Differential Effects of the Swedish Mutant Amyloid Precursor Protein on β-Amyloid Accumulation and Secretion in Neurons and Nonneuronal Cells
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.32247
ISSN1083-351X
AutoresMark S. Forman, David G. Cook, Susan Leight, Robert W. Doms, Virginia M.‐Y. Lee,
Tópico(s)Supramolecular Self-Assembly in Materials
ResumoExpression of the Swedish ΔNL mutation in the β-amyloid precursor protein (APPΔNL) dramatically increases Aβ generation in nonneuronal cell lines, although it is unclear whether intracellular levels of β-amyloid (Aβ) are also elevated after APPΔNL expression. Furthermore, the effects of expressing APPΔNL in neurons on the production and secretion of Aβ-(1–40) and Aβ-(1–42) are unknown. To address these issues, we examined the generation of both intracellular and secreted Aβ-(1–40) and Aβ-(1–42) in human neuronal NT2N cells, in primary rat astrocytes, and in Chinese hamster ovary cells engineered to express wild-type APP or APPΔNL using a recombinant Semliki Forest virus expression system. Expression of APPΔNL led to a marked increase in APPβ and the C-terminal fragment containing the entire Aβ sequence (C99) in all cells tested. However, a dramatic elevation of intracellular and secreted Aβ-(1–40) and Aβ-(1–42) was seen only in astrocytes and Chinese hamster ovary cells. The ΔNL mutation did not cause a significant increase in intracellular or secreted Aβ-(1–40) or Aβ-(1–42) in NT2N cells. Since NT2N cells expressing APPΔNL accumulate much higher levels of C99 than cells expressing wild-type APP, we conclude that the rate-limiting step in Aβ production could be the further processing of C99 by γ-secretase in these cells. These results show that the Swedish ΔNL mutation causes nonneuronal cells to process APP via pathways more in common with the metabolism of wild-type APP in neurons. Expression of the Swedish ΔNL mutation in the β-amyloid precursor protein (APPΔNL) dramatically increases Aβ generation in nonneuronal cell lines, although it is unclear whether intracellular levels of β-amyloid (Aβ) are also elevated after APPΔNL expression. Furthermore, the effects of expressing APPΔNL in neurons on the production and secretion of Aβ-(1–40) and Aβ-(1–42) are unknown. To address these issues, we examined the generation of both intracellular and secreted Aβ-(1–40) and Aβ-(1–42) in human neuronal NT2N cells, in primary rat astrocytes, and in Chinese hamster ovary cells engineered to express wild-type APP or APPΔNL using a recombinant Semliki Forest virus expression system. Expression of APPΔNL led to a marked increase in APPβ and the C-terminal fragment containing the entire Aβ sequence (C99) in all cells tested. However, a dramatic elevation of intracellular and secreted Aβ-(1–40) and Aβ-(1–42) was seen only in astrocytes and Chinese hamster ovary cells. The ΔNL mutation did not cause a significant increase in intracellular or secreted Aβ-(1–40) or Aβ-(1–42) in NT2N cells. Since NT2N cells expressing APPΔNL accumulate much higher levels of C99 than cells expressing wild-type APP, we conclude that the rate-limiting step in Aβ production could be the further processing of C99 by γ-secretase in these cells. These results show that the Swedish ΔNL mutation causes nonneuronal cells to process APP via pathways more in common with the metabolism of wild-type APP in neurons. The 4-kDa amyloid β peptide (Aβ) 1The abbreviations used are: Aβ, β-amyloid; AD, Alzheimer's disease; Aβ-(1–40), Aβ containing 40 amino acid residues; Aβ-(1–42), Aβ containing 42 amino acid residues; APP, β-amyloid precursor protein; APPwt, wild-type human APPwt protein, APPΔNL, human APPwt protein bearing the Swedish double mutation; APPS, N-terminal ectodomain of APP derivatives; APPα, α-secretase cleaved N-terminal ectodomain of APP; APPβ, β-secretase-cleaved N-terminal ectodomain of APP; APPβΔNL, β-secretase cleaved N-terminal ectodomain of APPΔNL; p3, Aβ fragments cleaved at amino acid residues 16 and 17 of Aβ; C99, C-terminal fragment containing the entire Aβ sequence; C83, C-terminal fragment containing only p3; NT2N cells, neurons derived from a human embryonal carcinoma cell line (NT2); CHO cells, Chinese hamster ovary cells; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; IC, intermediate compartment; SFV, Semliki Forest virus; PBS, phosphate-buffered saline; FBS, fetal bovine serum; MEM, modified Eagle's medium. 