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• W2023274935 endingPage "30806" @default.
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• W2023274935 abstract "β-Secretase (BACE1) is the rate-limiting protease for the generation of the amyloid β-peptide (Aβ) in Alzheimer disease. Mice in which the bace1 gene is inactivated are reported to be healthy. However, the presence of a homologous gene encoding BACE2 raises the possibility of compensatory mechanisms. Therefore, we have generated bace1, bace2, and double knockout mice. We report here that BACE1 mice display a complex phenotype. A variable but significant number of BACE1 offspring died in the first weeks after birth. The surviving mice remained smaller than their littermate controls and presented a hyperactive behavior. Electrophysiologically, subtle alterations in the steady-state inactivation of voltage-gated sodium channels in BACE1-deficient neurons were observed. In contrast, bace2 knockout mice displayed an overall healthy phenotype. However, a combined deficiency of BACE2 and BACE1 enhanced the bace1–/– lethality phenotype. At the biochemical level, we have confirmed that BACE1 deficiency results in an almost complete block of Aβ generation in neurons, but not in glia. As glia are 10 times more abundant in brain compared with neurons, our data indicate that BACE2 could indeed contribute to Aβ generation in the brains of Alzheimer disease and, in particular, Down syndrome patients. In conclusion, our data challenge the general idea of BACE1 as a safe drug target and call for some caution when claiming that no major side effects should be expected from blocking BACE1 activity. β-Secretase (BACE1) is the rate-limiting protease for the generation of the amyloid β-peptide (Aβ) in Alzheimer disease. Mice in which the bace1 gene is inactivated are reported to be healthy. However, the presence of a homologous gene encoding BACE2 raises the possibility of compensatory mechanisms. Therefore, we have generated bace1, bace2, and double knockout mice. We report here that BACE1 mice display a complex phenotype. A variable but significant number of BACE1 offspring died in the first weeks after birth. The surviving mice remained smaller than their littermate controls and presented a hyperactive behavior. Electrophysiologically, subtle alterations in the steady-state inactivation of voltage-gated sodium channels in BACE1-deficient neurons were observed. In contrast, bace2 knockout mice displayed an overall healthy phenotype. However, a combined deficiency of BACE2 and BACE1 enhanced the bace1–/– lethality phenotype. At the biochemical level, we have confirmed that BACE1 deficiency results in an almost complete block of Aβ generation in neurons, but not in glia. As glia are 10 times more abundant in brain compared with neurons, our data indicate that BACE2 could indeed contribute to Aβ generation in the brains of Alzheimer disease and, in particular, Down syndrome patients. In conclusion, our data challenge the general idea of BACE1 as a safe drug target and call for some caution when claiming that no major side effects should be expected from blocking BACE1 activity. Alzheimer disease (AD) 1The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β-peptide; APP, amyloid precursor protein; SFV, Semliki Forest virus; APPwt, wild-type APP; APPsw, Swedish APP mutation; APPfl, Flemish APP mutation; MEM, minimal essential medium; VSV, vesicular stomatitis virus. is the most common cause of dementia for which neither a good diagnostic test nor an effective treatment is available yet. The most widely accepted hypothesis states that AD is initially triggered by the abnormal accumulation and possibly deposition of the small amyloid β-peptide (Aβ) in different brain regions, which in turn initiates a pathogenic cascade that ultimately leads to neuronal death, AD pathology, and dementia. Aβ is cleaved from a long membrane-bound precursor, the amyloid precursor protein (APP), by two consecutive cleavages. β- and γ-secretases are the enzymes that liberate the N and C termini of Aβ, respectively, and are the subject of intense investigation because of their relevance as candidate therapeutic targets to treat AD. BACE1 and BACE2 are two highly homologous membrane-bound aspartyl proteases that can process APP at the β-secretase site (1Hussain I. Powell D. Howlett D.R. Tew D.G. Meek T.D. Chapman C. Gloger I.S. Murphy K.E. Southan C.D. Ryan D.M. Smith T.S. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 1999; 14: 419-427Crossref PubMed Scopus (1001) Google Scholar, 2Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar, 3Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. Jewett N. Keim P. Knops J. Lieberburg I. Power M. Tan H. Tatsuno G. Tung J. Schenk D. Seubert P. Suomensaari S.M. Wang S. Walker D. John V. Nature. 1999; 402: 537-540Crossref PubMed Scopus (1482) Google Scholar, 4Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Crossref PubMed Scopus (3308) Google Scholar, 5Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Crossref PubMed Scopus (1339) Google Scholar, 6Lin X. Koelsch G. Wu S. Downs D. Dashti A. Tang J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1456-1460Crossref PubMed Scopus (740) Google Scholar, 7Acquati F. Accarino M. Nucci C. Fumagalli P. Jovine L. Ottolenghi S. Taramelli R. FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar, 8Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (347) Google Scholar). Although both enzymes exhibit many of the characteristics expected for β-secretase, it has been quite convincingly demonstrated that BACE1 is in fact the major β-secretase responsible for Aβ generation in brain (9Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (954) Google Scholar, 10Roberds S.L. Anderson J. Basi G. Bienkowski M.J. Branstetter D.G. Chen K.S. Freedman S.B. Frigon N.L. Games D. Hu K. Johnson-Wood K. Kappenman K.E. Kawabe T.T. Kola I. Kuehn R. Lee M. Liu W. Motter R. Nichols N.F. Power M. Robertson D.W. Schenk D. Schoor M. Shopp G.M. Shuck M.E. Sinha S. Svensson K.A. Tatsuno G. Tintrup H. Wijsman J. Wright S. McConlogue L. Hum. Mol. Genet. 2001; 10: 1317-1324Crossref PubMed Google Scholar, 11Luo Y. Bolon B. Kahn S. Bennett B.D. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Martin L. Louis J.C. Yan Q. Richards W.G. Citron M. Vassar R. Nat. Neurosci. 2001; 4: 231-232Crossref PubMed Scopus (951) Google Scholar). Contrary to BACE1, BACE2 is more highly expressed in peripheral tissues, but also to some extent in brain (2Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar, 8Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (347) Google Scholar, 12Solans A. Estivill X. de La Luna S. Cytogenet. Cell Genet. 2000; 89: 177-184Crossref PubMed Scopus (82) Google Scholar, 13Bennett B.D. Babu-Khan S. Loeloff R. Louis J.C. Curran E. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 20647-20651Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar), raising the question of whether BACE2 could contribute to the generation of the brain Aβ pool. Both BACE1 and BACE2 can cleave APP in vitro not only at Asp1 (numbering considering the first amino acid of Aβ as position 1), but also at internal sites within the Aβ region. BACE1 cleaves between amino acids 10 and 11 of Aβ, resulting in an N-terminally truncated peptide that is considered more amyloidogenic and more neurotoxic than full-length Aβ (14Pike C.J. Overman M.J. Cotman C.W. J. Biol. Chem. 1995; 270: 23895-23898Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) and that has been observed in senile plaques (15Masters 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 (3668) Google Scholar, 16Naslund J. Schierhorn A. Hellman U. Lannfelt L. Roses A.D. Tjernberg L.O. Silberring J. Gandy S.E. Winblad B. Greengard P. Nordstedt C. Terenius L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8378-8382Crossref PubMed Scopus (370) Google Scholar). The internal BACE2 cleavage site is between amino acids 19 and 20 (8Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (347) Google Scholar, 17Fluhrer R. Capell A. Westmeyer G. Willem M. Hartung B. Condron M.M. Teplow D.B. Haass C. Walter J. J. Neurochem. 