Molecular Psychiatry (2004) 9, 594–602 & 2004 Nature Publishing Group All rights reserved 1359-4184/04 $30.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Baculoviruses expressing the human familial Alzheimer’s disease 1 mutation lacking exon 9 increase levels of an -like in Sf9 cells G Verdile1, D Groth1, PM Mathews2, P St George-Hyslop3,4, PE Fraser3,4, TV Ramabhadran5, JBJ Kwok6, PR Schofield6,7, T Carter8, S Gandy8 and RN Martins1 1Sir James McCusker Alzheimer’s Disease Research Unit, University of Western Australia, School of Psychiatry and Clinical Neurosciences, Hollywood Private Hospital, Nedlands, WA, Australia; 2Dementia Research Program, Nathan Kline Institute, Orangeburg, NY, USA; 3Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada; 4Departments of Medical Biophysics and Medicine (Neurology), University of Toronto, Toronto, Ontario, Canada; 5Tranzyme, Inc., Birmingham, AL, USA; 6Garvan Institute for Medical Research, Department of Biotechnology, Darlinghurst, NSW, Australia; 7University of New South Wales, Sydney, NSW, Australia; 8Farber Institute for Neurosciences at Thomas Jefferson University, Philadelphia, PA, USA

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-b protein (Ab) that is central to the pathogenesis of Alzheimer’s disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP- C99), a cleavage event that releases Ab following a reaction catalyzed by an termed ‘c-secretase’. The molecular mechanism of PS1-mediated, c-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the c-secretase or whether instead PS1 mediates c-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the ‘authentic’ protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Ab-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DE9 molecules expressed in Sf9 cells retain the ability to modulate Ab levels. Baculoviral-expressed PS1DE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting c-secretase activity from its individual purified component parts. Molecular Psychiatry (2004) 9, 594–602. doi:10.1038/sj.mp.4001458 Published online 2 March 2004 Keywords: Alzheimer’s disease; amyloid-b;Ab; amyloid precursor protein; presenilin-1; nicastrin; proteolytic processing; Spodoptera frugiperda cells; g-secretase

