PS2 mutation increases neuronal cell vulnerability to ...

©2005 FASEB

The FASEB Journal express article 10.1096/fj.05-4017fje. Published online October 25, 2005.

PS2 mutation increases neuronal cell vulnerability to neurotoxicants through activation of caspase-3 by enhancing of ryanodine receptor-mediated calcium release Sun Young Lee,* Dae Youn Hwang,† Young Kyu Kim,† Jae Woong Lee,* Im Cheul Shin,* Ki Wan Oh,* Myung Koo Lee,* Jong Seok Lim,‡ Do Young Yoon,‡ Se Jin Hwang,§ Jin Tae Hong* *College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Korea; †National Institute of Toxicological Research, Korea Food and Drug Administration, Eunpyung-gu, Seoul, Korea; ‡Laboratory of Cell Biology, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, Korea; and §College of Medicine, Hanyang University, Seoungdong-gu, Seoul, Korea Corresponding author: Jin Tae Hong, College of Pharmacy, Chungbuk National University 48, Gaesin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Korea. E-mail: [email protected] ABSTRACT Increase of neuronal cell vulnerability by presenilin-2 (PS2) mutation by increase of caspase-3 activity through ryanodine receptor-mediated perturbation of calcium homeostasis was investigated. Stably transfected PC12 cells and cortical neurons from transgenic mice expressing mutant PS2 (N141I) showed a significantly enhanced sensitivity to L-glutamate, Aβ25–35, and Aβ1–42 (ADDLs form) compared with cells expressing wild-type PS2. Consistent with this result, much greater intracellular calcium levels and caspase-3 activity were found in PC12 cells and cortical neurons expressing mutant PS2 after treatment with L-glutamate, Aβ25–35, and Aβ1–42. Double-labeling confocal micrographic and coimmunoprecipitation analyses showed that ryanodine receptor type 3 (RyR) and PS2 colocalize in the endoplasmic reticulum (ER) in PC12 cells and in the brain of transgenic mice. The expression of RyR was much higher in the neurons of cells expressing mutant PS2. Moreover, pretreatment with dantrolene, an agent that blocks calcium release from RyR, protected against the mutant PS2-enhanced neuronal cell death and caspase-3 activity. The present data indicate that activation of caspase-3 by RyR-mediated increase of intracellular calcium levels may be an important neurotoxic mechanism in the neuronal cells expressing mutant PS2. Key words: presenilin-2 mutation • calcium homeostasis • cell death

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lzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive loss of memory and cognitive functions, and it is frequently characterized by massive neuronal and synaptic loss and the appearance of apoptotic cells (1, 2). In many cases, AD is associated with mutations in the genes encoding presenilin-1 (PS1) on chromosome 14

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and presenilin-2 (PS2) on chromosome 1 (3–5). These two proteins share a high degree of homology and are located primarily in the endoplasmic reticulum (ER). It has been demonstrated that the PS1 mutation increases the production of amyloid β peptide (Aβ), which is initially deposited in senile plaque (6, 7) and that it modulates calcium homeostasis (8–10) and increases neuronal cell death. Neurons from PS1 mutant knock-in mice exhibit increased production of Aβ peptide and increased vulnerability to oxidative stress, excitotoxicity, and various neurotoxic stimuli (11–15), as well as to focal ischemia and to DNA-damaging agents (16, 17). The increase in intracellular calcium is a critical factor in the increased susceptibility of cultured neuronal cells, or of neurons from transgenic mice expressing mutant PS1, to neurotoxic stimuli (9, 18). The perturbation of intracellular calcium homeostasis by PS1 is related to disruptions in the ER calcium pool (19, 20). A direct interaction between PS1 and the ryanodine receptor (RyR) in the ER and a close relationship between an increase in RyR expression and calcium release have also been found in PC12 cells and cortical neurons expressing mutant PS1 (18). Although both PS1 and PS2 appear to be multifunctional and similarly involved in various cellular processes, and mutation of both proteins may be implicated in neurodegenerative disease, the increased vulnerability of neuronal cells conferred by the PS2 mutation and the mechanisms of PS2 mutation in the development of AD have been poorly investigated. A recent study demonstrated that the PS2 mutation modulates calcium signaling in Xenopus oocytes (21), and PS2 has been reported to interact with calcium binding proteins such as calsenilin and calmyrin in cells transiently transfected with PS2 cDNA (22). It was also found that sorcin, a calciumbinding protein, serves as a modulator of the ryanodine receptor in neuronal cell lines and brain tissue (23). We previously demonstrated that transgenic mice expressing a PS2 mutant (N141I) show elevated Aβ production, caspase-3 activity, and cell death, and that they exhibit behavioral dysfunction (24). We were interested in determining whether the PS2 mutation disrupts cellular calcium homeostasis and sensitizes neurons to neurotoxicants by disturbing calcium homeostasis, similar to what has been reported for the PS1 mutation. In this study, we investigated whether the PS2 mutation increases the vulnerability of neurons to cell death promoted by Aβ, a neural peptide linked to AD pathogenesis, and L-glutamate, an excitotoxic agent and whether this increased vulnerability is related to a disruption of calcium homeostasis in the ER mediated by RyR. Caspase-3 is involved in amyloid precursor protein processing, consistent with the elevation of amyloid formation in the neurons of AD patients (25) and a caspase-3 inhibitor blocks staurosporine-induced cell death in primary cortical neurons overexpressing PS2 (26). In addition, a caspase-3-like activity increases during the serum deprivation-induced apoptosis of SK-N-SH human neuroblastoma cells expressing mutant PS2 (N141I) (27). Moreover, a sustained elevation of intracellular calcium levels can activate calcium-sensitive caspase, which has been shown to have a critical role in the progression of apoptotic cell death (17). Therefore, a change in caspase-3 activity was investigated as a possible mechanism of cell death by the disruption of calcium homeostasis in the ER through RyR. MATERIALS AND METHODS Gene construction and stable transfection of mutant and wild-type PS2 into PC12 cells The plasmid pNSE-PS2m was constructed by ligating the NSE (neuron-specific enolase) promoter-containing Hind-EcoR1 fragment from pNSE-CAT to the PS2 mutant gene (N1411,

