Endoplasmic Reticulum Stress-inducible Protein, Herp, Enhances Presenilin-mediated Generation of Amyloid β-Protein
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m112372200
ISSN1083-351X
AutoresXiaorei Sai, Yuuki Kawamura, Koichi Kokame, Haruyasu Yamaguchi, Hirohisa Shiraishi, Ryo Suzuki, Toshiharu Suzuki, Masashi Kawaichi, Toshiyuki Miyata, Toshio Kitamura, Bart De Strooper, Katsuhiko Yanagisawa, Hiroto Komano,
Tópico(s)Cellular transport and secretion
ResumoPresenilin (PS) is essential for the γ-cleavage required for the generation of the C terminus of amyloid β-protein (Aβ). However, the mechanism underlying PS-mediated γ-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of γ-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of Aβ generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Aβ generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in Aβ accumulation, including the formation of senile plaques and vascular Aβ deposits. Presenilin (PS) is essential for the γ-cleavage required for the generation of the C terminus of amyloid β-protein (Aβ). However, the mechanism underlying PS-mediated γ-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of γ-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of Aβ generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Aβ generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in Aβ accumulation, including the formation of senile plaques and vascular Aβ deposits. Alzheimer disease presenilin amyloid β-protein β-amyloid precursor protein endoplasmic reticulum enzyme-linked immunosorbent assay human embryonic kidney radioimmune precipitation buffer Mutations in the presenilin genes, PS1 and PS2, cause early onset familial Alzheimer disease (AD)1 (1.Sherrington R. Rogaev E.I. Liang Y. Rogaeva E.A. Levesque G. Ikeda M. Chi H. Lin C. Li G. Holman K. Tsuda T. Mar L. Foncin J.-F. Bruni A. Montesi M.P. Sorbi S. Rainero I. Pinessi L. Nee L. Chumakov I. 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PS is required for intramembranous cleavage of APP (termed γ-cleavage) and Notch (5.De Strooper B. Saftig P. Craessaerts K. Vanderstichele H. Guhde G. Annaert W. Von Figura K. Van Leuven F. Nature. 1998; 391: 387-390Crossref PubMed Scopus (1552) Google Scholar, 6.De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1800) Google Scholar, 7.Herreman A. Serneels L. Annaert W. Collen D. Schoonjans L. De Strooper B. Nat. Cell. Bio. 2000; 2: 461-462Crossref PubMed Scopus (450) Google Scholar, 8.Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell. Bio. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar). Recently, ErbB4 has been found to be another natural substrate of the PS-mediated intramembranous proteolysis (9.Lee H.J. Jung K.M. Huang Y.Z. Bennett L.B. Lee J.S. Mei L. Kim T.W. J. Biol. Chem. 2002; 277: 6318-6323Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Interestingly, although the γ-cleavage of APP is a critical step toward the production of Aβ, the major intramembranous cleavage site of APP was found to be distinct from the γ-cleavage site (10.Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11.Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Crossref PubMed Scopus (430) Google Scholar). The mechanism underlying PS-mediated intramembranous proteolysis including γ-cleavage remains to be clarified. To date, a number of PS-interacting proteins have been identified, but no natural interactors with PS have been found to modulate Aβ generation, although some mutant forms of nicastrin, which was identified as a component of the PS complex, increase Aβ production (12.Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (824) Google Scholar). To elucidate the mechanism underlying the PS-mediated γ-cleavage, we have recently developed a new functional screening method for identifying cDNAs that increase the degree of γ-cleavage using a combination of γ-cleavage-dependent puromycin-resistant assay and Aβ quantitation (see "Experimental Procedures"; the details of this screening method will be described elsewhere). Using this method, we have identified Herp, which was originally identified as a homocysteine-induced protein (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Interestingly, elevated levels of homocysteine are correlated with multiple neurological disorders (14.Clarke R. Smith A.D. Jobst K.A. Refsum H. Sutton L. Ueland P.M. Arch. Neurol. 1998; 55: 1449-1455Crossref PubMed Scopus (1271) Google Scholar, 15.Gottfries C.G. Lehmann W. Regland B. J. Neural Transm. 