Introduction see Nunan and Small3). The initial processing of APP via a-secretases (TACE/ADAM10/ADAM17) or b- One of the key histopathological elements of Alzhei- secretase (BACE) liberates fragments of the APP mer’s disease is the accumulation of cerebral and ectodomain (soluble APPa or soluble APPb, respec- cerebrovascular amyloid deposits (for a review, see tively) leaving membrane-bound carboxyl-terminal 1 Selkoe ). The major protein component of these fragments, referred to as a-CTF (APP-C83) or b-CTF B deposits is a 4 kDa peptide (ranging from 39 to 43 (APP-C99), respectively.1 An activity designated residues in length) termed the amyloid-b-protein g-secretase is responsible for the cleavage of a-CTF (Ab), that is generated from the proteolytic processing to generate either a nonamyloidogenic fragment 2 of the amyloid precursor protein (APP; for a review, called p3 or, alternatively, g-secretase can cleave b-CTF to generate the Ab protein.4 Correspondence: R Martins, Sir James McCusker Alzheimer’s Mutations in presenilin 1 (PS1) account for the Disease Research Unit, Hollywood Private Hospital, The majority of inherited forms of early onset familial University of Western Australia Nedlands, Perth, WA 6009, 5 Australia. E-mail: [email protected] AD (FAD; for a review, see Verdile et al ). FAD-linked Received 17 July 2003; revised 13 October 2003; accepted 17 PS1 mutations result in increased generation of October 2003 the highly amyloidogenic Ab42.6–8 These findings PS1DE9 and c-secretase activity in Sf9 cells G Verdile et al 595 indicate that PS1 is intimately involved in Protein expression in SF9 cells Ab production, a conclusion that was definitively The baculoviruses containing APP-C99 were kindly established by the observation that PS1 deficiency provided by Dr Rachel Neve, Harvard Medical eliminates g-secretase activity.9 The identification of School, McLean Hospital, and have been described.19 g-secretase inhibitors that affinity-label PS110,11 A control baculovirus containing the unrelated strongly suggests that the PS1 polypeptide forms at protein tropomyosin (TPM3) was kindly provided least part of the catalytic site of g-secretase. Further by Dr Anthony Akkari (University of Western Aus- evidence is provided by other studies describing tralia). For the production of baculoviruses containing a physical interaction between PS1 and the APP PS1DE9 and nicastrin inserts, recombinant bacmid C-terminal fragments.12–14 DNA fragments containing the inserts were generated Recent studies have identified candidate , as per the manufacturer’s instructions using the BAC- nicastrin, APH-1a, APH-1b and PEN-2, that together TO-BACs system (Invitrogen, CA, USA). Serum- with PS1 are essential components for g-secretase adapted Sf9 insect cells (Invitrogen, CA, USA) were activity.15,16 However, it still remains to be determined then transfected with recombinant DNA. Sf9 cells if PS1 includes the catalytic site of the g-secretase were seeded at a density of 9 Â 105 cells per well of a protease or whether instead PS1 mediates g-secretase six-well tissue culture plate, in 2 ml of Sf-900 II SFM activity indirectly, perhaps by regulating the trafficking containing antibiotics (Invitrogen, CA, USA). The or presentation of substrates to the ‘authentic’ protease, cells were allowed to attach by incubating them at which may be a molecule distinct from PS1. Formal 271C for 1 h. Following a wash with 2 ml of Sf-900 II proof that PS1 is identical to g-secretase requires SFM without antibiotics, the cells were transfected reconstitution of the catalytic activity from its purified with a lipid–DNA mixture. For each transfection, 1 mg component part(s), a goal that has not yet been achieved. of baculovirus DNA, diluted in 100 ml of Sf-900 II SFM The aim of the present study was to determine without antibiotics, was combined with 9 mlof whether PS1 overexpressed in insect cells was CELLFECTINs reagent (Life Technologies, CA, competent to modulate Ab generation in intact cells. USA), diluted in 100 ml of Sf-900 II SFM without Spodoptera frugiperda (Sf9) cells generate abundant antibiotics and incubated at RT for 30 min. The C83 fragments from overexpressed human APP, combined mixture was diluted in 800 ml of Sf-900 II identical in sequence to the C83 produced by SFM without antibiotics, mixed gently and subse- mammalian cells.17 C99-like and Ab-like species have quently added to cells, following aspiration of wash been demonstrated immunochemically in Sf9 cells media. The cells were incubated for 5 h at 271C before infected with either holo APP of APP-C99, respec- the transfection mixture was removed and fresh Sf- tively.18,19 Here we demonstrate that levels of such an 900 II SFM containing antibiotics was added. The Ab-like species can be modulated by co-overexpres- cells were incubated for an additional 48 h before sion of APP-C99 together with PS1DE9, a pathogenic harvesting the newly synthesized virus. Sf9 cells were PS1 molecule lacking exon 9. Nicastrin or tropomyo- infected with baculoviruses at various multiplicities sin viruses either failed to elevate levels of the of infection (MOI), as described in Results. The MOIs ‘Ab-like’ species or else showed a modest lowering were determined using a viral plaque assay as in the ‘Ab-like’ levels under various contributors of described previously.23 single or multiple infections. This evidence that overexpressed recombinant PS1DE9 molecules are Western blotting competent to modulate g-secretase activity suggests Media from cells infected with APP-C99 alone or in that this is viable starting material for proceeding combination with PS1DE9 or nicastrin were subjected with efforts to reconstitute g-secretase in vitro follow- to immunoprecipitation for Ab as described below. ing purification of its recombinant components. Following washing in PBS, the cells were scraped into a culture tube using a rubber policeman. After centrifugation (500 g, 2 min at 41C), the cells were Methods resuspended in 500 ml of PBS, and centrifuged at 500 g Antibodies for 2 min at 41C. The cell pellet was sonicated in PBS, The human-specific PS1 monoclonal antibody NT1, and 10 mg of total protein was resuspended in sample directed against residues 41–49 of PS1, and the APP buffer (70 mM Tris–HCl, pH 6.8, containing 3.2% monoclonal antibody C1/1.6, directed against the last (w/v) SDS, 0.4 mM glycine, 6 M urea, 0.1 M DTT and 20 residues of APP, have been previously described.20 0.01% (w/v) phenol red) and separated using poly- Antibodies WO2, raised against amino-acid residues acrylamide gel electrophoresis. Following electro- 5–821 of the Ab domain of APP, 6E10, raised against phoresis and electrophoretic transfer, the residues 1–17 of Ab, the Ab42-specific antibody, membranes were immunoblotted using the antibodies 21F12 and 29AF (directed against residues 17–24 of as indicated, using standard techniques as de- Ab)22 were all kindly provided by Dr Dale Schenk scribed.12 from Athena Neurosciences. The rabbit polyclonal antibody NCT Affi, directed against residues 691–701 Immunoprecipitation for Ab of nicastrin, was purchased from Research Genetics The media harvested from infected Sf9 cells were (AL, USA). subjected to immunoprecipitation with antibodies

