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J. Biol. Chem., Vol. 279, Issue 27, 28733-28743, July 2, 2004

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Herp Stabilizes Neuronal Ca²⁺ Homeostasis and Mitochondrial Function during Endoplasmic Reticulum Stress^*

Sic L. Chan, Weiming Fu, Peisu Zhang, Aiwu Cheng, Jaewon Lee, Koichi Kokame¶, and Mark P. Mattson||

From the Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, Maryland 21224, the ^¶National Cardiovascular Research Institute, Suita, Osaka 565-8565, Japan, and the ^||Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, April 16, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In response to endoplasmic reticulum (ER) stress, cells launch homeostatic and protective responses, but can also activate cell death cascades. A 54 kDa integral ER membrane protein called Herp was identified as a stress-responsive protein in non-neuronal cells. We report that Herp is present in neurons in the developing and adult brain, and that it is regulated in neurons by ER stress; sublethal levels of ER stress increase Herp levels, whereas higher doses decrease Herp levels and induce apoptosis. The decrease in Herp protein levels following a lethal ER stress occurs prior to mitochondrial dysfunction and cell death, and is mediated by caspases which generate a 30-kDa proteolytic Herp fragment. Mutagenesis of the caspase cleavage site in Herp enhances its neuroprotective function during ER stress. While suppression of Herp induction by RNA interference sensitizes neural cells to apoptosis induced by ER stress, overexpression of Herp promotes survival by a mechanism involving stabilization of ER Ca²⁺ levels, preservation of mitochondrial function and suppression of caspase 3 activation. ER stress-induced activation of JNK/c-Jun and caspase 12 are reduced by Herp, whereas induction of major ER chaperones is unaffected. Herp prevents ER Ca²⁺ overload under conditions of ER stress and agonist-induced ER Ca²⁺ release is attenuated by Herp suggesting a role for Herp in regulating neuronal Ca²⁺ signaling. By stabilizing ER Ca²⁺ homeostasis and mitochondrial functions, Herp serves a neuroprotective function under conditions of ER stress.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The endoplasmic reticulum (ER)¹ is a unique cellular compartment simultaneously involved in the processes of protein synthesis and Ca²⁺ homeostasis. Various conditions, including oxidative and metabolic stress and Ca²⁺ overload can interfere with ER functions leading to the accumulation of misfolded proteins. Cells sense and respond to such ER stress by activating a signaling cascade termed the unfolded protein response, which results in the transcriptional up-regulation of stress proteins including members of the glucose-regulated protein (grp) family and other protein chaperones (calnexin, calreticulin, ERp72) that enhance the protein folding capability of the ER (1). ER stress has been documented in neurons in a variety of acute pathological conditions including cerebral ischemia and severe epileptic seizures (2). However, despite the fact that disruption of cellular Ca²⁺ homeostasis contributes to the death of neurons in these conditions, it is not known how molecular responses to ER stress modify cellular Ca²⁺ homeostasis and the cell death process. Studies of cultured cells suggest that ER stress can stimulate the expression of cytoprotective genes such as protein chaperones (3) but may also trigger a form of programmed cell death called apoptosis (4), which may involve activation of ER-associated caspases and transcription factors such as Gadd153. A better understanding of ER stress and its links to cell survival/death decisions is therefore needed.

Recent findings suggest that ER stress is also implicated in several chronic neurodegenerative disorders including Alzheimer's (5, 6), Parkinson's (7), and Huntington's (8) diseases. Alzheimer's disease (AD) results from altered proteolytic processing of the amyloid precursor protein (APP), resulting in aggregation of neurotoxic forms of amyloid -peptide (A) (9). Exposure of cultured neurons to A-peptide, and metabolic and oxidative insults can induce an ER stress response (6, 10). Moreover, mutations in presenilin-1 (PS1) that cause early-onset familial AD perturb ER Ca²⁺ homeostasis (11, 12) and impair the ability of neurons to engage a cytoprotective ER stress response (20). The adverse effects of A and PS1 mutations on ER function may sensitize neurons to excitotoxicity and apoptosis (11).

A novel 54 kDa protein called Herp (homocysteine-induced ER protein) was recently identified and characterized as a stress-responsive protein localized in the ER membrane; Herp contains a ubiquitin-like domain and resembles the human DNA excision repair protein hHR23 (13). The function of Herp is unknown. Sai et al. (14) showed that Herp binds to PS1 and alters APP processing in HEK-293 cells, although it is not known whether the interaction affects the PS1 role in regulating ER Ca²⁺ homeostasis, which might also contribute to altered APP processing in neurons (15). In the present study we demonstrate a role for Herp in stabilizing cellular Ca²⁺ homeostasis and preventing neuronal death following ER stress.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Neuronal Cell Cultures—Hippocampal and cortical cell cultures were established from 18 day Sprague-Dawley rat embryos as described previously (16). Briefly, intact hippocampi and neocortical fragments were trypsinized, and cells were dissociated by mild trituration using a Pasteur pipette with a fire-polished tip. Cells were seeded into polyethyleneimine-coated plastic 35- or 60-mm diameter plastic dishes or 22 mm² glass coverslips, and maintained at 37 °C in Neurobasal medium containing B-27 supplements (Invitrogen), 2 mM L-glutamine, 0.001% gentamycin sulfate and 1 mM HEPES (pH 7.2). All experiments were performed using 7–8-day-old cultures; greater than 90% of the cells in these cultures were neurons.