2002; 81: 1011-1020Crossref PubMed Scopus (100) Google Scholar, 18Yan R. Munzner J.B. Shuck M.E. Bienkowski M.J. J. Biol. Chem. 2001; 276: 34019-34027Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and the resulting Aβ has thus far not been found in senile plaques. Moreover, BACE2-transfected cells produce reduced levels of Aβ (2Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. Mol. Cell. Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (143) Google Scholar, 8Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (347) Google Scholar, 13Bennett B.D. Babu-Khan S. Loeloff R. Louis J.C. Curran E. Citron M. Vassar R. J. Biol. Chem. 2000; 275: 20647-20651Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 18Yan R. Munzner J.B. Shuck M.E. Bienkowski M.J. J. Biol. Chem. 2001; 276: 34019-34027Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and selective knockdown of endogenous BACE2 in human embryonic kidney 293 cells by RNA interference elevates Aβ secretion (19Basi G. Frigon N. Barbour R. Doan T. Gordon G. McConlogue L. Sinha S. Zeller M. J. Biol. Chem. 2003; 278: 31512-31520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). These observations led to the suggestion that BACE2 does not function as a β-secretase, but rather as an α-like secretase that precludes Aβ formation (17Fluhrer R. Capell A. Westmeyer G. Willem M. Hartung B. Condron M.M. Teplow D.B. Haass C. Walter J. J. Neurochem. 2002; 81: 1011-1020Crossref PubMed Scopus (100) Google Scholar, 18Yan R. Munzner J.B. Shuck M.E. Bienkowski M.J. J. Biol. Chem. 2001; 276: 34019-34027Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 19Basi G. Frigon N. Barbour R. Doan T. Gordon G. McConlogue L. Sinha S. Zeller M. J. Biol. Chem. 2003; 278: 31512-31520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 20Wong P.C. Price D.L. Cai H. Science. 2001; 293: 1434Crossref PubMed Google Scholar). However, these in vitro observations cannot rule out a possible contribution of BACE2 to the Aβ pool in brain, and it has even been suggested that BACE2-mediated APP cleavage might play a role in the development of AD in individuals carrying the Flemish familial AD mutation in APP (8Farzan M. Schnitzler C.E. Vasilieva N. Leung D. Choe H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9712-9717Crossref PubMed Scopus (347) Google Scholar) as well as in the AD-like disease associated with Down syndrome (12Solans A. Estivill X. de La Luna S. Cytogenet. Cell Genet. 2000; 89: 177-184Crossref PubMed Scopus (82) Google Scholar, 21Motonaga K. Itoh M. Becker L.E. Goto Y. Takashima S. Neurosci. Lett. 2002; 326: 64-66Crossref PubMed Scopus (35) Google Scholar). From a therapeutic point of view, there are increasing concerns with using γ-secretase inhibitors to treat AD. γ-Secretase processes a growing number of membrane proteins, and blocking their cleavage is likely to have toxic side effects. Indeed, administration of a potent γ-secretase inhibitor to mice results in marked defects in lymphocyte development and in intestinal villi and mucosa (22Wong G.T. Manfra D. Poulet F.M. Zhang Q. Josien H. Bara T. Engstrom L. Pinzon-Ortiz M.C. Fine J.S. Lee H.J. Zhang L. Higgins G.A. Parker E.M. J. Biol. Chem. 2004; 279: 12876-12882Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar), as was also observed in presenilin-deficient mice (23Tournoy J. Bossuyt X. Snellinx A. Regent M. Garmyn M. Serneels L. Saftig P. Craessaerts K. De Strooper B. Hartmann D. Hum. Mol. Genet. 2004; 13: 1321-1331Crossref PubMed Scopus (75) Google Scholar). In contrast, BACE1 appears to be a promising drug target because genetic ablation of the bace1 gene in mice does not seem to be associated with any gross abnormality (9Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (954) Google Scholar, 10Roberds S.L. Anderson J. Basi G. Bienkowski M.J. Branstetter D.G. Chen K.S. Freedman S.B. Frigon N.L. Games D. Hu K. Johnson-Wood K. Kappenman K.E. Kawabe T.T. Kola I. Kuehn R. Lee M. Liu W. Motter R. Nichols N.F. Power M. Robertson D.W. Schenk D. Schoor M. Shopp G.M. Shuck M.E. Sinha S. Svensson K.A. Tatsuno G. Tintrup H. Wijsman J. Wright S. McConlogue L. Hum. Mol. Genet. 