Herp Stabilizes Neuronal Ca2+ Homeostasis and Mitochondrial Function during Endoplasmic Reticulum Stress
2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês
10.1074/jbc.m404272200
ISSN1083-351X
AutoresSic L. Chan, Weiming Fu, Peisu Zhang, Aiwu Cheng, Jaewon Lee, Koichi Kokame, Mark P. Mattson,
Tópico(s)Nerve injury and regeneration
ResumoIn 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. 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. The endoplasmic reticulum (ER) 1The 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; Aβ, amyloid β; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase. 1The 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; Aβ, amyloid β; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase. 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 (1Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-355Crossref PubMed Scopus (669) Google Scholar). ER stress has been documented in neurons in a variety of acute pathological conditions including cerebral ischemia and severe epileptic seizures (2Paschen W. Frandsen A. J. Neurochem. 2001; 79: 719-725Crossref PubMed Scopus (208) Google Scholar). 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 (3Yu Z. Luo H. Fu W. Mattson M.P. Exp. Neurol. 1999; 155: 302-314Crossref PubMed Scopus (402) Google Scholar) but may also trigger a form of programmed cell death called apoptosis (4Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: 255-263Crossref PubMed Scopus (1284) Google Scholar), 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 (5Katayama T. Imaizumi K. Sato N. Miyoshi K. Kudo T. Hitomi J. Morihara T. Yoneda T. Gomi F. Mori Y. Nakano Y. Takeda J. Tsuda T. Itoyama Y. Murayama O. Takashima A. St George-Hyslop P. Takeda M. Tohyama M. Nat. Cell Biol. 1999; 1: 479-485Crossref PubMed Scopus (483) Google Scholar, 6Mattson M.P. LaFerla F.M. Chan S.L. Leissring M.A. Shepel P.N. Geiger J.D. Trends Neurosci. 2000; 23: 222-229Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar), Parkinson's (7Ryu E.J. Harding H.P. Angelastro J.M. Vitolo O.V. Ron D. Greene L.A J. Neurosci. 2002; 22: 10690-10698Crossref PubMed Google Scholar), and Huntington's (8Kouroku Y. Fujita E. Jimbo A. Kikuchi T. Yamagata T. Momoi M.Y. Kominami E. Kuida K. Sakamaki K. Yonehara S. Momoi T. Hum. Mol. Genet. 2002; 11: 1505-1515Crossref PubMed Scopus (168) Google Scholar) diseases. Alzheimer's disease (AD) results from altered proteolytic processing of the amyloid precursor protein (APP), resulting in aggregation of neurotoxic forms of amyloid β-peptide (Aβ) (9Mattson M.P. Physiol. Rev. 1997; 77: 1081-1132Crossref PubMed Scopus (872) Google Scholar). Exposure of cultured neurons to Aβ-peptide, and metabolic and oxidative insults can induce an ER stress response (6Mattson M.P. LaFerla F.M. Chan S.L. Leissring M.A. Shepel P.N. Geiger J.D. Trends Neurosci. 2000; 23: 222-229Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 10Nakagawa T. Zhu H. Morishima N. Li E. Xu J. Yankner B.A. Yuan J. Nature. 2000; 403: 98-103Crossref PubMed Scopus (2912) Google Scholar). Moreover, mutations in presenilin-1 (PS1) that cause early-onset familial AD perturb ER Ca2+ homeostasis (11Guo Q. Sopher B.L. Furukawa K. Pham D.G. Robinson N. Martin G.M. Mattson M.P J. Neurosci. 1997; 17: 4212-4222Crossref PubMed Google Scholar, 12Leissring M.A. Akbari Y. Fanger C.M. Cahalan M.D. Mattson M.P. LaFerla F.M. J. Cell Biol. 2000; 149: 793-798Crossref PubMed Scopus (284) Google Scholar) and impair the ability of neurons to engage a cytoprotective ER stress response (20Rao R.V. Hermel E. Castro-Obregon S. del Rio G. Ellerby L.M. Ellerby H.M. Bredesen D.E. J. Biol. Chem. 2001; 276: 33869-33874Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). The adverse effects of Aβ and PS1 mutations on ER function may sensitize neurons to excitotoxicity and apoptosis (11Guo Q. Sopher B.L. Furukawa K. Pham D.G. Robinson N. Martin G.M. Mattson M.P J. Neurosci. 1997; 17: 4212-4222Crossref PubMed Google Scholar). 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 (13Kokame K. Agarwala K.L. Kato H. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The function of Herp is unknown. Sai et al. (14Sai X. Kawamura Y. Kokame K. Yamaguchi H. Shiraishi H. Suzuki R. Suzuki T. Kawaichi M. Miyata T. Kitamura T. De Strooper B. Yanagisawa K. Komano K.H. J. Biol. Chem. 2002; 277: 12915-12920Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) 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 (15Yoo A.S. Cheng I. Chung S. Grenfell T.Z. Lee H. Pack-Chung E. Handler E. Shen J. Xia W. Tesco G. Saunders A.J. Ding K. Frosch M.P. Tanzi R.E. Kim T.W. Neuron. 2000; 27: 561-572Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). In the present study we demonstrate a role for Herp in stabilizing cellular Ca2+ homeostasis and preventing neuronal death following ER stress. Neuronal Cell Cultures—Hippocampal and cortical cell cultures were established from 18 day Sprague-Dawley rat embryos as described previously (16Mattson M.P. Barger S.W. Begley J.G. Mark R.J. Methods Cell Biol. 1995; 46: 187-216Crossref PubMed Scopus (310) Google Scholar). 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 (13Kokame K. Agarwala K.L. Kato H. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). 