Generation of DNA Constructs and Stably Transfected PC12 Cell Lines—Plasmids containing the full-length human Herp cDNA or full-length human Herp with an N-terminal c-Myc tag and a C-terminal FLAG tag were constructed as described previously (13). Site-directed mutagenesis was performed to generate by a PCR-based primer overlap extension method. In brief, same pair of flanking primers and two different mutant overlapping primers were synthesized. The flanking primers are 5'-CGCGGATCCTTTTTTAAAATGGAGTCCGAGACC-3'(forward) and 5'-CCGGAATTCTCAGTTTGCGATGG-3'(reverse) (the start and stop codons are underlined). To produce the mutant Herp D266E construct the overlapping mutant primers were 5'-AATCGAGATTGGTTGGAATGG-3'(forward) and 5'-TCCATTCCAACCAATCTCGATTTA-3' (reverse). The PCR products that contained the mutant sequence were subcloned into the PCR4 TOPO TA cloning vector (Stratagene), which was then amplified and digested with BamHI and EcoRII and subcloned into the pcDNA3.1 vector. The mutation was confirmed by automated DNA sequencing (ABI Prism 3700 DNA analyzer). Transfection of PC12 cells was carried out using the LipofectAMINE reagent using the manufacturer's protocol (Invitrogen). Stably expressing clones were obtained after selection for growth in the presence of geneticin (500 mg/liter) and characterized for Herp expression by immunoblot analysis.

Experimental Treatments—To induce ER stress, undifferentiated PC12 cells were treated with tunicamycin, thapsigargin, homocysteine, or 2-mercaptoethanol (Sigma) for various time points as indicated. At the end of each treatment, cultures were processed for biochemical analyses of levels of cytoplasmic and ER stress proteins, and for evaluating the extent of cell death. The caspase inhibitor zVAD-fmk (Bio-Mol) was prepared as a 500x stock in dimethyl sulfoxide. Amyloid -peptide-(1–42) (Bachem) was prepared as a 1 mM stock in water, which was incubated overnight at room temperature prior to dilution into culture medium.

siRNA Preparation and Nucleofection—Several 19-base pair (bp) sequences were selected from the rat Herp sequence and synthesized with an additional 2-bp overhang using the siRNA Construction kit (Ambion). siRNA duplexes targeting Herp (siRNA_Herp) were prepared as described. Briefly, 100 nM sense primers (5'-AAACAACCGGCTCTTCGTCdTdT-3' for siRNA_Herp1;5'-AAUCCUGACUUCCGGGAAAdTdT-3' for siRNA_Herp2; 5'-AAUCCUGACUUCCGGGAAAdTdT-3' for siRNA_Herp3) and corresponding antisense primers were mixed in 50 µl of water with 5 µlof10x annealing buffer (0.1 M potassium acetate, 30 mM HEPES-KOH, pH 7.2, 2 mM magnesium acetate). The mixture was heated to 95 °C for 5 min and then allowed to cool to 25 °C at a rate of 1 °C/min. The siRNA duplexes were diluted in RNase-free water and stored at -80 °C in multiple aliquots. Non-silencing control siRNA duplexes (siRNA_CTRL) were synthesized using scrambled sequences as described above. GenBank^TM search revealed no other known genes exhibiting sequence homology to these selected target sequences. After thorough mixing of the cells and 0.5–1 µg of siRNA duplexes, transfection was immediately carried out using the Cell Line Nucleofector Kit V according to Amaxa's optimized protocol for PC12 cells. Mock-transfected cells were nucleofected with vehicle (water).

RNA Isolation and Reverse Transcriptase-PCR—Total RNA was extracted from primary neuronal cultures using TRIzol reagent (Invitrogen). 2 µg of RNA was used for synthesis of cDNA using random primers and a first strand synthesis kit (Invitrogen). 200 ng of the cDNA was used in the PCR reaction using the following pairs of primers: Herp 5'-GAAGAAGATGAAATAAATCGAGAT-3'(forward) and 5'-TCAGTTTGCGATGGCTGGGGGG-3' (reverse); -actin 5'-TGTGATGGACTCCGGTGACGG-3' (forward) and 5'-ACAGCTTCTCTTTGATGTCACGC-3' (reverse). The optimized PCR conditions were 2 min at 94 °C, 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s, and 10 min at 72 °C. The PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized using a Fuji 3000 PhosphorImager.

Immunoblot Analysis—Methods for protein quantitation, electrophoretic separation, and transfer to nitrocellulose membranes were as described previously (17). Membranes were incubated in blocking solution (5% milk in Tween Tris-buffered saline; TTBS) overnight at 4 °C followed by a 1-h incubation in primary antibody diluted in blocking solution at room temperature. Membranes were then incubated for 1 h in secondary antibody conjugated to horseradish peroxidase, and bands were visualized using a chemiluminiscence detection kit (ECL, Amersham Biosciences). The primary antibodies included: rabbit polyclonal antibodies against Grp78, Grp94, and Hsp60 (StressGen), -tubulin, and -actin (Sigma), Herp (13), JNK (C-17, Santa Cruz Biotechnology) and phospho-c-Jun (Ser⁶³, Cell Signaling); mouse monoclonal antibodies against Hsp70 (Sigma), PS1 (Chemicon), p-JNK (G7, Santa Cruz Biotechnology), cytochrome c (PharMingen), and Bcl-2 (StressGen); chicken antibody against calreticulin (ABR); rat monoclonal antibody against caspase 12 (gift from J. Yuan, Harvard University); goat antibody against c-Jun (sc-45, Santa Cruz Biotechnology).

Immunoprecipitation—Aliquots of cell lysates or brain homogenates containing 100 µg of total protein were incubated with mouse monoclonal anti-c-Myc, anti-FLAG tag, or rabbit polyclonal anti-Herp antibodies in immunoprecipitation buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 2 µg/ml pepstatin A, 0.25 mM phenylmethylsulfonyl fluoride). Antigen-antibody complexes were precipitated with protein A or G for 1 h at 4 °C, washed three times in immunoprecipitation buffer, and solubilized by heating in Laemmli buffer containing 2-mercaptoethanol at 100 °C for 5 min.