1The abbreviations used are: Aβ, β-amyloid; AD, Alzheimer's disease; Aβ-(1–40), Aβ containing 40 amino acid residues; Aβ-(1–42), Aβ containing 42 amino acid residues; APP, β-amyloid precursor protein; APPwt, wild-type human APPwt protein, APPΔNL, human APPwt protein bearing the Swedish double mutation; APPS, N-terminal ectodomain of APP derivatives; APPα, α-secretase cleaved N-terminal ectodomain of APP; APPβ, β-secretase-cleaved N-terminal ectodomain of APP; APPβΔNL, β-secretase cleaved N-terminal ectodomain of APPΔNL; p3, Aβ fragments cleaved at amino acid residues 16 and 17 of Aβ; C99, C-terminal fragment containing the entire Aβ sequence; C83, C-terminal fragment containing only p3; NT2N cells, neurons derived from a human embryonal carcinoma cell line (NT2); CHO cells, Chinese hamster ovary cells; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; IC, intermediate compartment; SFV, Semliki Forest virus; PBS, phosphate-buffered saline; FBS, fetal bovine serum; MEM, modified Eagle's medium. is the principal proteinaceous component of senile plaques, the hallmark pathological feature of Alzheimer's disease (AD). The Aβ peptide varies in length from 39 to 43 amino acids (1Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3598) Google Scholar, 2Selkoe D.J. Neurobiol. Aging. 1986; 7: 425-432Crossref PubMed Scopus (87) Google Scholar, 3Yamaguchi H. Hirai S. Morimatsu M. Shoji M. Harigaya Y. Acta Neuropathol. 1988; 77: 113-119PubMed Google Scholar, 4Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 122: 1131-1135Crossref PubMed Scopus (1239) Google Scholar, 5Trojanowski J.Q. Shin R.W. Schmidt M.L. Lee V.M.-Y. Neurobiol. Aging. 1995; 16: 335-340Crossref PubMed Scopus (60) Google Scholar) and is derived from post-translational cleavage of the amyloid precursor protein (APP) (6Kang J. Lemaire H.-G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.-H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3915) Google Scholar, 7Robakis N.K. Ramakrishna N. 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For example, a portion of APP is processed by the α-secretase pathway in which APP is cleaved within the Aβ region at or near the plasma membrane, releasing a large N-terminal ectodomain fragment (APPα) (12Sisodia S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6075-6079Crossref PubMed Scopus (627) Google Scholar, 13Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Science. 1990; 248: 1122-1124Crossref PubMed Scopus (1193) Google Scholar), thereby precluding the formation of full-length Aβ. The utilization of this pathway appears to be preferred by transfected nonneuronal cells expressing wild-type APP (APPwt), since the N-terminal ectodomain containing the first 17 amino acids of Aβ (i.e. APPα) and p3, the Aβ fragment beginning at amino acid residue 17 of Aβ, are recovered at high levels from media conditioned by these transfected nonneuronal cells (13Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Science. 1990; 248: 1122-1124Crossref PubMed Scopus (1193) Google Scholar, 14Sisodia S.S. Koo E.H. Beyreuther K. Unterbeck A. Price D.L. Science. 1990; 248: 492-495Crossref PubMed Scopus (737) Google Scholar).Processing pathways that result in the constitutive production of Aβ have also been identified, although their utilization is relatively minor in nonneuronal cells (13Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Science. 1990; 248: 1122-1124Crossref PubMed Scopus (1193) Google Scholar, 15Haass C. Koo E.H. Mello A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 16Seubert P. Oltersdorf T. Lee M.G. Barbour R. Blomquist C. Davis D.L. Bryant K. Fritz L.C. Galasko D. Thal L.J. Lieberburg I. Schenk D.B. Nature. 1993; 361: 260-263Crossref PubMed Scopus (497) Google Scholar). Cleavage of APP by β-secretase at the N terminus of the Aβ sequence releases a soluble N-terminal fragment (APPβ) and generates a C-terminal fragment (C99) that contains the entire Aβ sequence. C99, but not APPβ, has been recovered from transfected nonneuronal cells expressing high levels of APPwt (15Haass C. Koo E.H. Mello A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 17Golde T.E. Estus S. Younkin L.H. Selkoe D.J. Younkin S.G. Science. 