Molecular Psychiatry PS1DE9 and c-secretase activity in Sf9 cells G Verdile et al 596 6E10 and 29AF as described.24 Following immuno- precipitation, the complexes were loaded onto 8–12% tris-tricine gels and separated using electrophoresis. Following electrophoresis, the gels were transferred to nitrocellulose membranes and immunoblotted with antibodies WO2 or 21F12, as described.12

To achieve better separation between the Ab40 and Ab42 species, supernatants were subjected to electro- phoresis using a 15% bis-bicine SDS-PAGE gel.25,26 In this method, the immunoprecipitates were resus- pended in 0.36 M Bis–Tris, containing 0.16 M Bicine, 1% (w/v) SDS, 15% (w/v) sucrose, 2.5% (v/v) b-mercaptoethanol and 0.004% bromophenol blue, and loaded onto a 15% bis-bicine SDS-PAGE gel. Electrophoresis was performed at 12 mA/gel constant current for 30 min and 24 mA/gel for 3–4 h in cathode buffer consisting of 0.2 M bicine, 0.25% SDS and 0.1 M NaOH and an anode buffer consisting of 1.6 M

Tris and 0.4 M H2SO4. Electrophoretic transfer and immunoblotting with WO2, 6E10 or 21F12 were Figure 1 Expressing PS1DE9 and Nicastrin in Sf9 cells. 12 performed as described above. (a) Sf9 cells were transfected with recombinant bacmid cDNA containing the PS1DE9 mutation insert (trans, lane 3). Results Media collected from transfected cells was incubated with untransfected Sf9 cells for 48 h (1st Amp, lane 2) then a Expression of recombinant PS1DE9 and nicastrin in Sf9 further 72 h (2nd Amp, lane 1) to amplify the baculovirus. cells Immunoblotting of cell lysates revealed that the expression B Recombinant bacmid DNA containing the nicastrin or of the 45 kDa PS1 holoprotein was the highest following a PS1DE9 insert was generated using the BAC-TO- second amplification process (2nd Amp, lane 1) compared to initial transfection (trans, lane 3), and amplification (1st BACs baculovirus expression system. Expression of B Amp, lane 2). The PS1 holoprotein was absent in cells the 45 kDa PS1DE9 holoprotein in insect cells was infected with bacmid DNA lacking the PS1DE9 insert. only seen following amplification of the virus by (b) Sf9 cells were transfected with four clones of bacmid incubating the Sf9 cells with virus for 72 h. Immuno- cDNA containing the nicastrin insert. Following a 48 h blotting cell lysates at 48 h post infection revealed a incubation, cells were harvested and lysed. Western B45 kDa protein representing the PS1DE9 holopro- immunoblotting (WB) using Affi-NCT antibody revealed a tein (Figure 1a, compare lanes 1 and 2). band of B110 kDa in all of the clones (lanes 3–6), which was Four clones of this DNA containing the nicastrin absent in uninfected cells (lane 2) or cells infected with insert, as well as bacmid DNA lacking the insert, were bacmid cDNA lacking the nicastrin insert (lane 1). (c) To used to infect Sf9 cells. Immunoblotting analysis of examine the maturation process of nicastrin in Sf9 cells, cell lysates from baculovirus-infected Sf9 cells and HEK-293 cell lysates at 48 h post infection revealed a B110 kDa cells overexpressing nicastrin were treated with endoglu- protein, representing nicastrin, in all of the four cosidases. Immature and mature nicastrin are present in the clones studied (Figure 1b, lanes 3–6). This species transfected HEK-293 cells (lane 4), while only the immature was absent in uninfected cells (Figure 1b, lane 2) and form is present in the infected insect cells (lane 1). An in cells infected with recombinant bacmid DNA 80 kDa band is seen following EndoH and PNGase treat- lacking the nicastrin insert (Figure 1b, lane 1). ments of both cell types (lanes 2, 3, 5 and 6), indicating that The N-glycosylated form of nicastrin has been nicastrin derived from insect or mammalian cells is fully shown to interact with PS1,27,28 and this interaction deglycosylated. The major form of nicastrin derived from may be required for g-secretase activity.29,30 Therefore, the insect cells following glucosidase treatment is the in order to determine the