In Vitro Protein Synthesis and Caspase Cleavage—Two oligonucleotides, 5'-GGGGTACCATGGAGTCCGAGACCGAAC-3' and 5'-CGGAATTCTCAGTTTGCGATGGCTGGG-3', were used to PCR amplify the entire open reading frame of Herp. The product, digested with KpnI and EcoRI, was ligated into the corresponding site of a plasmid vector, pZeoSV2(+) (Invitrogen), with a T7 priming site. Using the resultant plasmid DNA, pZeoSV2Herp, as a template, Herp was synthesized in vitro by the TNT T7 quick-coupled transcription/translation systems (Promega). [³⁵S]Met-labeled full-length Herp protein was obtained by in vitro transcription and translation using the Promega Coupled kit (Promega) and subjected to cleavage by purified caspase-3 (gift from G. Salvesen, The Burnham Institute) as described (18).

Immunofluorescence Confocal Microscopy—Following experimental treatments, cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 10 min, followed by a 30-min incubation in the presence of 5% nonimmune horse serum, and incubation in the presence of primary antibodies for 2 h or overnight. The primary antibodies included rabbit polyclonal antibodies against Herp (1:1000), Grp94, and Hsp60 (1:1000, StressGen), and mouse monoclonal antibodies against c-Myc (1:2000, Santa Cruz Biotechnology) and PS1 (1:500, Chemicon). Cells were then incubated for 1 h in PBS containing fluorescein isothiocyanate (FITC)-labeled horse anti-mouse IgG or Texas Red-labeled goat anti-rabbit secondary antibodies (1:50 dilution in PBS; Vector Laboratories). Cells were then washed with PBS, and images of fluorescence were acquired using the Zeiss LSM 510 confocal laser-scanning microscope.

Analyses of Cell Death and Mitochondrial Membrane Potential—Cell viability was assessed using the fluorescent DNA binding dye Hoechst 33342 or by the trypan blue exclusion method as described previously (18). Mitochondrial membrane potential was assessed using the fluorescent probe TMRE (Molecular Probes). Briefly, cells were incubated for 30 min in the presence of 100 nM TMRE, washed three times in fresh culture medium, and confocal images of cellular TMRE fluorescence were acquired using a confocal microscope (488 nm excitation and 510 nm emission). The average pixel intensity in individual cell bodies was determined using the software supplied by the manufacturer (Zeiss). Triplicate cultures were used for each condition, and all images were coded and analyzed without knowledge of experimental treatment history of the cultures.

Cytochrome c Release—Cytochrome c release was analyzed by Western blotting. Proteins were extracted in lysis buffer containing 250 mM sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EGTA, 1 mM EGTA, 1 mM dithiothreitol, and protease mixture inhibitor set (Roche Applied Science). Homogenates were centrifuged twice at 750 x g for 10 min at 4 °C, and the supernatants were centrifuged at 10,000 x g for 15 min at 4 °C to isolate the mitochondrial fraction. The resulting supernatants were further centrifuged at 100,000 x g for 1 h at 4 °C. The remaining supernatant represents the cytosolic fraction. Both the mitochondrial and cytosolic fractions were subjected to immunoblot analysis.

Caspase Activity Measurement—Caspase activity was assessed using a method described previously (11). Briefly, after exposure of cells to tunicamycin for designated time periods, cell membranes were permeabilized by incubation of cells in a solution of 0.01% digitonin in PBS for 5 min. Cells were then incubated for 30 min in PBS containing biotinylated DEVD-CHO (a substrate for caspase 3 as well as caspases 6, 7, 8, and 10) for 30 min. Cells were then fixed in a solution of 4% paraformaldehyde in PBS for 20 min, incubated for 5 min in PBS containing 0.2% Triton X-100, and then incubated for 30 min in PBS containing Oregon green-streptavidin (Molecular Probes). Images of fluorescence were captured using a confocal laser-scanning microscope, and the average pixel intensity in individual cell bodies was measured using software supplied by the manufacturer.

Calcium Imaging—Levels of intracellular free Ca²⁺ ([Ca²⁺]_i) were quantified by fluorescence imaging of the calcium indicator dye fura-2 as described previously (18). Briefly, cells were incubated for 30 min in the presence of 2 µM acetoxymethylester form of fura-2 (Molecular Probes) and then washed twice in Locke's buffer (mM: NaCl, 154; KCl, 5.6; CaCl₂, 2.3; MgCl_2, 1.0; NaHCO_3, 3.6; glucose, 10; Hepes 5, pH 7.2) and allowed to incubate an additional 20–30 min to allow complete deesterification of the dye. Measurement of ER Ca²⁺ content was performed by washing and imaging the cells in Ca²⁺-free Locke's buffer upon addition of 1 µM thapsigargin. Cells were imaged on a Zeiss Axiovert microscope (x40 oil immersion objective) coupled to an Attofluor imaging system. The average [Ca²⁺]_i in 40–60 cells per microscope field was quantified in four separate cultures per treatment condition.