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Volga German Families) containing the Hind-EcoR1 fragment from plasmid pUHD-10-3. The plasmids pNSE-CAT and pUHD10-3 were gifts from Dr. J. Gregor Sutcliffe, the Scripps Research Institute, and Dr. Tae-Wan Kim, Columbia University, respectively. The resulting construct pNSE-PS2m carries the 1.8-kb promoter fragment fused to a PS2m fusion gene, as described previously (24). PC12 cells, a rat cell line derived from a pheochromocytoma, were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and F-12 nutrients (GIBCO BRL, Gaithersburg, MD) supplemented with 10% heat-activated horse serum, 5% heat-activated fetal bovine serum, penicillin-streptomycin (10,000 units/ml) at 37°C under an atmosphere of 5% CO2 and 95% air. The culture medium was changed 3 times per week. Plasmids containing wild-type or mutant PS2 and a control plasmid containing the Neo construct alone were transfected into PC12 cells with lipofectAMINE PLUS in OPTI-MEN according to the manufacturer’s specification (Invitrogen, Carlsbad, CA). After 24 h, the culture medium was exchanged for medium containing geneticin (G418, 0.25 mg/ml) to select for colonies expressing PS2. Stably transfected clones were selected on the basis of the increased expression of a PS2-like holoprotein (54 kDa) and N- and C-terminal maturation fragments. Each type of cell line was selected that showed the highest PS2 expression level in the PC12 cells carrying wild-type (PC12/PS2wt h3) or mutant PS2 (PC12/PS2mt j3) was used for the following response and mechanism studies. Aβ25–35, the most toxic peptide fragment derived from amyloid precursor protein, was dissolved in deionized distilled water at 1 mM and stored at –20°C. Stock solutions were diluted to the desired concentration immediately before addition to the culture medium. Globular oligomers of Aβ1–42 (ADDLs) were prepared by the method of Klein (28). PS2 mutant transgenic mice and primary cortical neuron cell culture The targeting strategy used to generate mutant PS2 knock-in mice is detailed elsewhere (24). Mutant PS2 mice have no overt developmental abnormalities, but they exhibit increased levels of Aβ1–42 in brain tissue, an increased vulnerability of hippocampal neurons to apoptosis, and behavioral defects. Cultures of dissociated cortical cells were prepared from day 18 embryos of nontransgenic age-matched ICR mice, wild-type and homozygous PS2 knock-in mouse pups, using methods similar to those described previously (9, 17, 18). Briefly, cerebral cortices were removed and incubated for 15 min in Ca2+- and Mg2+-free Hanks' balanced saline solution (Life Technologies) 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 containing B27 supplements (Life Technologies). Experiments were performed with 6- to 8-day-old cultures; greater than 90% of the cells in these cultures were neurons, and the remainder were astrocytes, as judged by cell morphology and by immunostaining with antibodies against neurofilaments and glial fibrillary acidic protein. For calcium measurements, cortical neuronal cells were isolated from 1-day-old mice brain, and intracellular calcium levels were immediately determined. Measurement of cytotoxicity Cell viability was estimated by the propidium iodide (PI) fluorescence method described by Nieminien et al. (29). Briefly, PC12 or transfected PC12 cells (0.5×106 cells/cm2) were cultured for the indicated time, the medium was removed by aspiration, and the cells were permeabilized