2001; 10: 1317-1324Crossref PubMed Google Scholar, 11Luo Y. Bolon B. Kahn S. Bennett B.D. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Martin L. Louis J.C. Yan Q. Richards W.G. Citron M. Vassar R. Nat. Neurosci. 2001; 4: 231-232Crossref PubMed Scopus (951) Google Scholar). Moreover, BACE1 deficiency could prevent the learning and memory impairments and the cholinergic dysfunction observed in a transgenic mouse model for AD (24Ohno M. Sametsky E.A. Younkin L.H. Oakley H. Younkin S.G. Citron M. Vassar R. Disterhoft J.F. Neuron. 2004; 41: 27-33Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Although BACE1 function might still be required under particular conditions that may have escaped detection, these results highlight BACE1 as one of the best available drug targets for AD. At this point, however, it cannot be excluded that BACE1 has important functions in vivo and that the apparent lack of phenotype in bace1 knockout mice is due to the activation of compensatory mechanisms or to genetic redundancy. Because of their high homology, BACE2 is the best candidate protease to compensate for the absence of BACE1 function. Based on this homology, it is also likely that active-site inhibitors for BACE1 will affect, in addition, BACE2 protease activity. To better understand the biological functions of BACE1 and BACE2, to analyze possible overlapping functions of these two proteases, and to attempt to predict the consequences of blocking BACE function in vivo, we generated mice with inactivated bace1 and/or bace2 genes. Unexpectedly and in contrast to what has been published for bace1 knockout mice, we observed a phenotype associated with BACE1 deficiency, viz. a higher mortality rate early in life. bace2 knockout mice were fertile and viable, with no major phenotypic alteration. Most important, mice with inactivated bace1 and bace2 genes were fertile and viable, but presented neonatal mortality that was even higher than that of the monogenic bace1 line. These results suggest that BACE2 indeed partially compensates for the absence of BACE1 in bace1 knockout mice and that therapeutic inhibition of BACE function may result in adverse side effects. Antibodies—The C terminus-specific antibody for mouse BACE1 (B48) was raised in New Zealand White rabbits using synthetic peptide CLRHQHDDFADDISLLK. Rabbit antibodies B7/8 raised against Aβ (25De 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) and B63 raised against the C terminus of human APP (26Esselens C. Oorschot V. Baert V. Raemaekers T. Spittaels K. Serneels L. Zheng H. Saftig P. De Strooper B. Klumperman J. Annaert W. J. Cell Biol. 2004; 166: 1041-1054Crossref PubMed Scopus (152) Google Scholar) have been described. Anti-FLAG monoclonal antibody was from Sigma. The N terminus-specific antibody for human Aβ (82E1) was from IBL Co., Ltd. (Tokyo, Japan). Plasmid Construction—cDNAs to be expressed in non-neuronal cells were subcloned into a derivative of the eukaryotic expression vector pSG5 (Stratagene) that contains a larger polylinker (pSG5**; polylinker EcoRI, SpeI, SacII, HindIII, NotI, XhoI, SmaI, SacI, BamHI, and BglII). BACE1 cDNA was amplified from mouse brain RNA using primers 5′-GGATTCATGGCCCCAGCGCTGCACTGGCT-3′ and 5′-GAGCTCTCACTTGAGCAGGGAGATGTCATC-3′ (with the SacI site underlined) and directly cloned into pGEM-T (Promega). The SacI-SacII fragment was subsequently subcloned into the SacI-SacII sites of pSG5**. BACE2 cDNA was amplified from mouse pancreas cDNA using primers 5′-ATGGGCGCGCTGCTTCGAGCAC-3′ and 5′-TCATTTCCAGCGATGTCTGAC-3′ and cloned into the pGEM-T vector. The XmaIII fragment of pGEM-T-mBACE2 was subsequently subcloned into the SmaI site of pSG5**. For cloning of BACE2 cDNA containing a deletion of exon 6 (BACE2ΔE6), two subfragments of the cDNA were separately amplified using primers that contain the deletion. The 5′-fragment was amplified using T7 as the forward primer and 