Subcellular Fractionation—Pellets of frozen PC12 cells and cortical brain tissues were homogenized in ice-cold fractionation buffer pH 7.4 (20 mM HEPES, 10 mM KCl, 250 mM sucrose, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and a protease inhibitor mixture). Nuclei were pelleted by a 10-min spin at 750 x g, and the supernatant was recovered and centrifuged at 10,000 x g for 30 min. The mitochondrial pellet was resuspended in fractionation buffer, and the supernatant was recentrifuged for 1 h at 100,000 x g. The resulting supernatant contained the soluble cytosolic fraction, and the pellet constituted the microsomal fraction. The purity of the microsomal fraction was confirmed by the presence of grp94, an ER lumen protein.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Herp Is Present in the ER in the Developing and Adult Brain and Its Levels Are Markedly Increased in Neurons Subjected to ER Stress—We first examined Herp protein levels in the mouse brain during development. Herp was present at relatively high levels in the cerebral cortex during embryonic development from ages E11 to E15, but levels decreased between E15 and postnatal day 2, and were decreased further in adult mice (Fig. 1A). A developmental decrease in constitutive expression of Herp similar to that seen in the cerebral cortex also occurred in the hippocampus and cerebellum (data not shown). The differential expression of Herp from E11 to postnatal day 2 suggests that Herp may play an important role during CNS development. In the adult rat and mouse, Herp protein levels were similar in the cerebral cortex, hippocampus and cerebellum (Fig. 1B). Next, we investigated the subcellular localization of Herp in adult brain cells. Brain homogenates were fractionated into nuclear, mitochondrial, microsomal, and soluble fractions and analyzed by immunoblotting (Fig. 1C). Herp was predominantly found in the microsomal fraction along with grp94 suggesting that, as is the case in non-neuronal cells (13), Herp is also an ER-resident protein in neural cells. To determine whether Herp is induced by ER stress in neurons, we examined the time course of increase in Herp mRNA and protein levels in cultured primary rat cortical neurons after treatment with agents that induce ER stress. Basal levels of Herp mRNA and protein were very low in the cultured cortical neurons, but increased rapidly within 6 h of exposure to a subtoxic concentration tunicamycin (an agent that inhibits N-glycosylation) or thapsigargin (an inhibitor of the ER Ca²⁺-ATPase that induces depletion of ER Ca²⁺) (Fig. 1, D and E). Homocysteine (Fig. 1D) and 2-mercaptoethanol (data not shown), agents that modify the redox environment of the ER, also induced an elevation in Herp protein within 6 h. The up-regulation of Herp was specific for ER stress, because Herp levels did not increase in neurons subjected to serum deprivation, which induces apoptosis (data not shown).

View larger version (45K): [in this window] [in a new window]   FIG. 1. Herp is constitutively expressed in the brain where its levels change during development. A, immunoblot analysis showing relative levels of Herp and -tubulin proteins in samples of cerebral cortex from mice of the indicated ages from embryonic day 11 (E11) to postnatal day 2 (P2) and a 3-month-old adult. B, immunoblot showing Herp protein levels in the cerebral cortex, hippocampus, striatum, and cerebellum of three different 3-month-old adult mice and rats. C, immunoblot showing Herp and grp94 protein levels in nuclear, microsomal, mitochondrial, and cytosolic fractions of cerebral cortex tissue from an adult mouse. D, immunoblots showing levels of Herp protein in cultured rat primary cortical neurons before and after exposure to 1 µM thapsigargin, 1 µg/ml tunicamycin, or 100 µM homocysteine. For each immunoblot each lane was loaded with 50 µg of protein. E, levels of Herp mRNA in cultured rat primary cortical neurons before and after exposure to 1 µM thapsigargin and 1 µg/ml tunicamycin.

View larger version (30K): [in this window] [in a new window]   FIG. 2. Generation and characterization of PC12 cells stably overexpressing Herp proteins. A, schematic diagrams of Herp proteins expressed by constructs used in the transfection of PC12 cells. Ub, ubiquitin-like domain; TM, transmembrane domain. B, immunoblots showing basal levels of Herp protein in untransfected and vector-transfected (VT) PC12 cells and cells stably overexpressing Herp or c-Myc- and FLAG-tagged Herp (t-Herp). Whole cell lysates (50 µg/lane) were subjected to immunoblot analysis using the polyclonal rabbit anti-Herp (top panel) and monoclonal mouse anti-c-Myc antibodies (bottom panel). C, Herp proteins are localized to the ER in PC12 cells stably transfected with empty vector (VT), Herp or t-Herp. The staining with rabbit anti-Herp (Herp), mouse anti-c-Myc (for t-Herp), rabbit anti-grp94 (an ER protein), and rabbit anti-Hsp60 (a mitochondrial protein) was revealed by corresponding fluorescein isothiocyanate- and Texas Red-conjugated antibodies. D, immunoblots comparing protein levels of endogenous Herp in PC12 cells exposed to 5 and 20 µg/ml tunicamycin with that of ectopically expressed Herp used as positive control (CON). For each immunoblot each lane was loaded with an equal amount (50 µg) of total protein as confirmed by the actin band.

View larger version (35K): [in this window] [in a new window]   FIG. 3. Herp protects neural cells against ER stress-induced apoptosis. A and B, overexpression of Herp protects against ER stress-induced apoptosis. Cultures of vector-transfected (VT) and Herp-overexpressing PC12 cells were treated for various time points with (A) tunicamycin (Tun; 5 and 20 µg/ml) or (B) thapsigargin (100 nM), and the percentage of cells exhibiting condensed or fragmented nuclei in each culture was quantified. Values are the means and S.D. of three independent experiments. *, p < 0.01; **, p < 0.005 (ANOVA with Scheffe post-hoc tests) compared with VT PC12 cells. C and D, suppression of Herp induction by RNA interference sensitizes PC12 cells to ER-stress induced apoptosis. Small interfering RNA (siRNA) duplexes (for sequences and target mRNAs for each siRNA targeting Herp (siRNAHerp) or nonsilencing control siRNA (siRNACTRL) see "Experimental Procedures") were delivered in naive PC12 cells by nucleofection. One day after nucleofection, PC12 cells were exposed to 5 µg/ml Tun for the indicated time points and subsequently harvested for detection of endogenous levels of Herp, Grp78, and Grp94 by immunoblotting analysis (C) or fixed for quantitation of apoptotic cell death (D). Values are the means and S.D. of three dishes per group for each time point. *, p < 0.01 (ANOVA with Scheffe post-hoc tests) compared with mock-transfected cells and cells transfected with siRNACTRL.