1998; 105: 773-786Crossref PubMed Scopus (54) Google Scholar, 16.Miller J.W. Nutr. Rev. 1999; 57: 126-129PubMed Google Scholar). Plasma homocysteine levels have been reported to be elevated in some cases of AD (16.Miller J.W. Nutr. Rev. 1999; 57: 126-129PubMed Google Scholar) and could be an early marker of cognitive impairment in the elderly (17.Lehmann M. Gottfries C.G. Regland B. Dement. Geriatr. Cogn. Disord. 1999; 10: 12-20Crossref PubMed Scopus (232) Google Scholar). Herp mRNA is constitutively expressed in various tissues, but its expression is up-regulated by homocysteine or the inducers of ER stress such as 2-mercaptoethanol, A23187, and thapsigargin (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 18.Kokame K. Kato H. Miyata T. J. Biol. Chem. 2001; 276: 9199-9205Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Herp is a membrane protein localized in the ER, and it has an N-terminal ubiquitin-like domain; the function of this protein, however, is not known (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). In contrast to ER stress-induced molecular chaperones including BiP and GRP94 in the ER lumen, a major portion of Herp molecules are present in the cytoplasmic side of the ER membrane (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Herp may have a role distinct from that of molecular chaperones under ER stress. Here, we show that Herp enhances PS-mediated Aβ generation and that it interacts with PS. We designed a functional screening system for isolating cDNAs that increase the degree of γ-cleavage as follows. (The details of the screening system will be described elsewhere.) 2H. Komano, Y. Kawamura, H. Shiraishi, X. Sai, R. Suzuki, M. Kawaichi, T. Kitamur, and K. Yanagisawa, unpublished data. We established a cell line (designated as A5–9), BaF/3 cells (19.Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp. Hematol. 1996; 24: 324-329PubMed Google Scholar) stably transfected with pCxN-C53NICD (see below) and pHES1-pac (see below) in which an increase in the degree of γ-cleavage confers on the cells an increase in puromycin resistance. BaF/3 cells do not express endogenous APP. 3H. Komano and K. Yanagisawa, unpublished data. To initiate screening, a human hippocampus-derived cDNA library in a retroviral vector, pMX, was infected into A5–9 cells as reported previously (19.Onishi M. Kinoshita S. Morikawa Y. Shibuya A. Phillips J. Lanier L.L. Gorman D.M. Nolan G.P. Miyajima A. Kitamura T. Exp. Hematol. 1996; 24: 324-329PubMed Google Scholar, 20.Xu X. Leo C. Jang Y. Chan E. Padilla D. Huang B.C. Lin T. Gururaja T. Hitoshi Y. Lorens J.B. Anderson D.C. Sikic B. Luo Y. Payan D.G. Nolan G.P. Nat. Genet. 2001; 27: 23-29Crossref PubMed Scopus (88) Google Scholar). The infected A5–9 cells were treated with a lethal dose of puromycin. The level of Aβ40 secreted from surviving clones was measured using a highly sensitive immunoblotting method. The cDNAs from surviving clones expressing a higher level of Aβ40 as compared with that of parental cells were extracted and amplified by PCR using vector primers. One of these encoded Herp lacking Gln76–Lys100. Since we did not know whether this Herp variant is a natural form or a PCR artifact product, we recloned a previously reported Herp cDNA (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) into pcDNA3.1 or pMX. The effect of the Herp cDNA on Aβ generation from the full-length APP was then tested using HEK293 cells stably expressing APP. By the PCR method, we generated a chimeral cDNA encoding C53NICD that consists of the first Met plus the Asp596–Leu648fragment of APP695 (C100 (N-terminal truncated APP starting at the β-secretase site) lacking the intracellular domain) and the C-terminal intracellular domain (from Ser1745 to the C terminus) of mouse Notch-1. This chimeral cDNA was inserted into pCxN (21.Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4597) Google Scholar) and designated as pCxN-C53NICD. A plasmid named pHES1-pac, which contains a puromycin-resistant gene (pac) driven by the HES1 promoter (a DNA element responsible for Notch-dependent gene expression) (22.Takebayashi K. Sasai Y. Sakai Y. Watanabe T. Nakanishi S. Kageyama R. J. Biol. Chem. 1994; 269: 5150-5156Abstract Full Text PDF PubMed Google Scholar), was constructed as follows: (i) The HES1 promoter isolated by the PCR method from the mouse genomic library was inserted into a vector, PGV-B (TOYO B-Net Co., Ltd., Tokyo, Japan). This plasmid was designated as PGV-B-HES-1. (ii) pac of pPUR (CLONTECH) was inserted into PGV-B-HES-1. The final plasmid was designated as pHES-pac. We ensured that the cells expressing C53NICD, as well as the cells expressing APP, generate Aβ40 and Aβ42. We also confirmed that the expression of C53NICD in the cells induced the expression of the luciferase gene driven by the HES1 promoter. The retrovirus-mediated infection was carried out as reported previously (20.Xu X. Leo C. Jang Y. Chan E. Padilla D. Huang B.C. Lin T. Gururaja T. Hitoshi Y. Lorens J.B. Anderson D.C. Sikic B. Luo Y. Payan D.G. Nolan G.P. Nat. Genet. 2001; 27: 23-29Crossref PubMed Scopus (88) Google Scholar). The gene encoding Herp tagged with the N-terminal Myc and C-terminal FLAG was prepared as described previously (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). HEK293 cells stably transfected with APP695 were generated as reported previously (23.Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 19304-19310Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). HEK293 cells stably transfected with APP695 and human PS1 were generated from HEK293 cells stably expressing APP695 by the transfection of the cells with the pcDNA 3.1-Hygro vector (Invitrogen) carrying human PS1. Tissue from PS-deficient embryo (24.Herreman A. Hartmann D. Annaert W. Saftig P. Craessaerts K. Serneels L. Umans L. Schrijvers V. Checler F. Vanderstichele H. Baekelandt V. Dressel R. Cupers P. Huylebroeck D. Zwijsen A. Van Leuven F. De Strooper B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11872-11877Crossref PubMed Scopus (436) Google Scholar) was digested with collagenase and cultured in Dulbecco's modified Eagle's medium-F12 containing 10% fetal calf serum. Outgrowing cells were immortalized with large T antigen and split twice a week. An affinity-purified rabbit anti-Herp antibody was prepared as described previously (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). A rat anti-PS1 antibody (for the N-terminal fragment of PS1) and a rabbit anti-PS2 antibody (for the N-terminal fragment) were purchased from Chemicon International, Inc. (Temecula, CA) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. A monoclonal antibody 6E10 specific to human Aβ1–17 was purchased from Senetek (St. Louis, MO). BA27 specific for the Aβ40 terminal site, BC05 specific for the Aβ42 terminal site, BAN50 raised against Aβ1–6, and BNT77 raised against human Aβ11–28 have all been characterized previously (25.Asami-Odaka A. Ishibashi Y. Kikuchi T. Kitada C. Suzuki N. Biochemistry. 1995; 34: 10272-10278Crossref PubMed Scopus (150) Google Scholar). Affinity-purified rabbit antibody 369 was raised against the C-terminal residues of APP695 (26.Buxbaum J.D. Gandy S.E. Cicchetti P. Ehrlich M.E. Czernik A.J. Fracasso R.P. Ramabhadran T.V. Unterbeck A.J. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6003-6006Crossref PubMed Scopus (427) Google Scholar). Affinity-purified rabbit antibody B12/4 was raised against 20 C-terminal amino acid residues of APP695 (27.De Strooper B. Simons M. Multhaup G. Van Leuven F. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (162) Google Scholar). Cultured cells were lysed in RIPA buffer (150 mm NaCl, 10 mmTris/HCl pH 7.5, 1% Nonidet P-40, 0.1% SDS, and 0.2% sodium deoxycholate) containing a protease inhibitor mixture. The solubilized proteins were subjected to immunoprecipitation as described previously (28.Sudoh S. Kawamura Y. Sato S. Wang R. Saido T.C. Oyama F. Sakaki Y. Komano H. Yanagisawa K. J. Neurochem. 1998; 71: 1535-1543Crossref PubMed Scopus (62) Google Scholar). The precipitated proteins were resolved by SDS-PAGE on 4–20% gel for the detection of PS and Herp and on 7.5% gel for the detection of intracellular APP. Immunoblotting was performed as reported previously (28.Sudoh S. Kawamura Y. Sato S. Wang R. Saido T.C. Oyama F. Sakaki Y. Komano H. Yanagisawa K. J. Neurochem. 1998; 71: 1535-1543Crossref PubMed Scopus (62) Google Scholar). The secreted Aβ was immunoprecipitated and detected using a highly sensitive immunoblotting technique as described previously (28.Sudoh S. Kawamura Y. Sato S. Wang R. Saido T.C. Oyama F. Sakaki Y. Komano H. Yanagisawa K. J. Neurochem. 1998; 71: 1535-1543Crossref PubMed Scopus (62) Google Scholar). Intracellular C99 was immunoprecipitated with BAN50 and detected using a highly sensitive immunoblotting technique with B12/4. ELISA for Aβ was performed as described previously (25.Asami-Odaka A. Ishibashi Y. Kikuchi T. Kitada C. Suzuki N. Biochemistry. 