5′-AGAAAACTCTGGAATCTCTCTGCAGTCCAGGTTGAGGTTCTGG-3′ as the reverse primer. The 3′-fragment was amplified using primers 5′-CTGGACTGCAGAGAGATTCCAGAGTTTTCTGATGGCTTCTGGAC-3′ and 5′-GCTGCAATAAACAAGTTCTGCT-3′. The purified 5′- and 3′-subfragments were mixed together and PCR-amplified using the T7 and 5′-GCTGCAATAAACAAGTTCTGCT-3′ primers. The PCR product was digested with EcoRI and BamHI and cloned into the same sites of pSG5**. Cloning of bace2 and bace2ΔE6 containing a C-terminal FLAG epitope was done by PCR amplification on pSG5**BACE2 and pSG5**-BACE2ΔE6, respectively, using primers 5′-CGGAATTCCACCATGGGCGCGCTGCTTCGAGCA-3′ (with the EcoRI site underlined) and 5′-CGGGATCCTCATTTATCGTCGTCATCCTTGTAGTCTTTCCAGCGATGTCTGACTAGT-3′ (with the BamHI site underlined and the FLAG epitope in italics). PCR products were digested with EcoRI-BamHI and cloned into the same sites of the pSG5** vector. All constructs were verified by sequencing. For expression in neuronal and glial cells, cDNAs were cloned into Semliki Forest virus (SFV) type 1. Cloning of SFV-APPwt, SFV-APPsw, and SFV-APPfl has been described previously (27Simons M. De Strooper B. Multhaup G. Tienari P.J. Dotti C.G. Beyreuther K. J. Neurosci. 1996; 16: 899-908Crossref PubMed Google Scholar, 28Tienari P.J. De Strooper B. Ikonen E. Ida N. Simons M. Masters C.L. Dotti C.G. Beyreuther K. Cold Spring Harbor Symp. Quant. Biol. 1996; 61: 575-585Crossref PubMed Google Scholar). Primary Cultures and Cell Lines—Medium, serum, and supplements for maintenance of cells were obtained from Invitrogen. COS cells and adult mice fibroblasts were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F-12 (1:1) supplemented with 10% fetal calf serum. Primary neuronal cultures were generated from trypsinized brains obtained from day 14 embryos and maintained in Neurobasal medium (Invitrogen) supplemented with B27 and 0.5 μm l-glutamine. Cytosine arabinoside (5 μm) was added 24 h after plating to prevent non-neuronal (glial) cell proliferation. For glial cell cultures, Neuro-basal medium was replaced with minimal essential medium (MEM; Invitrogen) supplemented with 10% horse serum, 0.225% NaHCO3, 2 mm l-glutamine, and 0.6% glucose (MEM-HS). Cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere. DNA Transfer and Metabolic Labeling—COS cells were plated in 6-cm2 plates 1 day before transfection. Approximately 70–80% confluent cells were transfected with a total of 2 μg of DNA (1 μg of APP and 1 μg of BACE plasmids) and 6 μl of FuGene 6 (Roche Applied Science). Two days after transfection, cells were metabolically labeled with 100 μCi/ml [35S]methionine for 4 h; the conditioned medium was collected; and cells were directly lysed in double immunoprecipitation assay buffer (50 mm Tris-HCl (pH 7.8), 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS). Neurons were maintained in Neurobasal medium, and ∼48 h after the addition of cytosine arabinoside, they were infected with recombinant SFV. Glial cells were maintained for ∼1 week in MEM-HS, passaged at least once, and infected with recombinant SFV ∼48 h after trypsinization. (This treatment ensured the absence of neurons in the culture.) For both neurons and glial cells, a 10-fold dilution of SFV encoding APPwt, APPsw, or APPfl was added to the cultures, and infection was allowed to proceed for 1 h. The conditioned medium containing the virus was then replaced with fresh medium, and cells were further incubated for 2 h. Cells were metabolically labeled with 100 μCi/ml [35S]methionine for 4 h; the conditioned medium was collected; and the cells were directly lysed in double immunoprecipitation assay buffer. Mouse fibroblasts were plated in 12-well plates 1 day before infection (∼300,000 cells/well). A 1:4 dilution of adenovirus encoding APPsw was added to the medium, and cells were further incubated for 48 h. Metabolic labeling was subsequently done as described above. Analysis of APP Processing—Full-length APP and C-terminal fragments were immunoprecipitated from cell extracts using antibody B63. Aβ was immunoprecipitated from the conditioned medium using