Suppression of Herp expression with siRNA significantly increased sensitivity to tunicamycin-induced apoptosis. Reduction of basal Herp protein levels appeared to increase spontaneous apoptosis suggesting a central role for Herp in cell homeostasis (Fig. 3D). Cells transfected with the non-silencing control siRNA exhibited normal morphology (data not shown) and were not more sensitive to ER stress-induced apoptosis when compared with mock-transfected cells (Fig. 3D).

Overexpression of Herp Attenuates Activation of Downstream Caspases during ER Stress—In many cell types, including neurons, ER stress-induced death involves an apoptotic cascade involving activation of caspases (20). We found that tunicamycin induced an increase in caspase 3 activity in PC12 cells, and that that magnitude of caspase 3 activation was significantly decreased in cells overexpressing Herp (Fig. 4A). Apoptosis triggered by ER stress involves mitochondrial alterations that include membrane depolarization and release of cytochrome c (4, 21). Because Herp overexpression suppressed ER stress-induced caspase 3 activation, a caspase activated in response to cytochrome c release from mitochondria, we determined the effects of Herp overexpression on mitochondrial membrane potential and release of cytochrome c. TMRE fluorescence, an indicator of mitochondrial membrane potential, decreased in PC12 cells following exposure to tunicamycin for 24 h, and this decrease was significantly attenuated in cells overexpressing Herp (Fig. 4B). Cytochrome c release from the mitochondria, a central step in the apoptosis induced by many death stimuli, was also reduced in Herp-overexpressing cells compared with VT cells (Fig. 4C). Thus, Herp can maintain mitochondrial function and inhibit downstream events associated with ER stress-induced apoptosis such as mitochondrial depolarization and cytochrome c release.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Herp prevents mitochondrial dysfunction and caspase activation during ER stress. A, reduction of ER stress induced caspase-3 activity in Herp-overexpressing PC12 cells exposed to 20 µg/ml Tun. Quantitative analysis of caspase-3 activity measured using the fluorogenic DEVD-CHO caspase substrate. Values are the means and S.D. of three independent experiments. *, p < 0.01 (ANOVA with Scheffe post-hoc test). Comparable results were achieved in additional experiments utilizing two different PC12 clones overexpressing Herp. B, Herp attenuates the decrease in mitochondrial membrane potential following ER stress. Vector-transfected (VT) control cells and cells overexpressing Herp were exposed to 20 µg/ml Tun for the indicated time points and levels of TMRE fluorescence, a measure of mitochondrial membrane potential, were quantified. Values are the mean and S.D. of three separate experiments. *, p < 0.01, compared with Herp-overexpressing cells; #, p < 0.05 compared with non-stressed cells (ANOVA with Scheffe post-hoc test). C, Herp attenuates release of cytochrome c from mitochondria in ER-stressed cells. Cytochrome c release was assessed by immunoblotting of isolated mitochondrial and cytosolic fractions of cells exposed to 20 µg/ml Tun for the indicated time points. Comparable results were achieved in experiments utilizing an additional PC12 clone overexpressing Herp.

View larger version (60K): [in this window] [in a new window]   FIG. 5. Herp overexpression counteracts ER stress induced apoptotic signaling cascades involving JNK and caspase 12. A, left panels, immunoblots showing basal levels of grp78, hsp70, calreticulin, and Bcl-2 protein in vector-transfected (VT), and Herp-overexpressing cells. Right panels, immunoblots showing no changes in protein levels of Hsp70 and Bcl-2 in VT- and Herp-overexpressing cells treated with 20 µg/ml tunicamycin (Tun). For each immunoblot each lane was loaded with 50 µg of protein. The result shown is representative of three independent experiments. B, immunoblots showing the induction of several ER stress proteins in VT and Herp cells treated with 5 and 20 µg/ml Tun for the indicated hours. For each immunoblot, each lane was loaded with equal amount (50 µg) of total protein as confirmed by the actin band. Densitometric analyses (bottom panels) were performed on the immunoblots obtained from two independent studies. The ratio of the signal of the protein indicated on the x-axis and that of actin is shown on the y-axis in arbitrary units. Comparable results were achieved using a different PC12 clone overexpressing Herp. C, immunoblots showing the time course of Tun-induced phosphorylation of JNK/c-Jun in VT and Herp cells. Blots were first probed with antibodies directed against phospho-JNK or phospho-c-Jun (Ser63) and reprobed with antibodies against JNK1 and c-Jun to show that the overall levels of these proteins were not affected by Tun. Comparable results were achieved using a different PC12 clone overexpressing Herp. D, immunoblot showing the time course of caspase 12 activation in VT and Herp-overexpressing cells. The membrane was first reacted with an antibody that recognizes full-length (fl) caspase 12 and its proteolytic active fragment (indicated by asterisk) and reprobed with an antibody directed against JNK1 (not shown) to indicate equality of total protein loading.

In addition to the pro-apoptotic activation of JNK, ER stress induced by tunicamycin also promotes the activation of the ER-localized pro-caspase 12 (20). Fig. 5D shows that Herp attenuated the proteolytic activation of pro-caspase 12 which, compared with caspase-3 activation (Fig. 4A), also occurs very rapidly in VT cells. Sai et al. (14) recently showed that Herp physically interacts with PS1, an ER integral membrane protein that has been implicated in ER Ca²⁺ homeostasis (11, 12). Since mobilization of the ER Ca²⁺ store and increase in the intracellular Ca²⁺ concentration could play a role in the activation of JNK (28) and caspase 12 (29), we performed a series of studies to determine whether Herp affects ER Ca²⁺ homeostasis under conditions associated with ER stress.