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with 350 μM digitonin and resuspended in 1 ml fresh media containing 30 μM PI. After 30 min at 37°C, the initial fluorescence was measured using a FLUO star (BMG LabTechnologies, Durham, NC) using 560 nm excitation and 645 nm emission filters. Percentage viability (V) was calculated as V= 100(X–A)/(B–A), where A is the initial fluorescence, B is the fluorescence after the addition of digitonin, and X is the fluorescence of the designated sample. Cell viability was also determined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, after 72 h culture with L-glutamate and Aβ. Cells were washed once with PBS, and 1 ml DMEM containing 0.05 mg/ml MTT solution was added. After incubation at 37°C for 1 h, the medium was removed, and formazan crystals in the cells were solubilized in 1 ml DMSO for determination of OD at 570 nm using a spectrophotometer. Cortical neuronal necrosis was evaluated 24 h after the addition of Aβ25–35 and L-glutamate. Cultures were incubated with 0.1% Trypan blue in PBS 5 min at 22°C, rinsed with PBS, and fixed in 4% paraformaldehyde in 0.1 M sodium phosphate. Trypan blue-positive and -negative cells were counted by an unbiased observer. More than 300 cells were evaluated in each experimental group (3 cultures per group). Measurement of apoptotic cell death Apoptotic PC12 cells were identified by morphological changes after 4,6-diamino-2phenylindole (DAPI) staining and observation by fluorescence microscopy (DAS Microscope LEICA DAR, ×100 or ×200). Cells were fixed with paraformaldehyde (4%) at room temperature for 30 min, washed twice with PBS, and incubated at room temperature with DAPI for 1 min. Dishes were then coverslipped using Vectashield medium (Vector, Biosys, Compiegne, France). For each determination, three separate 100-cell counts were made. Apoptosis was expressed as a percentage, calculated as the number of cells with apoptotic nuclear morphology divided by the total number of cells. Measurement of intracellular calcium Intracellular calcium was measured by fluorescence ratio imaging with the calcium indicator dye fura-2 AM. Briefly, cells (1.5×106 cells/ml) were treated with 5 μM fura-2 AM at 37°C for 1 h and imaged with a Delta Scan System (Photon Technology International; Princeton, NJ), and the average calcium concentration was expressed as a ratio of the fluorescence emissions obtained using excitation wavelengths of 340 and 380 nm. The fluorescence intensities were calibrated to provide an estimation of the relative change in the intracellular calcium concentration in a solution containing EGTA (no calcium level) or digitonin (saturating level of calcium) using the following equation: [Ca2+]i = Kd [(R – Rmin)/(Rmax – R)](Fo/Fs). Flow cytometry analysis PC12 cells were harvested with trypsin-EDTA solution and fixed in ice-cold 70% ethanol. At least 1–2 h before flow cytometry analysis, the cells were resuspended in 1 ml modified Vindelov’s DNA staining solution (10 μg/ml RNase A and 5 μg/ml PI in PBS). Analysis was performed with a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Cells in the G1, S, and G2/M phases were identified with Modifit LT (Verity House Software, Topsham, ME).

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Immunocytochemical staining and double-labeling immunohistochemistry Cells (0.5×106 cells/cm2) were cultured on a chamber slide (Lab-Tak II chamber slider system, Nalge Nunc Int., Naperville, IL), fixed in 4% paraformaldehyde, membrane-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 rabbit polyclonal antibody for active caspase-3 (1:500 dilution) overnight at 4°C, followed by an anti-rabbit biotinylated secondary antibody for 1 h at room temperature before addition of the ABC reagent for 30 min at room temperature. The cells were incubated with diaminobenzidine tetrahydrochloride solution until the development of desired stain intensity. For the analysis of colocalization of PS2 and RyR, cells were fixed in 4% paraformaldehyde, membrane-permeabilized by exposure for 5 min to 0.2% Triton X-100 in phosphate-buffered saline, and placed in blocking serum containing 5% horse or goat serum in phosphate-buffered saline. Cells were then exposed to primary antibodies (1:100 dilutions of rabbit polyclonal N- or C-terminal PS2 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 antimouse secondary antibodies (Molecular Probes, Eugene, OR). Immunofluorescence images were acquired using a confocal laser scanning microscope (dual-wavelength scan, MRC1024, Bio-Rad, Hercules, CA) with a ×360 oil immersion objective. Double fluorescence images were captured and saved in TIFF format. Final images were composed with Corel Draw (version 10 Corel, Ontario, Canada). Measurement of caspase-3 activity Cells (0.5×106 cells/cm2) were cultured in 6-well plates for 24 h and treated with drugs for an additional 24 h in the presence or absence of 50 μM Z-VAD-FMK. Caspase-3 activity was measured by proteolytic cleavage of the caspase-3 substrate Ac-DEVD-pNA, using the CaspACE Assay system, according to the manufacturer’s instructions (Promega, Pittsburgh, PA). Western blotting After culture, cells (0.5×106 cells/cm2) were homogenized with lysis buffer [50 mM Tris pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 1 mM PMSF, 10 μl/ml aprotinin, 1% igapel 630 (Sigma), 10 mM NaF, 0.5 mM EDTA, 0.1 mM EGTA and 0.5% sodium deoxycholate] and centrifuged at 23,000 g for 1 h. Equal amount of proteins (20 μg) were separated on a SDS/12% polyacrylamide gel and transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, Piscataway, NJ). Blots were blocked for 2 h at room temperature with 5% (w/v) nonfat dried milk in Tris-buffered saline solution [10 mM Tris (pH 8.0) and 150 mM NaCl] containing 0.05% Tween-20. The membrane was incubated for 3 h at room temperature with specific antibodies. Rabbit polyclonal antibodies against caspase-3 (1:1000 dilution) and Nand C-terminal fragments of PS2 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA) were used in this study. The blot was then incubated with the corresponding conjugated anti-rabbit immunoglobulin G-horseradish peroxidase (Santa Cruz Biotechnology). Immunoreactive proteins were detected with the ECL Western blotting detection system. Membranes were routinely stripped and reprobed with a β-actin control. The relative density of the protein bands was quantified by densitometry using the Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, Rochester, NY).