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 500× stock in dimethyl sulfoxide. Amyloid β-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 (siRNAHerp) were prepared as described. Briefly, 100 nm sense primers (5′-AAACAACCGGCTCTTCGTCdTdT-3′ for siRNAHerp1;5′-AAUCCUGACUUCCGGGAAAdTdT-3′ for siRNAHerp2; 5′-AAUCCUGACUUCCGGGAAAdTdT-3′ for siRNAHerp3) and corresponding antisense primers were mixed in 50 μl of water with 5 μlof10× 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 (siRNACTRL) were synthesized using scrambled sequences as described above. GenBank™ 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); β-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 (17Chan S.L. Mayne M. Holden C.P. Geiger J.D. Mattson M.P. J. Biol. Chem. 2000; 275: 18195-18200Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). 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), β-tubulin, and α-actin (Sigma), Herp (13Kokame K. Agarwala K.L. Kato H. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), 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 (18Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (204) Google Scholar). 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 (18Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (204) Google Scholar). 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 × g for 10 min at 4 °C, and the supernatants were centrifuged at 10,000 × g for 15 min at 4 °C to isolate the mitochondrial fraction. The resulting supernatants were further centrifuged at 100,000 × 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 (11Guo Q. Sopher B.L. Furukawa K. Pham D.G. Robinson N. Martin G.M. Mattson M.P J. Neurosci. 1997; 17: 4212-4222Crossref PubMed Google Scholar). 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 (18Ellerby L.M. Hackam A.S. Propp S.S. Ellerby H.M. Rabizadeh S. Cashman N.R. Trifiro M.A. Pinsky L. Wellington C.L. Salvesen G.S. Hayden M.R. Bredesen D.E. J. Neurochem. 1999; 72: 185-195Crossref PubMed Scopus (204) Google Scholar). 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; MgCl2, 1.0; NaHCO3, 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 (×40 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 × g, and the supernatant was recovered and centrifuged at 10,000 × g for 30 min. The mitochondrial pellet was resuspended in fractionation buffer, and the supernatant was recentrifuged for 1 h at 100,000 × 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. 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 (13Kokame K. Agarwala K.L. Kato H. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), 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). Overexpression of Herp Protects PC12 Cells against ER Stress-induced Cell Death—To determine the consequences of an increase in Herp protein levels during the ER stress response, we overexpressed Herp, or Herp-tagged with an N-terminal c-Myc tag and a C-terminal FLAG tag, in PC12 cells (Fig. 2A). Several clones stably overexpressing Herp were selected, and the level of Herp overexpression and its subcellular localization were analyzed by immunoblot and immunocytochemical analyses. The levels of Herp and tagged Herp (t-Herp) proteins in extracts of stably transfected PC12 were 4–8-fold higher than the endogenous level of Herp in VT cells (Fig. 2B). Confocal images of Herp immunoreactivity revealed localization to reticular structures in the cytoplasm, which were also stained with the anti-grp94 antibody demonstrating that Herp was mainly localized in the ER (Fig. 2C) as previously reported (13Kokame K. Agarwala K.L. Kato H. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). The tags did not affect protein localization, because the staining pattern of t-Herp was identical to that of the untagged protein. The levels of ectopic expression of Herp protein were approximately 2–6-fold higher than the total inducible Herp protein levels achieved in cells following exposure to 5 and 20 μg/ml tunicamycin, respectively, for 12 h (Fig. 2D), a time point at which greater than 80% of the cells exposed to 20 μg/ml tunicamycin were still viable (see also Fig. 3A). The effect of increased Herp levels on the vulnerability of cells to ER stress-induced death was determined by exposing Herp-overexpressing and VT cells to increasing concentrations of tunicamycin. Compared with VT cells, Herp-overexpressing cells were significantly more resistant to death induced by tunicamycin (Fig. 3A). After 24 h of exposure to a toxic concentration of tunicamycin (20 μg/ml), 77% of the VT cells were apoptotic compared with 42% of cells overexpressing Herp. Herp-overexpressing cells were also more resistant to ER stress-induced apoptosis following a prolonged exposure to a subtoxic concentration of tunicamycin (5 μg/ml). Similar results were obtained when thapsigargin (100 nm) was applied as the ER stressor (Fig. 3B). We also tested several other clones overexpressing Herp, and they were consistently more resistant to cell death induced by ER stress but were not more resistant to death induced by serum withdrawal (data not shown).Fig. 3Herp 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.View Large Image Figure ViewerDownload (PPT) siRNA Inhibition of Herp Expression Increases Vulnerability of Neurons to ER Stress-induced Cell Death—If induction of Herp expression during the ER stress response is critical for maintaining neuronal surv
Referência(s)