glycosylation status of deglycosylated form (lanes 2 and 3), while both immature and deglysosylated forms are present in the mammalian nicastrin derived from insect cells, lysates from Sf9 HEK-293 cells (lanes 5 and 6). cells infected with nicastrin and HEK-293 cells overexpressing nicastrin were treated with the en- EndoH or PNGase (Figure 1c; compare lanes 4–6 vs doglucosaminidases EndoH and PNGase. The lanes 1–3). These results indicate that the majority of B 110 kDa nicastrin band was present in untreated nicastrin derived from the baculovirus-infected insect lysates from both cell lines (Figure 1c, lanes 1 and 4). cells are still in the high-mannose form (Man9- Deglycosylation of the nicastrin with EndoH or GlcNAc ), lacking oligosaccharides such as galactose, B 2 PNGase resulted in the conversion of the 110 kDa N-acetyl glucosamine or sialic acid. protein into an B80 kDa protein (Figure 1c, lanes 2, 3, 5 and 6), representing the nascent unmodified An ‘Ab-like’ species secreted by APP-C99-expressing protein. Compared to mammalian cells, the majority Sf9 cells of nicastrin expressed in Sf9 cells is immature A previous report indicates that Sf9 cells can generate nicastrin, which is completely sensitive to either a cell-associated, Ab-like peptide from APP-C99

Molecular Psychiatry PS1DE9 and c-secretase activity in Sf9 cells G Verdile et al 597 baculoviruses.19 For the next series of experiments, from noninfected cells or those infected with the we sought to determine the appearance of this peptide bacmid lacking the APP-C99 insert (Figure 2a, lanes 7 in the supernatant as well as to determine the effect of and 8, respectively). These data indicate that the multiplicity-of-infection (MOI) on levels of this Ab- overexpression of APP-C99 in Sf9 cells results in the like species. Serum-free media were harvested from generation of an ‘Ab-like’ species and that the levels Sf9 cells infected with APP-C99 at MOI ranging from of this species vary according to the APP-C99 MOI. 1 to 10 (Figure 2a). The media samples were This variation corresponded directly to the variation immunoprecipitated with a mixture of monoclonal in APP-C99 levels (Figure 2b). antibodies 6E10 (directed against residues 1–17 of Ab) and 29AF (directed against residues 17–24) Dual expression of PS1DE9 and APP-C99 elevates subjected to SDS-PAGE, and immunoblotted using relative levels of Ab-like species antibody WO2 (with an epitope spanning residues A standard MOI of 5 was used for APP-C99 expres- 5–8 of Ab), revealing a B4 kDa protein (Figure 2a, sion in the next series of experiments. An escalating lanes 1–4, 6) that co-migrated with synthetic Ab and 40 dose of PS1DE9 (MOI 1–100) was added to the Sf9 Ab peptides (Figure 2a, lanes 10–12) and with the 42 cells in combination with the standard dose of APP- same Ab peptides when media from bacmid-infected C99 virus, and revealed that the viral titre at which cells were spiked with synthetic peptides (Figure 2a, PS1DE9 protein levels were expressed optimally was lane 9). The protein species was present in the at an MOI of 10 (data not shown). Compared to APP- precipitates from cells infected with APP-C99 at C99 expression alone, expressing PS1DE9 at an MOI the highest MOI (MOI 9 and 10; Figure 2a, lanes 1 of 10 diminished the expression of APP-C99 (Figure and 6, respectively) but not at the lowest MOI (MOI 1; 3b and c, compare lane 12 with lane 1). However, the Figure 2a, lane 5). The Ab-like species was absent fractional levels of Ab-like species (per arbitrary unit of APP-C99 immunoreactive) were elevated (Po0.05) when compared to the fractional levels of Ab-like species (per arbitrary unit of APP-C99 immunoreac- tivity) observed in the presence of APP-C99 alone (Figure 3a, compare lane 1 vs lane 12 and 3b). Expression of wild-type PS1 at MOIs ranging from 0.001 to 10 did not have any effect on levels of APP- C99 (data not shown). These data indicate that PS1DE9 viruses facilitated metabolism of APP-C99 to yield an Ab-like species.