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Statistics Data were analyzed using one-way ANOVA followed by Bonferroni’s test as a post hoc test. Differences were considered significant at P < 0.05. RESULTS Expression of PS2 protein in PC12 and cortical neuron cells Wild-type PS2, PS2 constructs containing the 141 Asn-Ile mutation (N141I), or vector alone were transfected into PC12 cells. RT-PCR analysis showed that PS2 mRNAs were expressed at variable levels in each of the stably transfected cell lines (data not shown). Clonal lines expressing moderately high levels of the apparent molecular weight (54 kDa) PS2-like holoprotein and the N- and C-terminal PS2 fragments were established, as were cell lines transfected with the vector and an untransfected parental cell line. PS2 protein expression was determined by Western blot analysis of lysates of cells transfected with PS2 wild-type (PC12/PS2wt) or mutant PS2 N141I (PC12/PS2mt) constructs or vector alone (PC12/neo). Similar to previous results showing that the expression of PS2 and mutant PS2 was increased in the cortex and hippocampus of transgenic mice (24), densitometric analyses of Western blots showed that the levels of the (54 kDa) PS2-like protein, and of the N- and C-terminal PS2 fragments, were greater in three wild-type PS2 clones (PC12/PS2wth1, PC12/PS2wth2, and PC12/PS2wth3) and in three mutant PS2 clones (PC12/PS2mtj1, PC12/PS2mtj2, and PC12/PS2mtj3) than in PC12 and PC12/neo cells (Fig. 1A and B). Increased vulnerability of PC12 cells and cortical neurons expressing mutant PS2 to Lglutamate and Aβ Because mutation of PS1 has been implicated in the neurotoxic mechanism of AD, we next investigated whether mutant PS2 has similar effects on the pathophysiology of AD by enhancing the vulnerability of neuronal cells. We therefore first assessed the viability of individually selected PC12 cell lines 72 h after exposure to L-glutamate (30 mM). Cell viability was more significantly decreased by L-glutamate in PC12 cells expressing wild-type PS2 (average of two clones, 61±7.1%) or mutant PS2 (average of three clones, 42±5.1%) than in PC12/neo (72±3.3%) or PC12 cells (74±7.9%) (data not shown). We next assessed cell viability 24, 48, and 72 h after exposure to L-glutamate and Aβ25–35 in representative PC12 clones expressing either wild-type (PC12/PS2wt) or mutant PS2 (PC12/PS2mt), as compared with PC12 cells expressing the vector alone. Viability was decreased by L-glutamate (Fig. 2A and C) and Aβ25–35 (Fig. 2B and D) in PC12 cells expressing wild-type (Fig. 2A and B) or mutant PS2 (Fig. 2C and D) in a dose- and time-dependent manner. The viability of PC12 cells and of PC12 cells expressing vector alone, wild-type PS2, or mutant PS2 72 h after treatment with 50 μM Aβ25–35 was ~62, 56, 48, and 42%, respectively; for that of the untreated control, and for treatment with L-glutamate, the viabilities were 73, 69, 53, and 41%, respectively, and similar trend in cell viability was also found after treatment with Aβ1–42 (ADDLs form) (Fig. 2E and F). Similar to the results obtained with PC12 cells, treatment with the two agents resulted in a progressive but similar overall decrease in the survival of cortical neurons prepared from 18-day-old embryos of wild-type and mutant PS2 transgenic mice after 24 h culture. However, cortical neurons from mutant PS2 transgenic mice were significantly more vulnerable compared with cortical neurons from wildtype PS2 mice, as determined by Trypan blue staining (Fig. 2G and H). Under normal culture