antibody B7/8. Protein G-Sepharose beads (Amersham Biosciences) were added to the mixtures, followed by overnight incubation at 4 °C with rotation. The immunoprecipitates were washed five times with double immunoprecipitation assay buffer and once with 0.3× Tris-buffered saline and then solubilized with NuPAGE lithium dodecyl sulfate loading buffer. Samples were heated for 10 min at 70 °C and electrophoresed on 4–12% precast gels (Novex). Radiolabeled bands were detected using a PhosphorImager (Amersham Biosciences). Analysis of APP Processing Using Antibody 82E1—Neurons and glial cells were infected with recombinant SFV for 1 h as described above. The medium was subsequently replaced with Neurobasal medium (neurons) or MEM-HS (glial cells), and cells were further incubated for 6 h. Cells were lysed in phosphate-buffered saline containing protease inhibitors (Trasylol, 1 μg/ml pepstatin, and 5 mm EDTA) and 1% Triton X-100. Samples of cell extracts were resolved by SDS-PAGE and probed with antibody B63. Aβ was immunoprecipitated from the conditioned medium using antibody B7/8 and detected by Western blotting using antibody 82E1. Fluorescence Resonance Energy Transfer Analysis—COS cells were transfected with 2 μg of either empty vector or vector encoding BACE1-FLAG, BACE2-FLAG, or BACE2ΔE6-FLAG using 6 μl of FuGene 6. Forty-eight hours after transfection, cells were scraped in buffer containing 5 mm Tris (pH 7.4), 250 mm sucrose, 1 mm EGTA, and 1% Triton X-100, and protein concentration was determined using Bio-Rad protein assay dye reagent. Proteins (∼400 μg) were subsequently incubated overnight at 4 °C with antibody B48 (BACE1-transfected cells) or anti-FLAG antibody (BACE2-transfected cells) and protein G-Sepharose beads. The immunoprecipitates were washed three times with Tris-buffered saline containing 0.1% Triton X-100 and twice with Tris-buffered saline. BACE activity was subsequently measured in an in vitro assay (Panvera P2985) by fluorescence resonance energy transfer according to the manufacturer's instructions. Briefly, an APP-based peptide substrate carrying the Swedish mutation and containing a fluorescence donor and a quencher acceptor at each end was used. The intact substrate is weakly fluorescent and becomes highly fluorescent upon enzymatic cleavage. BACE immunoprecipitates were directly resuspended in 20 μl of assay buffer provided with the kit, and after substrate addition, excitation and emission were measured using VICTOR2 (PerkinElmer Life Sciences Model 1420 multilabel counter). Pup Exchange—A total of eight BACE1 homozygous and eight wild-type couples were used for the experiment. Coupling was synchronized, and pups were exchanged during the first day of birth. The number of pups was followed until weaning. Electrophysiological Recordings—Acutely isolated pyramidal cell somata were prepared from the sensorimotor cortex of anesthetized and then decapitated wild-type and bace1–/– mice (23–30 days of age) using an established method of combined enzymatic/mechanic dissociation (29Alzheimer C. J. Physiol. (Lond.). 1994; 479: 199-205Crossref Scopus (49) Google Scholar). Briefly, freshly prepared neocortical slices were incubated for 30 min in warmed (29 °C) artificial cerebrospinal fluid and then maintained at room temperature. Artificial cerebrospinal fluid was constantly gassed with 95% O2 and 5% CO2 and contained 125 mm NaCl, 3 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 1.25 mm NaH2PO4, 25 mm NaHCO3, and 10 mm d-glucose (pH 7.4). Small pieces of sliced tissue (∼2 × 2 mm) were incubated for 45 min at 29 °C in HEPES-buffered saline (150 mm NaCl, 3 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, and 10 mm d-glucose (pH 7.4)) containing 19 units/ml papain. All recordings were made at room temperature (19–20 °C). Current signals from acutely isolated pyramidal cell somata recorded in whole cell voltage clamp mode were sampled at 20 kHz and filtered at 5 kHz (–3 dB) using an Axopatch 200B amplifier in conjunction with a Digidata 1322A interface and pClamp 9 software (all