Effects of nicastrin on APP-C99 metabolism Unlike the results following PS1DE9 expression, the addition of baculoviruses containing the nicastrin insert appeared to inhibit the generation of the Ab- like species (Figure 3a, compare lanes 8 and 9 vs lanes 12 and 3b). Since the effect of nicastrin viruses on Figure 2 Secretion of an ‘Ab-like’ species from Sf9 cells APP-C99 metabolism was strikingly different from the expressing APP-C99. (a) Media from infected cells were effect of the PS1DE9 viruses, we tested the effect of an immunoprecipitated with Ab N-terminal antibodies 29AF unrelated molecule, tropomyosin (TPM3), in order to and 6E10. Subsequent immunoblotting with antibody WO2 B assess the specificity. The addition of TPM3 viruses, detected a 4 kDa protein in immunoprecipitates from cells like the addition of nicastrin viruses, was associated infected with APP-C99. The protein species co-migrated with Ab40 (lanes 10, 11) and Ab42 synthetic peptides (lane with lowering of the stoichiometry of Ab-like peptide 12) and with Ab-spiked media from bacmid-infected cells per unit APP-C99 (Figures 3a, lanes 5–6 and 3b), (lane 9). High levels of the Ab-like species were secreted while the levels of APP-C99 were similar to those from cells infected with APP-C99 at MOIs of 10, 9 and 7 when cells were infected with APP-C99 alone (Figure (lanes 1, 2 and 6) and low levels at MOIs of 5, 3 and 1 (lanes 3f, compare lanes 5–7 to lane 12). With triple 3, 4 and 5). The protein species was absent from expression co-infections, where either nicastrin or noninfected cells (lane 7) or cells infected with bacmid TPM3 reduced the levels of APP-C99 (Figure 3f, lanes virus lacking the APP-C99 insert (lane 8). (b) The variation 2–4, 10 and 11) or Ab-like species (Figure 3a, lanes in levels of the Ab-like species corresponded directly to the 2–4, 10 and 11) when combined with PS1D9 and APP- variation in APP-C99 levels. Sf9 cells were infected with C99 compared to levels seen in cells infected with APP-C99 at MOI ranging from 1 to 10. The APP-C99 fragments ranging from B6 to 14 kDa were present in all APP-C99 only (Figure 3a and f, lane 12). These results samples, with the exception of lysates from uninfected cells indicate that both nicastrin and TPM3 viruses or cells infected with baculovirus lacking the APP-C99 probably acted as nonspecific inhibitors of generation insert (lanes 7, 8 respectively). APP-C99 expression of the Ab-like species, while PS1DE9 viruses were decreased as the MOI was reduced (lanes 1–5), with the unusual in their ability to elevate levels of the Ab-like highest expression being at an MOI of 10 (lane 6). species.