1992; 255: 728-730Crossref PubMed Scopus (618) Google Scholar). A second proteolytic activity termed γ-secretase, cleaves APP at the C-terminal end of the Aβ sequence, releasing Aβ-(1–40) or Aβ-(1–42) (18Haass C. Selkoe D.J. Cell. 1993; 75: 1039-1042Abstract Full Text PDF PubMed Scopus (736) Google Scholar). Although secreted Aβ-(1–40) and Aβ-(1–42) are present in media conditioned by APPwt-transfected nonneuronal cells, intracellular Aβ has not been detected (19Martin B.L. Schrader-Fischer G. Busciglio J. Duke M. Paganetti P. Yankner B.A. J. Biol. Chem. 1995; 270: 26727-26730Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 20Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 21Perez R.G. Squazzo S.L. Koo E.H. J. Biol. Chem. 1996; 271: 9100-9107Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar).Unlike transfected nonneuronal cells expressing APPwt, postmitotic neurons such as human NT2N cells predominantly utilize the β-secretory pathway at the expense of the α-secretory pathway to process endogenous APP (22Wertkin A.M. Turner R.S. Pleasure S.J. Golde T.E. Younkin S.G. Trojanowski J.Q. Lee V.M.-Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9513-9517Crossref PubMed Scopus (195) Google Scholar, 23Turner R.S. Suzuki N. Chyung A.S.C. Younkin S.G. Lee V.M.-Y. J. Biol. 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Chyung A.S.C. Younkin S.G. Lee V.M.-Y. J. Biol. Chem. 1996; 271: 8966-8970Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 24Chyung A.S.C. Greenberg B.D. Cook D.G. Doms R.W. Lee V.M.-Y. J. Cell Biol. 1997; 138: 671-680Crossref PubMed Scopus (136) Google Scholar). At least one intracellular location for β-cleavage is within the endoplasmic reticulum/intermediate compartment (ER/IC) (24Chyung A.S.C. Greenberg B.D. Cook D.G. Doms R.W. Lee V.M.-Y. J. Cell Biol. 1997; 138: 671-680Crossref PubMed Scopus (136) Google Scholar,25Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (424) Google Scholar). Importantly, APP processed by the ER/IC pathway in NT2N cells produced only Aβ-(1–42) but not Aβ-(1–40) (25Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (424) Google Scholar, 26Wild-Bode C. Tamazaki T. Capell A. Leimer U. Steiner H. Ihara Y. Haass C. J. Biol. Chem. 1997; 272: 16085-16088Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Thus, it is evident that APP processing is cell-type-specific and that neuronal cells process APP differently from nonneuronal cells.Studies of a Swedish family with familial AD identified a double mutation immediately flanking the N terminus of the Aβ domain (APPΔNL (27Mullan M. Crawford F. Axelman K. Houlden H. Lilius L. Winblad B. Lannfelt L. Nat. Genet. 1992; 1: 345-347Crossref PubMed Scopus (1167) Google Scholar)) that results in elevated plasma levels of Aβ-(1–40) and Aβ-(1–42) (28Scheuner D. Eckman C. Jensen M. Song X. Citron M. Suzuki N. Bird T.D. Hardy J. Hutton M. Kukull W. Larson E. Levy-Lahad E. Viitanen M. Peskind E. Poorkaj P. Schellenberg G. Tanzi R. Wasco W. Lannfelt L. Selkoe D. Younkin S. Nat. Med. 1996; 2: 864-870Crossref PubMed Scopus (2249) Google Scholar). Moreover, nonneuronal cells transfected with APPΔNL secrete three to six times more Aβ-(1–40) and Aβ-(1–42) than cells transfected with APPwt (29Cai X.-D. Golde T.E. Younkin S.G. Science. 1993; 259: 514-517Crossref PubMed Scopus (826) Google Scholar, 30Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1517) Google Scholar). Unlike nonneuronal cells expressing APPwt, nonneuronal cells transfected with APPΔNL produce intracellular Aβ and APPβΔNL (19Martin B.L. Schrader-Fischer G. Busciglio J. Duke M. Paganetti P. Yankner B.A. J. Biol. Chem. 1995; 270: 26727-26730Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 20Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 21Perez R.G. Squazzo S.L. Koo E.H. J. Biol. 