1995; 34: 10272-10278Crossref PubMed Scopus (150) Google Scholar). The capture antibody used was BNT77. Detector antibodies were horseradish peroxidase-conjugated BA27 (for Aβ40) and horseradish peroxidase-conjugated BC05 (for Aβ42). ELISA data were statistically analyzed by analysis of variance (ANOVA) using StatView-J.4.11. Acetone-fixed cryostat sections or Kryofix-fixed, paraffin-embedded serial sections were prepared from five nondemented and five AD brains. Cryostat sections were used only for the staining of neurons with the anti-Herp antibody since the staining signals were weak in ordinary formalin-paraffin sections. Tissue sections were blocked with 5% normal goat (for anti-Herp antibody) or horse (for anti-HLA-DP, DQ, and DR antibody) serum and then reacted with the anti-Herp antibody (5 μg/ml) or the anti-HLA-DP, DQ, and DR (major histocompatibility complex class II antigen, 3 μg/ml; DAKO, CR3/43) antibody followed by biotinylated secondary antibodies. Positive signals were visualized by incubating the sections in a diaminobenzidine-H2O2solution. For the double immunofluorescent study, after pretreatment with 0.1% Sudan black B in 70% ethanol for 7 min to mask autofluorescence, Kryofix paraffin sections were reacted with a primary antibody mixture (anti-Herp, 5 μg/ml and anti-Aβ, 4G8, monoclonal, 1:1,000) and then reacted with a mixture of fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (40 μg/ml, Jackson ImmunoResearch Laboratories) and Texas red-conjugated goat anti-mouse IgG (20 μg/ml, Jackson). For the control study, anti-Herp antibody was preabsorbed with 10-fold by weight of recombinant Herp. We first investigated whether transfection of Herp cDNA increases the extent of Aβ generation from full-length APP using HEK293 cells stably expressing APP. We found that a stably high expression level of Herp increased the extent of Aβ40 generation (Fig. 1a). Statistical analysis of ELISA results using the cells stably expressing Herp showed that the increase in the Aβ40 level caused by the high expression of Herp was ∼1.8-fold (Fig. 1b). In contrast, the effect of Herp on the Aβ42 level was not significant (Fig. 1b). We next tested whether an increase in the level of Aβ generation by Herp expression can occur in PS-deficient cells since it is not conclusive yet whether PS itself is a γ-secretase. As shown in Fig. 1c, no Aβ was detected in PS-deficient fibroblasts expressing Herp, whereas Herp expression in wild-type fibroblasts increased the Aβ level. We also confirmed that the intracellular APP level was not changed by the high expression level of Herp (Fig. 1c). These results indicate that Herp enhances PS-mediated Aβ40 generation. In addition, the level of intracellular β-secretase-cleaved C-terminal APP fragment (C99) was significantly reduced by Herp transfection, whereas the level of soluble α-secretase-cleaved N-terminal APP fragment (α-APPs) was not changed (Fig. 1d). These results strongly suggest that Herp preferentially increases a degree of γ-secretase cleavage. Since both PS and Herp reside in the ER, it is likely that Herp interacts with PS, resulting in an increase in the extent of Aβ generation. Therefore, we next investigated the interaction of PS with Herp by coimmunoprecipitation studies using HEK293 cells stably expressing PS1 plus APP. An anti-PS1 antibody coimmunoprecipitated with Herp (Fig. 2a). The level of the coimmunoprecipitated Herp was approximately equivalent to that of Herp contained in the 10% starting lysate used for immunoprecipitation (Fig. 2a). Considering that the efficiency of immunoprecipitation by the anti-PS1 antibody is about 10% (data not shown), it is likely that almost the entire amount of Herp interacts with PS1. PS undergoes endoproteolysis, forming N- and C-terminal fragments (29.Haass C. Neuron. 