Molecular Psychiatry PS1DE9 and c-secretase activity in Sf9 cells G Verdile et al 598

Figure 3 Effects of nicastrin, PS1DE9 and tropomyosin on APP-C99 and the Ab-like species. (a) Media harvested from infections were immunoprecipitated and subsequently immunoblotted with WO2. The B4 kDa protein species migrated to a similar position to that in immunoprecipitated media spiked with synthetic Ab peptide (lane 14) and was absent in cells infected with the tropomyosin baculovirus only (lane 13). (b) The mean densities of the APP-C99 fragments and the B4 kDa Ab-like species shown in (a) were measured from two separate experiments, performed in duplicate. The levels of APP-C99 and the Ab-like species from co-infected cells are shown as a percentage of their respective levels from cells infected with APP-C99 alone. All data represent mean7SEM (n ¼ 4). *, ** Values were significantly reduced compared to those for cells infected with APP-C99 alone. (c) Levels of nicastrin decreased with reduction of MOI in cells co-expressing nicastrin and APPC99 (lanes 8, 9). Addition of PS1DE9 into the baculovirus mixture further reduced nicastrin levels (lanes 10, 11). (d) Levels of the B36 kDa tropomyosin protein increased with higher MOI (lanes 2, 3, 4, 5, 6, 7). Relative to cells expressing tropomyosin only (lane 13), cells co-expressing tropomyosin and APP-C99 (lanes 5, 6, 7) or PS1DE9, tropomyosin and APP- C99 (lanes 2, 3, 4) had reduced levels of the B36 kDa protein. (e) PS1 levels remained unchanged for either co-expression of PS1DE9 and APP-C99 (lane 1) or PS1DE9, tropomyosin and APP-C99 (lanes 2, 3, 4), but were slightly reduced with co- expression of PS1DE9, nicastrin and APP-C99 (lanes 10, 11). Collectively, these data suggest that expression of multiple proteins causes nonspecific reductions in protein levels. Nonetheless, when compared to other duplex combinations (e.g. APP-C99 and tropomyosin or APP-C99 and nicastrin), co-expressing PS1DE9 and APP-C99 specifically reduced the levels of APP-C99 and increased secretion of Ab-like species, suggesting that PS1DE9 cleaves the APP-C99 to the Ab-like species.

Characterization of Ab-like species Media collected from Sf9 cells expressing APP-C99 APP-C99 alone or in combination with PS1DE9 alone (Figure 4a, lane 1) or PS1 and APP-C99 (Figure (Figure 4b, lanes 1 and 2, respectively). The Ab-like 4A, lane 2), were immunoprecipitated. The immuno- species was immunoreactive with antibodies WO2 precipitates underwent electrophoresis using the (residues 5–8 of Ab; Figure 4b, lanes 1 and 2) and to a urea/bicine/bistris/tris/sulfate SDS-PAGE system lesser extent 6E10 (residues 1–17 of Ab; Figure 4c, and, after electrophoretic transfer, the membranes lanes 1 and 2). The species migrated to a similar 25,26 were immunoblotted. As reported previously, position as the synthetic Ab40 peptide (Figure 4b and

synthetic Ab peptide was separated into Ab40 and c, lanes 4–6) and is nonimmunoreactive with Ab42 species (Figure 4b, lanes 4–7). The secreted Ab42-specific antibody 21F12 (Figure 4d, lanes 1 Ab-like species was present following infection with and 2) or the non-Ab antibody C1/1.6 (Figure 4e,

Molecular Psychiatry PS1DE9 and c-secretase activity in Sf9 cells G Verdile et al 599 lanes 1 and 2). Furthermore, Ab42 could not be Overall, the above findings indicate that the detected in the media from infected cells using the secreted Ab-like species may have a C-terminus

ELISA method (data not shown). identical to that of typical Ab40. However, the lack of detection of Ab42 in this system may possibly reflect a limitation in the sensitivity of the assays employed in this study.