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However, this mutation does lead to greatly increased levels of intracellular and secreted Aβ-(1–40) and Aβ-(1–42) in both primary astrocytes and nonneuronal cell lines. These data provide evidence that the consequences of this familial AD-associated mutation on APP processing in nonneuronal cells is to increase the utilization of the β-secretory pathways at the expense of the α-secretory pathway such that they resemble the processing pathways in postmitotic neurons. Finally, we also provide evidence that γ-secretase, but not β-secretase, is a rate-limiting step in the production of Aβ in NT2N neuronal cells.DISCUSSIONExpression of APPΔNL in nonneuronal cells leads to a 5–10-fold increase in the amount of secreted and intracellular Aβ relative to that seen after expression of APPwt, perhaps explaining how this mutation results in accelerated AD pathology (19Martin B.L. Schrader-Fischer G. Busciglio J. Duke M. Paganetti P. Yankner B.A. J. Biol. Chem. 1995; 270: 26727-26730Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 20Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 21Perez R.G. Squazzo S.L. Koo E.H. J. Biol. Chem. 1996; 271: 9100-9107Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 29Cai X.-D. Golde T.E. Younkin S.G. Science. 1993; 259: 514-517Crossref PubMed Scopus (826) Google Scholar, 30Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1517) Google Scholar, 31Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar, 45Johnston J.A. Cowburn R.F. Norgren S. Wiehager B. Venizelos N. Winblad B. Vigo-Pelfrey C. Schenk D. Lannfelt L. O'Neill C. FEBS Lett. 1994; 354: 274-278Crossref PubMed Scopus (111) Google Scholar, 46Iizuka T. Shoji M. Kawarabayashi T. Sato M. Kobayashi T. Tada N. Kasai K. Matsubara E. Watanabe M. Tomidokoro Y. Hirai S. Biochem. Biophys. Res. Commun. 1996; 218: 238-242Crossref PubMed Scopus (29) Google Scholar). However, expression of APPΔNL in NT2N neurons resulted in only a minimal increase (<35%) in the amount of both secreted and intracellular Aβ-(1–40) and no increase in Aβ-(1–42) production. This differential effect of the Swedish ΔNL mutation on APP processing and Aβ production in NT2N neurons as compared with nonneuronal cell lines demonstrates that cells can process APPΔNL differently, provides insight into the mechanisms by which Aβ is generated from APPΔNL, and raises the question as to the cell type(s) specifically affected by this mutation in vivo that contribute the most to the formation of senile plaques.The failure of the ΔNL mutation to result in greatly enhanced Aβ production in NT2N neurons could be the result of several factors, including reduced processing by β- or γ-secretases. However, we found that expression of APPΔNL in all cell types examined, including NT2N cells, caused a marked shift from the α-secretory to the β-secretory pathway as evidenced by reduced secretion of APPα and increased secretion of APPβΔNL. The expression of APPΔNL also shifted the production of the C-terminal fragments from C83 (generated by α-secretase cleavage) to the C99 fragment (generated by β-secretase cleavage). This shift was especially pronounced in CHO cells, which produced little or no C99 after expression of APPwt. A similar though less dramatic effect on C99 production was observed in NT2N cells after APPΔNL expression, again indicating that processing by β-secretase(s) is increased in NT2N cells expressing APPΔNL. Thus, the ΔNL mutation leads to increased processing of APP by β-secretase(s) in both neuronal and nonneuronal cells.The increased processing by β-secretase(s) observed in all cell types after APPΔNL expression could be the result of several factors. At present, at least three different β-secretase processing pathways have been reported. The endosomal/lysosomal pathway, which processes APP after re-internalization from the cell surface into endosomes and lysosomes, is the most ubiquitous since both neurons and nonneuronal cells utilize this pathway to produce Aβ. However, the contribution of this pathway to the overall production of Aβ is relatively minor since nonneuronal cells transfected with APPwt produce mostly p3 and very little Aβ (15Haass C. Koo E.H. Mello A. Hung A.Y. Selkoe D.J. Nature. 1992; 357: 500-503Crossref PubMed Scopus (768) Google Scholar, 20Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 47Koo E.H. Squazzo S. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar, 48Lai A. Sisodia S.S. Trowbridge I.S. J. Biol. Chem. 1995; 270: 3565-3573Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In addition, it is unlikely that APPΔNL expression increases Aβ production via this pathway, since the expression of an APPΔNL construct lacking the cytoplasmic tail, which eliminates re-internalization of cell surface APPΔNL, does not reduce Aβ secretion (31Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar, 49Essalmani R. Macq A.-F. Mercken L. Octave J.-N. Biochem. Biophys. Res. Commun. 1996; 218: 89-96Crossref PubMed Scopus (23) Google Scholar). A second β-secretory pathway, active primarily in Golgi-derived vesicles, is more likely to process APPΔNL (31Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar). Previous studies have suggested that nonneuronal cells utilize this pathway at the expense of the α-secretory pathway when APPΔNL is expressed (31Haass C. Lemere C.A. Capell A. Citron M. Seubert P. Schenk D. Lannfelt L. Selkoe D.J. Nat. Med. 1995; 1: 1291-1296Crossref PubMed Scopus (437) Google Scholar). However, it is unclear whether Aβ-(1–40) and Aβ-(1–42) are both produced by this route, since a recently identified β-secretory pathway localized to the ER/IC results in exclusive production of Aβ-(1–42) (25Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (424) Google Scholar, 26Wild-Bode C. Tamazaki T. Capell A. Leimer U. Steiner H. Ihara Y. Haass C. J. Biol. Chem. 1997; 272: 16085-16088Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Since the secretion of both Aβ-(1–40) and Aβ-(1–42) is elevated in nonneuronal cells expressing APPΔNL, it is possible that both the Golgi-associated and the ER β-secretase pathways are affected by this mutation. Alternatively, it is possible that both Aβ-(1–40) and Aβ-(1–42) can be produced by Golgi-derived vesicles and that the ΔNL mutation only affects this pathway selectively.The increased processing of APPΔNL by the β-secretase pathway in all cells tested coupled with markedly increased levels of Aβ production in all cells except NT2N neurons suggests that γ-secretase processing may be rate-limiting for Aβ production in NT2N cells by one of several mechanisms. For example, C99 may be more rapidly degraded in NT2N cells than in nonneuronal cells, precluding or minimizing the possibility of γ-secretase cleavage of C99 and concomitant Aβ production. However, if this were true, we would not expect the observed increase in the C99 fragment after APPΔNL expression in NT2N cells. Another possibility is that the high levels of expression resulting from the SFV vector system saturates the γ-secretase. However, we found no difference in Aβ levels between NT2N cells expressing APPwt or APPΔNL at early time points when APP expression levels were low. In addition, De Strooper et al.(43De Strooper B.D. Simons M. Multhaup G. Leuven F.V. Beyreuther K. Dotti C.G. EMBO J. 1995; 14: 4932-4938Crossref PubMed Scopus (161) Google Scholar) found that expression of APPΔNL in rat hippocampal neurons caused only a 2-fold increase in Aβ levels relative to APPwt as judged by immunoprecipitation. Although larger than the increase we observed in this study using a more quantitative ELISA approach, the increase in Aβ was still significantly less than the 5–10-fold increase observed in nonneuronal cells. Finally, the intracellular processing pathways utilized by the NT2N cells may be distinct from that of the nonneuronal cells (12Sisodia S.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6075-6079Crossref PubMed Scopus (627) Google Scholar, 13Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Science. 1990; 248: 1122-1124Crossref PubMed Scopus (1193) Google Scholar, 22Wertkin A.M. Turner R.S. Pleasure S.J. Golde T.E. Younkin S.G. Trojanowski J.Q. Lee V.M.-Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9513-9517Crossref PubMed Scopus (195) Google Scholar, 23Turner R.S. Suzuki N. Chyung A.S.C. Younkin S.G. Lee V.M.-Y. J. Biol. Chem. 1996; 271: 8966-8970Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 25Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (424) Google Scholar). Thus, the possibility exists that the γ-secretase did not have access to the carboxyl-terminal fragments generated when APPΔNL was expressed. Identification of the γ-secretase(s) will help distinguish these possibilities.Several studies suggest that the cellular source of Aβ deposition in senile plaques in nonfamilial cases of AD is the neuron (50Cras P. Kawai M. Siedlak S. Mulvihill P. Gambetti P. Lowery D. Gonzalez-DeWhitt P. Greenberg B. Perry G. Am. J. Pathol. 