Herp Stabilizes ER Ca²⁺ Homeostasis in Neural Cells Subjected to ER Stress—The ER plays a critical role in maintaining and regulating levels of intracellular Ca²⁺, a messenger critical for diverse cellular functions and regulator of neuronal cell survival and death (6, 30). Recent findings suggest that ER Ca²⁺ stores are also targets of diverse apoptotic stimuli, such that stress-induced dysregulation of ER Ca²⁺ homeostasis plays a major role in triggering apoptosis (2, 6). We therefore determined if and how ER stress affects cellular Ca²⁺ homeostasis and whether Herp modifies Ca²⁺ homeostasis and signaling in neurons. First, we investigated the effects of overexpressing Herp on ER Ca²⁺ release and content in PC12 cells. The basal [Ca²⁺]_i in VT and Herp overexpressing cells was similar, averaging 50–60 nM (Fig. 6A). Bradykinin (BK), an agonist that activates cell surface receptors coupled to IP₃ production, evokes a rapid and transient increase of [Ca²⁺]_i, the amplitude of which was significantly decreased by 44% in cells overexpressing Herp compared with VT cells (Fig. 6A). The ER Ca²⁺ store was measured as the rapid increase in [Ca²⁺]_i on addition of thapsigargin to cells incubated in Ca²⁺-free medium. The peak [Ca²⁺]_i elevation induced by thapsigargin was attenuated by 49% in cells overexpressing Herp, suggesting that Herp reduces the total pool of ER Ca²⁺ available for release (Fig. 6B). Next, we examined the effects of Herp overexpression on ER Ca²⁺ stores under conditions associated with ER stress. In cells stressed for 8 and 16 h with tunicamycin, the total pool of ER Ca²⁺ available for release was decreased by 62 and 40%, respectively, in cells overexpressing Herp compared with VT cells (Fig. 6C). This is the first report showing that tunicamycin caused a significant increase in ER Ca²⁺ storage and release in a manner analogous to overexpression of sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) (31) and calreticulin (32) proteins, and that Herp overexpression effectively attenuated this increase. Lastly, we examined the effect of Herp inhibition on ER Ca²⁺ release induced by thapsigargin. All procedures were performed as in prior experiments except that PC12 cells were transfected with either siRNA_Herp2 or siRNA_CTRL 24 h prior to induction of ER stress with tunicamycin. The resting [Ca²⁺]_i and the peak [Ca²⁺]_i elevation induced by thapsigargin was significantly higher in ER-stressed PC12 cells transfected with the siRNA_Herp2 compared with that in mock-transfected cells or cells transfected with the siRNA_CTRL (Fig. 6D). Collectively, these data suggest that Herp regulates total ER Ca²⁺ load and release under conditions associated with ER stress.

View larger version (28K): [in this window] [in a new window]   FIG. 6. Herp stabilizes ER Ca2+ homeostasis during ER stress. A, representative recording showing levels of intracellular Ca2+ ([Ca2+]i) before and after addition of 10 µM bradykinin (BK; arrow indicates time of addition) to vector-transfected (VT) and Herp-overexpressing PC12 cell clones. Values are the mean and S.D. of determinations made in four separate cultures (40–60 cells assessed/culture). B, representative recording showing levels of [Ca2+]i prior to and after exposure of VT and Herp-overexpressing cells to 1 µM thapsigargin (Thap; arrow indicates time of addition). Values are the mean and S.D. of determinations made in four separate cultures. C, quantitative data from three different experiments showing the peak amplitude of the Thap-induced rise in [Ca2+]i in vehicle-treated cells (Con) and cells treated with 20 µg/ml tunicamycin for 8 and 16 h. *, p < 0.01; **, p < 0.005 (ANOVA with Scheffe post-hoc tests). D, quantitative data showing the resting levels of [Ca2+]i and the peak amplitude of the Thap-induced rise in [Ca2+]i in mock-transfected PC12 cells (vehicle) or cells transfected with the siRNAHerp2 or siRNACTRL. All cultures were exposed to 5 µg/ml tunicamycin for 8 h prior to measurements of [Ca2+]i. *, p < 0.01; **, p < 0.005 (ANOVA with Scheffe post-hoc tests).