Discussion The elusive enzyme g-secretase catalyzes the terminal reaction in the proteolytic processing of APP-C99 into Ab and g-CTF. Evidence to date has established that PS1 facilitates g-secretase activity.9,11,31–33 However, it remains to be elucidated whether PS1 is a protease or is instead a cofactor controlling substrate trafficking and/or presentation. To address this question, PS1 and its recently identified partner, nicastrin, were expressed in a baculovirus-insect cell expression system, along with the g-secretase substrate APP-C99. The baculovirus-insect cell expression system has been used for the expression of large quantities of recombinant proteins, including human APP.34,35 Moreover, human APP in insect cells is cleaved by an a-secretase-like activity to liberate secreted sAPP35,36 and to generate APP carboxyl-terminal fragments similar or identical to the ‘APP-C99’ and ‘APP-C83’ fragments produced in mammalian cells.18 The ‘APP-C99-like’ fragments generated in these cells have been shown to contain amyloidogenic epi- topes18,37 and the APP-C83-like fragment generated by processing of human APP has an amino terminus identical to that of its mammalian counterpart.17 Soluble APP released from infected insect cells demonstrates protease inhibitory activity identical to that of mammalian sAPP.34 Collectively, these studies indicate that the baculovirus–insect cell system is a relevant model to investigate some aspects of APP biology and its processing by secretases. A 4 kDa species, with a migration pattern identical to that of synthetic Ab, is generated by insect cells

Figure 4 The baculovirus Ab species migrates to a similar position as Ab40 on bicine/bis/tris/urea SDS-PAGE. Media were immunoprecipitated from cells infected with APP-C99 (a, lane 1) or PS1D9 (a, lane 3) separately or in combination (a, lane 2). Immunoprecipitates underwent bicine/bis/tris/ urea SDS-PAGE and immunoblotting with antibodies (b) WO2, (c) 6E10, (d) 21F12 or (e) C1/1.6. The Ab species was immunoreactive with antibodies WO2 (b, lanes 1, 2) and, to a lesser extent, 6E10 (c, lanes 1, 2) and nonreactive with the non-Ab antibody C1/1.6 (e, lanes 1, 2). The species migrated to a similar position to synthetic Ab40 peptide (b, c, lanes 4, 5, 6, open rectangle), and was not immunoreactive with the Ab42-specific antibody 21F12. Collectively, the data con- firm that the Ab species was not Ab42. An B10 kDa species was strongly immunoreactive with antibody WO2 (b, lanes 1, 2) and, to a lesser extent, antibody 6E10 (c, lanes 1, 2) and nonreactive with antibodies 21F12 and C1/1.6 (d, e, lanes 1, 2). This species was only present in media from cells expressing APP-C99 and PS1 or APP-C99 alone (b, c, lanes 1, 2), suggesting the species is a secreted cleavage product of APP-C99.

Molecular Psychiatry PS1DE9 and c-secretase activity in Sf9 cells G Verdile et al 600 that overexpress APP-C99. In the previous study of Compared with mammalian cells, all nicastrin Ab generation in insect cells, an intracellular Ab derived from baculovirus-infected Sf9 cells was more species was generated from APP-C99 only after sensitive to EndoH and PNGase treatment. This aggregation and digestion by proteinase K, suggesting indicates that, in insect cells, nicastrin exists, exclu-

that nonspecific cleavage of APP-C99 was responsible sively, as a high-mannose immature form (Man9- 19 for generating the Ab-like species in that case. The 3GlcNAc2), and there is no addition of oligosacchar- current study is the first to report the release of an Ab- ides such as GlcNAc, galactose or sialic acid to form a like species by Sf9 cells; therefore, in addition to the mature protein. The mature nicastrin protein has been evidence for endogenous a-secretase and b-secretase shown to preferentially bind PS1.27,28,30 Furthermore, activity in Sf9 cells,17,18 the current study provides a recent report has shown that only nicastrin contain- evidence for a possible endogenous g-secretase activ- ing N-linked complex oligosaccharides is present in ity in insect cells, although nonspecific cleavage due active g-secretase complexes.29 Thus, it is conceivable to a high-level overexpression cannot be completely that the suppression of Ab production in Sf9 cells excluded. The existence of an endogenous g-secretase expressing nicastrin may be related to its inability to activity is not entirely unexpected, since there exists a undergo full maturation in Sf9 cells. Drosophila homolog of PS1.38 In summary, PS1, APP-C99 and nicastrin were Since it was unknown whether Sf9 cells would successfully expressed in Sf9 insect cells infected contain the ‘presenilinase’ enzyme(s) required to with recombinant baculoviruses. Under some condi- cleave PS1 into the appropriate N- and C-terminal tions, we could recover an Ab-like species from the fragments required for g-secretase activity, the con- media of cells infected with APP-C99, and the levels stitutively active PS1DE9 mutation was used to of this species were enhanced by the addition of FAD