1990; 137: 241-246PubMed Google Scholar, 51Procter A.W. Francis P.T. Holmes C. Webster M.-T. Qume M. Stratmann G.C. Doshi R. Mann D.M.A. Harrison P.J. Pearson R.C.A. Bowen D.M. Acta Neuropathol. 1994; 88: 545-552Crossref PubMed Scopus (16) Google Scholar). Our data supports this hypothesis because only neuronal cells constitutively generate Aβ. This production of Aβ is accompanied by the generation of intracellular APPβ and C99 in NT2N cells. By contrast, intracellular APPβ and C99 were absent from nonneuronal cells expressing APPwt. This suggests that commitment to neuronal differentiation results in the acquisition of the ability to utilize the β pathway of APP processing, a necessary prerequisite for Aβ production. However, expression of APPΔNL led to markedly increased production of Aβ, APPβ, and C99 in nonneuronal cells, indicating that the Swedish ΔNL mutation alters APP processing in nonneuronal cells such that APP is now processed in a more "neuronal" fashion. NT2N cells may not exhibit such marked alterations in APP and Aβ metabolism after APPΔNL expression, because they are already committed to processing APP in pathways that favor increased production of both intracellular and secreted Aβ. The relatively modest effects of this mutation on Aβ production in NT2N neurons raises the question as to what role neurons play in elevating Aβ levels in individuals with the ΔNL mutation. It is possible that in such individuals cell types other than neurons, such as astrocytes, may significantly contribute to the marked increases in central nervous system Aβ levels and the formation of senile plaques. The 4-kDa amyloid β peptide (Aβ) 1The abbreviations used are: Aβ, β-amyloid; AD, Alzheimer's disease; Aβ-(1–40), Aβ containing 40 amino acid residues; Aβ-(1–42), Aβ containing 42 amino acid residues; APP, β-amyloid precursor protein; APPwt, wild-type human APPwt protein, APPΔNL, human APPwt protein bearing the Swedish double mutation; APPS, N-terminal ectodomain of APP derivatives; APPα, α-secretase cleaved N-terminal ectodomain of APP; APPβ, β-secretase-cleaved N-terminal ectodomain of APP; APPβΔNL, β-secretase cleaved N-terminal ectodomain of APPΔNL; p3, Aβ fragments cleaved at amino acid residues 16 and 17 of Aβ; C99, C-terminal fragment containing the entire Aβ sequence; C83, C-terminal fragment containing only p3; NT2N cells, neurons derived from a human embryonal carcinoma cell line (NT2); CHO cells, Chinese hamster ovary cells; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; IC, intermediate compartment; SFV, Semliki Forest virus; PBS, phosphate-buffered saline; FBS, fetal bovine serum; MEM, modified Eagle's medium. 1The abbreviations used are: Aβ, β-amyloid; AD, Alzheimer's disease; Aβ-(1–40), Aβ containing 40 amino acid residues; Aβ-(1–42), Aβ containing 42 amino acid residues; APP, β-amyloid precursor protein; APPwt, wild-type human APPwt protein, APPΔNL, human APPwt protein bearing the Swedish double mutation; APPS, N-terminal ectodomain of APP derivatives; APPα, α-secretase cleaved N-terminal ectodomain of APP; APPβ, β-secretase-cleaved N-terminal ectodomain of APP; APPβΔNL, β-secretase cleaved N-terminal ectodomain of APPΔNL; p3, Aβ fragments cleaved at amino acid residues 16 and 17 of Aβ; C99, C-terminal fragment containing the entire Aβ sequence; C83, C-terminal fragment containing only p3; NT2N cells, neurons derived from a human embryonal carcinoma cell line (NT2); CHO cells, Chinese hamster ovary cells; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; IC, intermediate compartment; SFV, Semliki Forest virus; PBS, phosphate-buffered saline; FBS, fetal bovine serum; MEM, modified Eagle's medium. is the principal proteinaceous component of senile plaques, the hallmark pathological feature of Alzheimer's disease (AD). The Aβ peptide varies in length from 39 to 43 amino acids (1Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3598) Google Scholar, 2Selkoe D.J. Neurobiol. Aging. 1986; 7: 425-432Crossref PubMed Scopus (87) Google Scholar, 3Yamaguchi H. Hirai S. Morimats
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