View larger version (45K): [in this window] [in a new window]   FIG. 7. Herp is a substrate of caspases activated in response to ER stress. A, schematic diagram of Herp showing the locations of putative caspase cleavage sites near the transmembrane and C-terminal regions. B, immunoblot (50 µg protein/lane) showing time-dependent decrease of full-length Herp protein levels and corresponding increase in levels of the 30- and 48-kDa Herp fragments (*) in PC12 cells following induction of ER stress with 20 µg/ml tunicamycin (Tun). C, immunoblot showing inhibition of Tun-induced Herp proteolysis by the pan-caspase inhibitor zVAD-fmk (100 µM). As control, the samples were immunoblotted using an antibody against PARP (PARP is cleaved by caspase 3 into a 85-kDa fragment). D, left panel, autoradiogram showing the time course of in vitro generation of the 48- and 30-kDa Herp cleavage fragments (indicated by asterisks) from 35S-labeled Herp by purified recombinant caspase-3. Right panels, immunoblots (50 µg protein/lane) showing cross-reactivity of the 30-kDa Herp cleavage fragment (indicated by arrowhead) with the anti-Herp and anti-cMyc antibodies (top). Immunoblots showing the presence of the 30-kDa Herp fragment (indicated by arrowhead) in immunoprecipitated materials, which immunoreacted with the anti-cMyc but not anti-FLAG antibodies (bottom). IgGH and IgGL denote the heavy and light chains of the immunoglobulin G protein, respectively. E, double labeling confocal images showing release of Herp fragments from the ER into the cytosol during ER stress induced by 20 µg/ml Tun. PC12 cells stably expressing t-Herp were stained with mouse anti-c-Myc (red fluorescence) and rabbit anti-presenilin (green fluorescence), as the ER marker, before and 24 h following exposure to 20 µg/ml Tun. F, comparison of the time course of Herp protein degradation and appearance of Herp cleavage fragments (indicated by asterisks) in cells stably expressing either mutagenized D266E or wild-type Herp following a short (left panel) and prolonged (right panel) exposure to 20 µg/ml Tun. G, reduced vulnerability of cells expressing mutagenized D266E Herp to death following prolonged exposure to 5 and 20 µg/ml Tun. Cultures of wild-type and mutagenized D266E Herp overexpressing PC12 cells were treated with Tun for 12 and 24 h (short ER stress) or for 48 and 72 (prolonged ER stress), and cell viability was assessed by the trypan blue dye exclusion method. Values are the mean and S.D. of three independent experiments. *, p < 0.05 (ANOVA with Scheffe post-hoc tests).

View larger version (34K): [in this window] [in a new window]   FIG. 8. Herp stabilizes cellular Ca2+ homeostasis and prevents cell death in neural cells exposed to amyloid peptide. A, vector-transfected (VT) control and Herp-overexpressing cells were exposed to the indicated concentrations of A-(1–42) for the indicated time periods and neuronal survival was quantified by counting trypan blue-positive cells. Values are the mean and S.D. of four separate experiments. *, p < 0.01; **, p < 0.001 compared with value for VT control cells (ANOVA with Scheffe post-hoc tests). B, VT control and Herp-overexpressing cells were exposed for 2 h to 10 µM A-(1–42). Intracellular Ca2+ levels were then measured, by imaging of the Ca2+ indicator dye fura-2, prior to and during exposure to either bradykinin (BK;10 µM) or thapsigargin (Thap;1 µM). Representative experiments are shown in the left and middle graphs (each trace represents the mean intracellular Ca2+ concentration of measurements made in 40–60 cells), and summary results from four separate experiments are shown in the graph at the right. Values are the mean and S.D. *, p < 0.01 compared with value for VT control cells; ANOVA with Scheffe post-hoc tests. C, primary rat cortical cultures were exposed to 10 µM A-(1–42) for the indicated time periods. Proteins in cell lysates were subjected to immunoblot analysis using antibodies against Herp and actin. Immunoblot shows increased levels of the 30-kDa Herp cleavage product (*) with increasing time following exposure to A-(1–42).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ability of Herp to prevent ER stress-induced death was correlated with its ability to stabilize cellular Ca²⁺ homeostasis. Dysregulation of ER Ca²⁺ homeostasis occurs as an early event during many forms of apoptosis and has been implicated in the pathophysiology of several neurodegenerative diseases including Alzheimer's, Huntington's, and prion diseases (6, 34, 35). Several different agents that evoke ER stress including Ca²⁺ ionophores and thapsigargin have been shown to disrupt intracellular Ca²⁺ homeostasis, suggesting a central role for Ca²⁺ in ER stress-induced apoptosis. ER stress increases the basal ER Ca²⁺ content (36), which is associated with a marked increase in Ca²⁺ fluxes across the ER membrane, decreased mitochondrial membrane potential and increased vulnerability of the cells to apoptosis (37, 38). In this regard, several members of the pro-apoptotic Bcl-2 family such as Bax and BIK, act on the ER to promote ER Ca²⁺ mobilization into the mitochondria (39, 40). Indeed, dantrolene and other agents that block ER Ca²⁺ release effectively suppress apoptosis induced by tunicamycin, oxidative stress and other apoptotic stimuli (3, 11, 18). Collectively, the available data suggest that increased Ca²⁺ release from the ER could serve as the primary trigger for ER stress-induced apoptosis. Our findings show that Herp plays a pivotal neuroprotective role under conditions of ER stress and exposure to A, and that this beneficial function of Herp is mediated, in part, by stabilization of ER Ca²⁺ homeostasis.

The mechanism by which Herp maintains ER Ca²⁺ homeostasis appears to be different from the proposed anti-apoptotic action of Bcl-2. Bcl-2 may reduce ER Ca²⁺ content and release (41–43), either directly by enhancing the permeability of the ER membrane to Ca²⁺ (44) or indirectly by down-regulating the transcription of genes encoding for proteins that are involved in ER Ca²⁺ re-uptake and sequestration such as SERCA2b and calreticulin (45). In addition, Bcl-2 transfected cells also exhibit reduced capacitative Ca²⁺ entry (46), a process whereby the depletion of Ca²⁺ from intracellular stores activates plasma membrane Ca²⁺ channels. However, in our study both the basal and ER stress-induced levels of Bcl-2 and calreticulin in Herp and VT cells were not significantly different (Fig. 5, A and B) and capacitative Ca²⁺ entry was not reduced in Herp-overexpressing cells.²

