia Haematologica, April 1, 2008; 93(4): 533 - 542. [Abstract] [Full Text] [PDF]

				G. Liang, Q. Wang, Y. Li, B. Kang, M. F. Eckenhoff, R. G. Eckenhoff, and H. Wei A Presenilin-1 Mutation Renders Neurons Vulnerable to Isoflurane Toxicity Anesth. Analg., February 1, 2008; 106(2): 492 - 500. [Abstract] [Full Text] [PDF]

				G. E. Stutzmann The Pathogenesis of Alzheimers Disease Is It a Lifelong "Calciumopathy"? Neuroscientist, October 1, 2007; 13(5): 546 - 559. [Abstract] [PDF]

				R. Bull, J. P. Finkelstein, A. Humeres, M. I. Behrens, and C. Hidalgo Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex Am J Physiol Cell Physiol, July 1, 2007; 293(1): C162 - C171. [Abstract] [Full Text] [PDF]

				M. S Parihar and G. J. Brewer Mitoenergetic failure in Alzheimer disease Am J Physiol Cell Physiol, January 1, 2007; 292(1): C8 - C23. [Abstract] [Full Text] [PDF]

				C. Supnet, J. Grant, H. Kong, D. Westaway, and M. Mayne Amyloid-beta-(1-42) Increases Ryanodine Receptor-3 Expression and Function in Neurons of TgCRND8 Mice J. Biol. Chem., December 15, 2006; 281(50): 38440 - 38447. [Abstract] [Full Text] [PDF]

				G. E. Stutzmann, I. Smith, A. Caccamo, S. Oddo, F. M. LaFerla, and I. Parker Enhanced ryanodine receptor recruitment contributes to Ca2+ disruptions in young, adult, and aged Alzheimer's disease mice. J. Neurosci., May 10, 2006; 26(19): 5180 - 5189. [Abstract] [Full Text] [PDF]

				S. Camandola, R. G. Cutler, D. S. Gary, O. Milhavet, and M. P. Mattson Suppression of Calcium Release from Inositol 1,4,5-Trisphosphate-sensitive Stores Mediates the Anti-apoptotic Function of Nuclear Factor-{kappa}B J. Biol. Chem., June 10, 2005; 280(23): 22287 - 22296. [Abstract] [Full Text] [PDF]

				G. E. Stutzmann Calcium Dysregulation, IP3 Signaling, and Alzheimer's Disease Neuroscientist, April 1, 2005; 11(2): 110 - 115. [Abstract] [PDF]

				A. Verkhratsky Physiology and Pathophysiology of the Calcium Store in the Endoplasmic Reticulum of Neurons Physiol Rev, January 1, 2005; 85(1): 201 - 279. [Abstract] [Full Text] [PDF]

				R. A. Christensen, A. Shtifman, P. D. Allen, J. R. Lopez, and H. W. Querfurth Calcium Dyshomeostasis in {beta}-Amyloid and Tau-bearing Skeletal Myotubes J. Biol. Chem., December 17, 2004; 279(51): 53524 - 53532. [Abstract] [Full Text] [PDF]

				S. L. Chan, W. Fu, P. Zhang, A. Cheng, J. Lee, K. Kokame, and M. P. Mattson Herp Stabilizes Neuronal Ca2+ Homeostasis and Mitochondrial Function during Endoplasmic Reticulum Stress J. Biol. Chem., July 2, 2004; 279(27): 28733 - 28743. [Abstract] [Full Text] [PDF]

				B. O. Popescu, A. Cedazo-Minguez, E. Benedikz, T. Nishimura, B. Winblad, M. Ankarcrona, and R. F. Cowburn {gamma}-Secretase Activity of Presenilin 1 Regulates Acetylcholine Muscarinic Receptor-mediated Signal Transduction J. Biol. Chem., February 20, 2004; 279(8): 6455 - 6464. [Abstract] [Full Text] [PDF]

				K.-C. Suen, M.-S. Yu, K.-F. So, R. C.-C. Chang, and J. Hugon Upstream Signaling Pathways Leading to the Activation of Double-stranded RNA-dependent Serine/Threonine Protein Kinase in {beta}-Amyloid Peptide Neurotoxicity J. Biol. Chem., December 12, 2003; 278(50): 49819 - 49827. [Abstract] [Full Text] [PDF]

				C. H. George, G. V. Higgs, J. J. Mackrill, and F. A. Lai Dysregulated Ryanodine Receptors Mediate Cellular Toxicity: RESTORATION OF NORMAL PHENOTYPE BY FKBP12.6 J. Biol. Chem., August 1, 2003; 278(31): 28856 - 28864. [Abstract] [Full Text] [PDF]

				A. Cedazo-Minguez, B. O. Popescu, M. Ankarcrona, T. Nishimura, and R. F. Cowburn The Presenilin 1 Delta E9 Mutation Gives Enhanced Basal Phospholipase C Activity and a Resultant Increase in Intracellular Calcium Concentrations J. Biol. Chem., September 20, 2002; 277(39): 36646 - 36655. [Abstract] [Full Text] [PDF]