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conditions, PC12 cells and cortical neurons expressing wild-type PS2 or the mutant PS2 used in the present study did not show any cytotoxic effect with respect to cell growth. To examine whether glutamate-increased cell vulnerability is mediated by glutamate receptor, we employed glutamate receptor antagonist MK801 (4 h pretreatment) into PC12 cells and cortical neurons carrying mutant PS2. The glutamate (10, 30, 50 mM)-increased cell death was not reversed by treatment of MK801 (1–100 μM) (data not shown). Increased apoptotic cell death of PC12 cells and cortical neurons expressing mutant PS2 treated with L-glutamate and Aβ A significant number of cells expressing mutant PS2 underwent apoptotic cell death, as determined by DAPI staining (Fig. 3A). The basal level of apoptosis was about twofold higher in PC12 cells expressing mutant PS2 than in other cell types. Treatment with L-glutamate (30 mM)induced apoptotic cell death in PC12 cells expressing wild-type (28±4.6%) or mutant PS2 (35±3.9%), compared with PC12/neo cells (32±5.1%), PC12 cells (21±5.3%), and the untreated control (6.1±0.8%) (Fig. 3B). Apoptosis of PC12 cells was also significantly and dose dependently increased by Aβ25–35; however, more PC12/PS2mt cells (58±3.9%) underwent apoptosis compared with PC12/neo (39±4.8%) or PC12/PS2wt cells (42±4.1%). Similar increase of cell vulnerability in PC12/PS2mt cells against Aβ1–42 was also found (Fig. 3C). We also analyzed the cell cycle distribution of these cells to determine whether mutant PS2 causes them to accumulate in the sub-G1 phase, which is characteristic of apoptotic cell death. In agreement with the induction of apoptotic cell death as determined by DAPI staining, a greater number of PC12/PS2mt cells arrested in sub-G1 after treatment with L-glutamate and Aβ25–35 for 72 h, compared with PC12, PC12/neo, and PC12/PS2wt cells (Fig. 3D). A greater extent of apoptosis occurred in cortical neurons from PS2 mutant transgenic mice, either untreated or treated with Lglutamate (30 mM, 31±3.1%) and Aβ25–35 (50 μM, 39±10%), compared with that in wild-type PS2 transgenic (28±2.0%, 31±3.0%) or control mice (13±2.0%, 21±6.0%) (Fig. 3E). Calcium release promoted by L-glutamate and Aβ is enhanced in PC12 cells and cortical neurons expressing mutant PS2 It has been suggested that the perturbation of intracellular calcium homeostasis is a critical contributor to the increased vulnerability of cells expressing mutant PS1 (9, 16–18). To determine whether PS2 mutations also increase calcium release and thereby contribute to an increased vulnerability to L-glutamate and Aβ25–35, we measured the intracellular calcium levels of PC12 cells compared with that in PC12/neo, PC12/PS2wt, and PC12/PSmt cells. The intracellular calcium level was elevated in PC12 cells expressing wild-type PS2 (Fig. 4A and B) or mutant PS2 (Fig. 4C and D) by L-glutamate (5–30 mM Fig. 4A and C) and Aβ25-35 (10–50 μM, Fig. 4B and D) in a dose-dependent manner. The basal levels of intracellular calcium were similar in PC12 cells, PC12 cells expressing vector alone, and cells expressing wild-type and mutant PS2 (105±7 nM, Fig. 4E and F). However, treatment with L-glutamate (30 mM) and Aβ25–35 (50 μM) resulted in a two- to threefold increase in the intracellular calcium level of PC12 cells, PC12/neo cells, and PC12/PS2wt cells, but a four- to fivefold increase in PC12/PS2mt cells (Fig. 2E and F). More interestingly, the elevated intracellular calcium level of PC12 cells expressing mutant PS2 was maintained after treatment with L-glutamate (Fig. 4C) and Aβ25–35 (data not shown). We also observed elevated intracellular calcium levels in cortical neurons isolated from transgenic mice. Soon after isolating cortical neurons (3×106 cells/ml), we determined the released intracellular calcium. The endogenous calcium level (511±270 nM) was