Activation of ER stress-induced apoptotic signaling cascades has been associated with dysregulated Ca²⁺ release from the ER (6, 29, 34). Agents that induce ER stress including tunicamycin and A can specifically activate ER-associated caspase 12 (10, 20). Activation of caspase 12, which may occur very early prior to any detectable changes in mitochondrial function, appears to be required for ER stress-induced apoptosis, because neurons from caspase 12-deficient mice are resistant to apoptosis induced by A (10). One mode of caspase 12 activation is by cleavage of procaspase 12 by calpain in response to elevation of intracellular Ca²⁺ levels (29). Calpain is one of the most abundant neutral proteases in the nervous system and its expression is increased in neuropathological conditions such as AD and Parkinson's disease (47), suggesting a role for this Ca²⁺-activated protease in stress responses associated with neurodegenerative disorders. Our data showing that Herp overexpression can normalize ER Ca²⁺ homeostasis and prevent Ca²⁺ overload during ER stress suggest that Herp can counteract the activation of calpain/caspase12 and downstream executor caspases. Another pro-apoptotic signaling cascade induced by ER stress is the activation of JNK/c-Jun, which is attenuated by Herp. Since activation of these cascades is involved in A-induced neurotoxicity (10, 27, 49), Herp may protect cells exposed to A by suppressing caspase 12 and JNK activation.

Herp maintains mitochondrial function during ER stress. The Ca²⁺ released from the ER is rapidly redistributed into the mitochondria (38) and accumulation of Ca²⁺ in this organelle may cause mitochondrial membrane depolarization and release of cytochrome c, which induces caspase 3 activation (4, 21). By limiting this pro-apoptotic ER-mitochondrial Ca²⁺-dependent cross-talk, Herp may attenuate mitochondrial membrane depolarization, cytochrome c release, and activation of caspase 3 (Fig. 4).

We found that Herp protein is rapidly cleaved by caspase 3 activated during ER stress-induced apoptosis. Cleavage of Herp releases the N-terminal fragment that contains the ubiquitin-like domain into the cytosol. Site-directed mutagenesis of the main caspase cleavage site prevented generation of the N-terminal fragment and significantly enhanced the neuroprotective function of Herp. Although the present study did not address possible cytotoxic properties of the released Herp fragments, it is more likely that cleavage reduces its intrinsic anti-apoptotic function at the ER. In this regard, it is noteworthy that the N-terminal ubiquitin-like domain of Parkin, a cytosolic protein, which also confers cytoprotection toward ER stress (50), is similarly cleaved and removed by caspases (51). As Herp has no demonstrable E3 ubiquitin-protein ligase activity, it is unlikely that Herp plays a direct role in protein substrate ubiquitination. On the other hand, the N-terminal ubiquitin-like domain of Herp may function as a proteasome-interacting domain, as has recently been demonstrated for Parkin (52) and other ubiquitin-like domain-containing proteins (53). Our findings that ER-stressed cells overexpressing Herp exhibited reduced ER Ca²⁺ release suggest that Herp may bind to and facilitate targeting of ER resident proteins involved in ER calcium regulation such as SERCA and the ER Ca²⁺ release channels for proteasome-mediated degradation (54–56). Studies employing specific proteasomal inhibitors will further elucidate possible mechanisms by which Herp stabilizes ER calcium homeostasis under conditions associated with ER stress. Alternatively, Herp may inhibit the sustained ER Ca²⁺ release during ER stress by inhibiting the association of the ER Ca²⁺ release channels with pathophysiological ligands including the huntington-associated protein 1 (35) and cytochrome c (57).

A recent study by Sai et al. (14) showed that Herp physically interacts with PS1, an ER integral membrane protein that has been linked to APP metabolism, ER Ca²⁺ regulation, and apoptosis (6). The contribution of this interaction to ER Ca²⁺ homeostasis may be dependent on the expression of APP as its processed carboxyl APP derivatives have been shown to regulate the expression of genes involved in ER Ca²⁺ homeostasis (58). In this study, we did not examine levels of the APP derivatives nor their downstream nuclear signaling activities (59). Clearly, further insight into Herp function will emerge from a better understanding of its interaction with PS1 and other ER resident proteins and how these interactions affect ER Ca²⁺ homeostasis and downstream cellular Ca²⁺ signaling that has been shown to impact APP processing (14, 15) and neuronal vulnerability to ER stress (11). PS1 mutations that cause AD perturb ER Ca²⁺ regulation in a manner that results in overfilling of ER Ca²⁺ stores (11, 12). This alteration in ER Ca²⁺ stores renders neurons vulnerable to death induced by excitatory amino acids, oxidative stress and other apoptotic stimuli (6). The PS1 mutations can exacerbate mitochondrial dysfunction and apoptotic cascades and these adverse effects of the mutations can be attenuated by agents that inhibit release of Ca²⁺ from the ER (11). The ability of Herp to protect neurons against A toxicity, and the apparent increase in proteolytic cleavage of Herp following prolonged exposure to A suggests a possible contribution of reduced Herp function to the neurode-generative process in AD. A better understanding of the mechanisms that regulate Herp expression, and of the ways in which Herp stabilizes cellular calcium homeostasis and suppresses apoptotic cascades, may therefore reveal novel preventative and therapeutic strategies for neurodegenerative disorders.

	FOOTNOTES

 
^* These studies were supported by the NIA, National Institutes of Health and by a grant (to M. P. M.) from the Alzheimer's Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Neurosciences, Gerontology Research Center, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8601; Fax: 410-558-8323; E-mail: chanst{at}grc.nia.nih.gov.

¹ The abbreviations used are: ER, endoplasmic reticulum; PBS, phosphate-buffered saline; JNK, Jun N-terminal kinase; ANOVA, analysis of variance; grp, glucose-regulated protein; AD, Alzheimer's disease; APP, amyloid precursor protein; Herp, homocysteine-induced ER protein; z, benzyloxycarbonyl; fmk, fluoromethylketone; PS1, presenilin-1; A, amyloid ; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase.

² S. Chan and M. P. Mattson, unpublished data.

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