1997; 18: 687-690Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Interestingly, an anti-Herp antibody immunoprecipitated the full-length PS1, but it only immunoprecipitated a small amount of the N-terminal fragment of PS1 (Fig. 2b). These results indicate that Herp mainly interacts with the full-length PS1. The study of PS2 and Herp interaction was also performed using HEK293 cells that transiently expressed PS2 and FLAG-epitope-tagged Herp. As shown in Fig. 2c, full-length PS2 coprecipitated with FLAG-epitope-tagged Herp. Thus, Herp interacts with both PS1 and PS2, and the major PS molecule that interacts with Herp was the full-length PS. The expression of Herp did not alter the steady-state levels of full-length PS and N-terminal fragment (Fig. 2, b and d), suggesting that Herp does not enhance the endoproteolysis of PS. APP and Herp interaction was also studied. Herp only coprecipitated APP at less than 1% of the total APP in the cells (data not shown). Therefore, in all probability, APP does not specifically interact with Herp. Thus, it is likely that the direct interaction of the full-length PS with Herp causes an increase in the degree of PS-mediated γ-cleavage of APP. Alternatively, at present, we cannot exclude the possibility that Herp indirectly affects the Aβ level, for example, by regulating the intracellular calcium level, since it was suggested that the intracellular calcium level modifies the Aβ level (30.Querfurth H.W. Selkoe D.J. Biochemistry. 1994; 33: 4550-4561Crossref PubMed Scopus (217) Google Scholar). Herp expression is up-regulated by calcium ionophores (13.Kokame K. Agarwala K.L. Kato H. Miyata T. J. Biol. Chem. 2000; 275: 32846-32853Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and Herp may potentially regulate the intracellular calcium level. Immunohistochemical analysis of Herp in a human brain section was performed to identify the cell types expressing a high level of Herp in the brain; these cells are considered to generate a high level of Aβ. As shown in Fig. 3a, only neurons were positive for Herp in the parenchyma cerebral cortex of a nondemented human brain. Interestingly, microglia in senile plaques in the brain of AD patients were strongly stained (Fig. 3b). The staining of HLA-DP, DQ, and DR (markers of activated microglia (31.Itagaki S. McGeer P.L. Akiyama H. Zhu S. Selkoe D. J. Neuroimmunol. 1989; 24: 173-182Abstract Full Text PDF PubMed Scopus (767) Google Scholar)) and Herp in serial sections (Fig. 3c) indicates that some of the activated microglia in the center of senile plaques are highly immunoreactive with the anti-Herp antibody. Double immunostaining with anti-Herp and anti-HLA-DP, DQ, and DR antibodies also confirmed that activated microglia are strongly positive for Herp (see supplemental data). In addition, smooth muscle cells in meningeal arteries and arterioles were strongly stained (Fig. 3d). One hallmark of AD is the accumulation of Aβ, although the cause of AD may be multifactorial. Here, we showed that ER stress-inducible Herp enhances Aβ40 generation. Since a significant increase in the level of Aβ42 was not observed, the high expression level of Herp appears to mainly enhance 40-γ-secretase cleavage. A recent report showed that some nonsteroidal anti-inflammatory drugs preferentially decreased the generation of Aβ42 (32.Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1333) Google Scholar). Taken together with our result, it appears that the regulation of the generation of Aβ42 is distinct from that of Aβ40, even though the generation of both Aβ forms requires PS. Herp was originally discovered as a homocysteine-induced protein. It is noteworthy that a high plasma homocysteine level was observed in some cases of AD (14.Clarke R. Smith A.D. Jobst K.A. Refsum H. Sutton L. Ueland P.M. Arch. Neurol. 1998; 55: 1449-1455Crossref PubMed Scopus (1271) Google Scholar). At present, we cannot determine whether Herp directly or indirectly activates γ-secretase. We have shown that Herp interacts with the full-length PS rather than with its endoproteolytic products. A transient overexpression of Herp did not enhance the endoproteolytic cleavage of PS nor change the PS level. Since endoproteolytic cleavage of PS is not essential for the biological function of PS or Aβ generation (34.Saura C.A. Tomita T. Soriano S. Takahashi M. Leem J.Y. Honda T. Koo E.H. Iwatsubo T. Thinakaran G. J. Biol. Chem. 2000; 275: 17136-17142Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), one interpretation for the enhancement of Aβ generation caused by Herp expression is that Herp directly activates the full-length PS-mediated γ-activity by binding to the full-length PS, possibly through a change in the conformation or the trafficking of PS. The physical interaction of Herp with PS also raises the possibility that the function of PS is modulated by ER stress through Herp expressed at high levels. It was reported that cells exposed to inducers of ER stress, such as calcium ionophore, A23187, or thapsigargin, have increased the extracellular Aβ level, suggesting that the intracellular calcium level regulates Aβ generation (30.Querfurth H.W. Selkoe D.J. Biochemistry. 