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much greater in neurons isolated from mutant PS2 mice than from wild-type PS2 (312±190 nM) or nontransgenic mice (215±7 nM). Calcium release from cortical neurons after L-glutamate (30 mM), Aβ25–35 (50 μM), and Aβ1–42 (10 μM) treatment was also examined and found to be significantly greater (two- to threefold increase, 915±301 nM) in cortical neurons from mutant PS transgenic mice compared with wild-type (745±336 nM) or nontransgenic mice (457±126 nM, Fig. 4G). An even greater increase in the intracellular calcium level was observed in cortical neurons isolated from transgenic mice expressing wild-type (1373±336 nM, 1050±300 nM) or mutant PS2 (2387±406 nM, 2022±250 nM) after treatment with Aβ25–35 or Aβ1–42 (10 μM) compared with nontransgenic mice (757±273 nM, 454±110 nM, respectively). PS2 colocalizes with RyR PS is an integral protein in the ER, and PS may be important for regulating ER calcium signals, which have been implicated in neuronal cell death by mutation of presenilin (14–17). Therefore, we determined whether PS2 is coexpressed or colocalizes with neuronal type RyR (type 3) in PC12 cells and cortical neurons. Double-labeling immunostaining confocal analysis showed that the PS2-reactive compartment in the ER of PC12 cells was also RyR-positive and that the expression of RyR increased to a greater extent in PC12 cells, expressing mutant PS2 than in cells expressing wild-type PS2 (Fig. 5A). We also found that the expression of PS2 and RyR in PC12 cells was increased by treatment with L-glutamate (30 mM), Aβ25–35 (50 μM), and Aβ1–42 (10 μM), as demonstrated by immunostaining confocal analysis (Fig. 5A) and Western blotting (Fig. 5B). Colocalization of PS2 and RyR was also observed in the cortex and hippocampus of transgenic mice expressing PS2 in the ER (Fig. 6A), and the expression of RyR and PS2 (full length, N- and C-terminal PS2) was greater in the cortex and hippocampus of PS2 mutant transgenic mice than in wild-type PS2 transgenic or age-matched nontransgenic mice (Fig. 6B). Coimmunoprecipitation analysis showed that the relative amounts of PS2 (N-terminal PS2) protein in RYR precipitates was greater in PC12/PS2mt cells (Fig. 5C) and in the cortex and hippocampus of transgenic mice expressing mutant PS2 (Fig. 6C). Similarly, immunoprecipitates of lysates with the N-PS2 antibody immunoreacted with the RyR antibody, and much greater coexpression was found in PC12/PS2mt cells and in the brain of transgenic mice expressing wild-type and mutant PS2 (Fig. 5C and 6C). Prevention of neuronal cell vulnerability by inhibition of calcium release from the ER Next, we speculated that mutation of PS2 may increase cell vulnerability by increasing the intracellular calcium level in a RyR-dependent manner. To investigate this possibility, we used the specific RyR inhibitor dantrolene in PC12/PS2mt cells and monitored cell viability and the induction of apoptosis. Pretreatment with dantrolene 30 min before treatment with L-glutamate (30 mM) and Aβ25–35 (50 μM) caused a reduction in cell viability in a dose-dependent manner (Fig. 7A and B) and diminished the induction of apoptotic cell death in PC12/PS2mt cells treated with L-glutamate (30 mM) and Aβ25–35 (50 μM) in a dose-dependent manner (Fig. 7C and D). Similar to what was observed for PC12 cells, pretreatment with dantrolene (50 μM) also reduced L-glutamate (30 mM)-, Aβ25–35 (50 μM)-, and Aβ1–42 (10 μM)-induced apoptotic cell death in cortical neuronal cells expressing wild-type and mutant PS2 (Fig. 7E).

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Increased caspase-3 activity in PC12 cells and cortical neurons expressing mutant PS2, and preventive effect of dantrolene, a RyR inhibitor Agents that disrupt ER-mediated calcium regulation are known to activate the apoptotic protein caspase-3, which is well known to promote cell death. Our previous study showed that caspase-3 is activated in transgenic mice expressing mutant PS2 (24). To determine whether there is a causal relationship between the RyR-mediated increase of calcium release and the increased caspase-3 activity and apoptotic cell death of PC12 cells expressing mutant PS2, we assayed caspase-3 activity by Western blot analysis and biochemical assay and by an immunostaining method with a corresponding antibody. Caspase-3 activity measured by the biochemical assay was much greater in PC12/PS2mt cells exposed to L-glutamate and Aβ25–35 than in other cell types (Fig. 8A and B). Expression of active caspase-3 was detectable in untreated PC12, PC12/neo, PC12/PS2wt, and PC12/PS2mt cells; however, treatment with L-glutamate, Aβ25–35, and Aβ1–42 caused a gradual increase in the expression of active caspase-3, and a much greater increase in expression was observed in PC12/PS2mt cells than in PC12, PC12/neo, or PC12/PS2wt cells (Fig. 8C). In agreement with the Western blot analysis, immunostaining against active caspase-3 was found to be more extensive and intense in PC12/PS2mt cells (Fig. 8D), and the number of caspase-3-reactive cells was greatly increased in the PC12/PS2mt cell line after treatment with L-glutamate and Aβ25–35, as compared with other cells (Fig. 8E). Like PC12 cells, cortical neuron cells from mutant PS2 transgenic mice exhibited much higher caspase-3 activity compared with cortical neurons from wild-type transgenic mice after Lglutamate (30 mM), Aβ25–35 (50 μM), and Aβ1–42 (10 μM) treatment (Fig. 8E). To further confirm the significance of intracellular calcium changes by RyR in the activation of caspase-3, we pretreated PC12 cells and cortical neurons expressing mutant PS2 with dantrolene and then measured caspase-3 activity. Pretreatment with dantrolene prevented the L-glutamate- and Aβ25– 35- or Aβ1–42-induced increase caspase-3 activity in PC12 cells (Fig. 9A) and in cortical neurons expressing mutant PS2 (Fig. 9B). DISCUSSION Several lines of evidence from in vivo and in vitro experiments have demonstrated that mutation of PS1 is implicated in oxidative or excitotoxic stress-induced neuronal cell death through the elevation of intracellular calcium levels (9, 18–20, 30, 31). Similar to what is observed for the PS1 mutation, PC12 cells and cortical neurons expressing mutant PS2 (N141I) were more vulnerable to L-glutamate and Aβ25–35 or Aβ1–42 compared with those expressing wild-type PS2. The increased vulnerability conferred by mutant PS2 is associated with an increased intracellular calcium level in both cell types. We also found a much higher level of type 3 RyR in the ER of PC12 cells and in the brain of transgenic mice overexpressing mutant PS2. Moreover, treatment of PC12 cells with L-glutamate, Aβ25–35 and Aβ1–42 resulted in a strongly enhanced expression of RyR. However, treatment with dantrolene, which blocks RyR, protected cells from death induced by L-glutamate and Aβ. These results demonstrate that the PS2 mutation can enhance cell vulnerability by increasing intracellular calcium levels and that the RyR level in the ER may be important in the mechanism of neuronal cell death associated with the PS2 mutation. However, in the present study, the magnitude of the increase in intracellular calcium levels caused by the PS2 mutation is lower (a two- to threefold increase) than that caused by the PS1 mutation (M146V and L286V, a four- to eightfold increase) in the same PC12 cells (18). However, the increase of intracellular calcium in cortical neurons isolated from transgenic mice expressing mutant PS2 was much greater than in cortical neurons expressing mutant PS1. This difference