from Axon Instruments, Inc., Foster City, CA). Access resistance in the whole cell configuration was 10–15 megaohms before series resistance compensation (75–80%). To improve voltage control, Na+ currents were investigated in a low Na+ bathing solution containing 15 mm NaCl, 115 mm choline chloride, 3 mm KCl, 2 mm MgCl2, 1.6 mm CaCl2, 0.4 mm CdCl2, 10 mm HEPES, and 10 mm d-glucose (pH 7.4). Patch pipettes were filled with 105 mm CsF, 20 mm triethanolamine chloride, 3 mm KCl, 1 mm MgCl2, 8 mm HEPES, 9mm EGTA, and 2 mm Na2ATP (pH 7.2 adjusted with CsOH). Data are presented as means ± S.E. Data were statistically analyzed (Student's t test, significance set at p < 0.05) using Origin Pro7 software. Substances were purchased from Sigma. Animals—A panel of 69 male mice (25 wild-type, 23 heterozygous, and 21 bace1 knockout littermate mice, aged 3–9 months) was used to assess anxiety-related behavior in the open field test and elevated zero maze and depression-related behavior in the tail suspension test and forced swim test. Animals were individually housed and kept under a 12-h light/12-h dark cycle (lights on at 6:00 a.m.) in a temperature- and humidity-controlled room with food and water ad libitum. All experiments were conducted during the light phase of the light/dark cycle with 1 week between experiments. Open Field Test—Locomotor activity was monitored using a Truscan© system (Coulbourn Instruments Inc., Allentown, PA). The animal was placed in the center of the activity field arena, which is a transparent plexiglas cage (260 (width) × 260 (depth) × 400 (height) mm) equipped with two photo beam sensor rings to register horizontal and vertical activities. Testing lasted 30 min. Elevated Zero Maze—Elevated zero maze testing was performed as described by Crawley (30Crawley J.N. What's Wrong with My Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice. John Wiley & Sons, Inc., New York2000Google Scholar). The zero maze consists of an annular platform (diameter, 50 cm; and width, 5 cm). The animals were allowed to freely explore the maze for 5 min, and their behavior was recorded and analyzed using the Ethovision Pro video tracking system (Noldus Information Technology, Wageningen, The Netherlands). Tail Suspension Test—Mice were suspended by their tail on a hook in a test chamber using adhesive tape. Total duration of immobility was measured over a period of 6 min using the VideoTrack system (Viewpoint, Champagne au Mont d'Or, France). Mice that curled up toward their tail or that fell off during testing were excluded from analysis. Forced Swim Test—A mouse was placed in a cylinder (inner diameter, 10 cm) filled with water to a height of 10 cm at a temperature of 25 ± 1 °C. The mouse was exposed to swim stress for 6 min. Total duration of immobility was measured using the VideoTrack system. One animal was excluded from analysis because it had a very high fat mass and had difficulties staying afloat. Statistical Analysis—Data were analyzed by one-way analysis of variance or by Kruskal-Wallis analysis of variance on ranks in case data were not normally distributed, followed by post hoc Tukey's test (oneway analysis of variance) or Dunn's method (Kruskal-Wallis analysis of variance on ranks) if appropriate. Lethal Phenotype in bace1 Knockout Mice—Several groups have reported the generation of bace1 knockout mice (9Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C. Nat. Neurosci. 2001; 4: 233-234Crossref PubMed Scopus (954) Google Scholar, 10Roberds S.L. Anderson J. Basi G. Bienkowski M.J. Branstetter D.G. Chen K.S. Freedman S.B. Frigon N.L. Games D. Hu K. Johnson-Wood K. Kappenman K.E. Kawabe T.T. Kola I. Kuehn R. Lee M. Liu W. Motter R. Nichols N.F. Power M. Robertson D.W. Schenk D. Schoor M." @default.
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• W2023274935 date "2005-09-01" @default.
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• W2023274935 title "Phenotypic and Biochemical Analyses of BACE1- and BACE2-deficient Mice" @default.
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