1994; 33: 4550-4561Crossref PubMed Scopus (217) Google Scholar). Taken together with our results, it is most likely that these inducers cause a Herp-mediated increase in the Aβ level since these inducers strongly up-regulate Herp expression. Our present study showed that, in the normal brain, Herp was expressed in neurons and highly expressed in vascular cells, suggesting that these cells are the major source of Aβ in the normal brain. Interestingly, Herp expression was up-regulated in activated microglia in senile plaques in the AD brain. Activated microglia are thought to play a role in the clearance of Aβ. However, some activated microglia, which strongly express Herp, may generate a high level of Aβ and function in the formation of Aβ deposits. Vascular Aβ deposits known as amyloid angiopathy are also one of the major signs of AD pathology (4.Selkoe D.J. Nature. 1999; 399: 23-31Crossref PubMed Scopus (1526) Google Scholar, 33.Yamada M. Neuropathology. 2000; 20: 8-22Crossref PubMed Scopus (165) Google Scholar). The high expression level of Herp in vascular cells may be related to amyloid angiopathy. The cells expressing Herp at high levels could contribute to Aβ accumulation, including the formation of senile plaques or vascular Aβ deposits, in patients with AD. In particular, it is interesting that Herp has an N-terminal ubiquitin-like domain. Several proteins were reported to have a ubiquitin-like domain in eukaryotic cells, but the biological significance of this domain still remains to be clarified. In some cases, such as ubiquitin itself, a ubiquitin-related domain is involved in targeting proteins for protein degradation (35.Kawakami T. Chiba T. Suzuki T. Iwai K. Yamanaka K. Minato N. Suzuki H. Shimbara N. Hidaka Y. Osaka F. Omata M. Tanaka K. EMBO J. 2001; 20: 4003-4012Crossref PubMed Scopus (274) Google Scholar), but other functions such as chaperon function or protein-protein interaction were also reported (36.Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science. 1995; 269: 1439-1443Crossref PubMed Scopus (292) Google Scholar). Presenilin undergoes degradation via the ubiquitin/proteosome pathway (29.Haass C. Neuron. 1997; 18: 687-690Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). However, the steady-state level of PS in cells expressing Herp was not changed as compared with that in nontransfected cells, strongly suggesting that the high expression level of Herp is not involved in the degradation of PS. Mutations in the PARKIN gene were discovered in families with an autosomal recessive form of Parkinson disease (37.Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4199) Google Scholar). Parkin also has the ubiquitin-like domain in the N-terminal site, although the function of the domain is not known, and it is up-regulated by ER stress (38.Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 39.Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar). Recent reports indicate that parkin is involved in the ubiquitination of Pael receptor and α-synuclein (39.Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar, 40.Shimura H. Schlossmacher M.G. Hattori N. Frosch M.P. Trockenbacher A. Schneider R. Mizuno Y. Kosik K.S. Selkoe D.J. Science. 2001; 293: 263-269Crossref PubMed Scopus (949) Google Scholar). Since a strong ubiquitin immunoreactivity is associated with the pathology of both AD and Parkinson disease, the complex of presenilin and Herp with a ubiquitin-like domain may also function in the elimination of misfolded proteins via the ubiquitin/proteasome pathway as parkin does. Further exploration of the biological significance of the interaction of Herp with PS will not only help determine the pathways by which γ-secretase activity is regulated by PS but may also further clarify the role of PS under ER stress or in AD pathogenesis. We thank G. P. Nolan (Stanford School of Medicine) for providing packaging cells (Phoenix cells); J. Miyazaki (Osaka University Medical School) for providing plasmid pCXN2; T. Tomita (University of Tokyo), K. Maruyama (Saitama medical school), and T. Iwatsubo (University of Tokyo) for providing PS2 cDNA; and S. Gandy (New York University School of Medicine) for providing antibody 369. Download .pdf (.05 MB) Help with pdf files
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