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may be due to differences in the source of cortical neurons and stimuli. That is, we isolated cortical neurons from the brain of 1-day-old mice, but in the other study, neurons were isolated from the embryonic brain of 18-day-old transgenic mice expressing mutant PS1. Also, the stimulant used in the PS1 mutation study was caffeine, but we used Aβ25–35 and L-glutamate. Calcium levels may change more significantly in response to caffeine than to Aβ25–35 or Lglutamate, since caffeine induces calcium release from ryanodine-sensitive stores. In fact, consistent with the much greater calcium release, the previous study in PC12 cells showed that caffeine enhanced cellular vulnerability more than Aβ25–35 or L-glutamate did. Although the relevance of the different magnitudes of the influence of mutant PS2 and mutant PS1 on intracellular calcium release is not clear, the present data support the notion that increases in intracellular calcium may be a common mechanism in the PS1 and PS2 mutation-induced increase of cell vulnerability. Increased calcium release mediated by RyR has received attention as a critical factor in the pathogenic mechanism of PS mutation-induced neuronal cell death (9, 14–20). This interpretation is further supported by our observations; first, the protein level of RyR was much greater in PC12 cells and cortical neurons expressing mutant PS2 than in those expressing vector alone or wild-type PS2, and the level of RyR in PC12 cells expressing mutant PS2 was greatly enhanced by treatment with L-glutamate and Aβ. Second, we also found that the RyR inhibitor dantrolene attenuated L-glutamate- and Aβ-induced cell death in PC12 cells and in cortical neurons expressing mutant PS2. Previous studies have reported that dantrolene prevents neuronal cell death promoted by metabolic and excitotoxic insults. For example, dantrolene, which blocks the release of Ca2+ stores from the ER, partially protects SH-SY5Y cells from oxygen-glucose deprivation toxicity and cell death induced by sarcoendoplasmic reticular Ca2+-ATPase pump inhibitors (32, 33). Administration of dantrolene to gerbil neonatal brain provides protection from ischemic neuronal cell death, as well as from ischemia/reperfusion-induced CA1 neuronal cell death (34, 35). These data suggest that changes in RyR expression in the ER may be associated with the increased vulnerability of cells expressing mutant PS2 through the perturbation of calcium homeostasis. This result is in accord with speculations by other investigators who have reported that the perturbation of intracellular calcium homeostasis through RyR may be critical for mutant PS1-induced PC12 and cortical neuronal cell death in response to various stimuli and conditions (9, 16–18). Similarly, the increased vulnerability of the cells by the elevated intracellular calcium from RyR was also found in the SH-SY5Y neuroblastoma cells, as well as neurons carrying mutant PS1 (36, 37). Schneider et al. (38) also reported that neuronal cell death of PS2 mutant mice was also prevented with dantrolene. We also found that PS2 directly interacts with RyR. Double-labeled confocal analysis and coimmunoprecipitation studies using N-terminal PS2 and RyR antibodies showed that PS2 and RyR colocalize in the ER, as has also been reported for PS1 and RyR (18). However, in contrast to mutant PS1, which does not affect the level of RyR, PC12 cells expressing mutant PS2 exhibited an increase in RyR, which was enhanced by treatment with L-glutamate and Aβ. An interaction between PS2 and RyR was also found in the brain of transgenic mice; this interaction was stronger in mice expressing mutant PS2. The mechanism by which mutation of PS2 increases RyR expression and interaction between the two proteins is not clear. However, it is noteworthy that the expression of RyR is induced by several mitochondrial metabolic inhibitors that can induce apoptosis (39). It has also been suggested that the increases in reactive oxygen species and in calcium, which are key consequences of the PS mutation, could promote RyR expression. Therefore, it is possible that Page 10 of 24 (page number not for citation purposes)

PS2 mutation-increased induction of reactive oxygen species and of intracellular calcium may result in increased RyR expression. In fact, a much higher level of reactive oxygen species with or without treatment of apoptotic agents was seen in PC12/PS2mt cells (data not shown). Kelliher and colleagues (40) reported that the levels of radiolabeled ryanodine binding were significantly increased in the CA1 hippocampal region of the brain of early-stage AD patients. It has been demonstrated that PS1 and PS2 interact with other calcium regulatory proteins such as the Ca2+-binding protein calsenilin (22), the Ca2+-dependent protease calpain (41), and sorcin, a RyR modulator (42). Mutation of presenilins (both PS1 and PS2) can alter calcium regulatory protein(s) or calcium-dependent proteases, resulting in an elevation of intracellular calcium. Therefore, it is possible that elevated levels of PS2 protein cause an initial increase in RyR expression in the ER, which may be critical in promoting neuronal cell death through the perturbation of calcium signals in the early stages of AD. The molecular mechanism by which mutant PS2 induces increases in RyR-mediated cell death is not clear. Here, we show that the level of expression of an active form of caspase-3, and the level of caspase-3 activity, was much higher in PC12 cells and cortical neuronal cells expressing mutant PS2. We also found a direct linkage between the activation of caspase-3 and PS2 mutation-induced calcium release, as evidenced by the finding that blockage of changes in the intracellular calcium level by pretreatment with dantrolene inhibited caspase-3 activity and reversed neuronal cell death induced by L-glutamate and Aβ (43). This result is consistent with our previous study demonstrating that large areas showing caspase-3-positive staining are observed in the hippocampus-dentate gyrus and in the cerebral cortex of transgenic mice carrying mutant PS2 (24). Similar results showing a significant role for caspase-3 activation in mutant PS2-induced neuronal cell death have been reported (11, 12, 14, 15, 17). Caspase-3 is involved in amyloid precursor protein processing, consistent with the elevation of amyloid formation in the neurons of AD patients (24, 42), Taken together, a possible molecular mechanism of mutant PS2-induced cell death is that increases in intracellular calcium levels promoted by mutant PS2 may activate caspase-3, which, in turn, may implement activation of the apoptotic pathway (24, 40, 44, 45). Directly excitotoxic cell death may be assumed as another possible mechanism in the case of glutamate treatment. However, we did not see any preventive effect by glutamate receptor antagonist (MK-801, pretreatment) in glutamate-induced cell death (data not shown), suggesting that glutamate receptor-mediated mechanism may not be related with the cell death of neuronal cells carrying presenilin-2 (or mutated presenilin-2) although the relatively high doses of glutamate were needed to induce cell death. Collectively, mutant PS2 increases the vulnerability of PC12 cells and cortical neuronal cells, and this effect may be associated with the increased release of intracellular calcium level through the RyRsensitive calcium pool, and activation of caspase-3 may be a significant in the pathway of mutant PS2-induced neuronal cell death. ACKNOWLEDGMENTS This work was supported by grant (R05-2002-000-00644-0) from the Basic Research Program of the Korea Science and Engineering Foundation and by Research Fund from the Korea Research Institute of Bioscience and Biotechnology (2003–2005).

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Fig. 1

Figure 1. Expression of PS2 in PC12 cells expressing wild-type and mutant PS2. A) Western blot analysis of the expression of PS2 protein in PC12 cells, and in PC12 cells with vector alone (PC12/neo), wild-type PS2 (PC12/PS2wt) or mutant PS2 (N141I) (PC12/PS2mt). Equivalent amounts of protein (50 µg/lane) from homogenates prepared from the indicated cell lines were separated by SDS-PAGE, transferred to nitrocellulose, and probed with C-terminal and N-terminal PS2 antibodies. The N-terminal PS2 antibody also recognizes full-length PS2 (54-kDa band). B) Densitometric analysis of the relative levels of putative PS2 protein in selected PC12 clones stably expressing wild-type PS2 or mutant PS2.

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Fig. 2

Figure 2. Viability of PC12, PC12/neo, PC12/PS2wt, and PC12/PS2mt cells, and of cortical neurons expressing wild-type and mutant PS2, after treatment with L-glutamate and Aβ. Cell viability was determined by the MTT assay with representative clones of PC12/PS2wt cells (A, C) or PC12/PS2mt cells (B, D) after treatment with different doses of Lglutamate (A, C) and Aβ25−35 (B, D) for 24, 48 and 72 h or Aβ1−42 (for 72 h) (F). PC12/PS2mt cells showed enhanced sensitivity to L-glutamate (E) and Aβ25-35 (F) compared with PC12/PS2wt or PC12/neo cells 72 h after treatment (*P