circumvent the presenilinase processing reaction. mutant PS1 lacking exon 9. No Ab42 could be detected The PS1DE9 molecule is catalytically active without by immunoblotting with an Ab42-specific antibody or requiring cleavage into the N- and C-terminal frag- by using the bicine/bis/tris SDS-PAGE system that 39 ments. separates Ab40 and Ab42 and allows visualization of Like other FAD-linked mutations, the deletion both Ab species on the same gel. However, the of exon 9 in PS1 typically results in increased peptides shared some electrophoresis and immuno- 39 Ab42 levels in transfected cell lines and in brain chemical properties with authentic human Ab40. tissue from FAD patients harboring the exon 9 Collectively, these data suggest that the PS1DE9 deletion.40 Thus, it might be expected that the mutation per se or together with immature nicastrin PS1DE9 mutation would be distinguished by genera- is not sufficient to generate a full complement of Ab

tion of Ab42 from the infected Sf9 cells. However, no species from APP-C99 and that other partners (eg pen- 46 Ab42 could be identified in the supernatant of infected 2, aph-1 ) may be required to generate this longer cells. The immunochemical and electrophoretic form of Ab in reconstituted systems. Most notably, the

properties indicated that Ab40-like peptides were apparent bioactivity of the recombinant PS1DE9 generated. provides support for its use as starting material in Previous studies have used Sf9 cells to co-express an effort to reconstitute functional g-secretase from its wild-type PS1 and full-length APP41,42 and have failed purified component parts. Efforts in using baculoviral to observe Ab production. The differences observed derived human C99 and PS1D9 to reconstitute g- between these studies and the current study may be secretase activity are currently underway. Current due to technical differences or the use of wild-type data indicate that another proteolytic product of APP, rather than mutant PS1. The studies by Octave et al41 the APP intracellular domain (AICD), is generated and Pitsi et al42 showed data for only one MOI point from PS1D9 cleavage of C99 (Carter et al, unpublished (MOI 10), which may not be optimal for active protein results). Furthermore, mass spectrometry of the AICD production. In our study, a range of MOI was used to has revealed the presence of Ab fragments (Carter et determine optimal protein expression. In addition, al, unpublished results), suggesting that Ab can be several N-terminal fragments were seen with the generated from insect cells expressing human PS1D9 expression of wild-type PS1,41,42 which indicates that and C99. Reconstituting the active catalytic domain of wild-type PS1 in Sf9 cells may undergo nonspecific g-secretase and determining the role of each of its cleavage into nonactive fragments. To circumvent this components would be important considerations in problem, we used the PS1D9 mutation, which is not therapeutic strategies directed at reducing the toxicity processed but still active.39 induced by increased Ab levels in Alzheimer’s Several reports have provided strong evidence that disease. nicastrin is a component of the PS1 complex.27,28,43 Recent studies indicate that nicastrin is required for Acknowledgements the stabilization of PS1/g-secretase activity44,45 and is essential for g-secretase activity. However, the addi- This research was supported by grants from the tion of nicastrin to the baculovirus system at MOIs of McCusker Foundation for Alzheimer’s Disease 5 and 10 resulted in suppression of levels of the Ab- Research, Department of Veterans Affairs, National like species associated with expression of APP-C99 Health and Medical Research Council, Alzheimer’s alone or in combination with PS1DE9. Association Australia and Hollywood Private

Molecular Psychiatry PS1DE9 and c-secretase activity in Sf9 cells G Verdile et al 601 Hospital (RNM). Support was also provided by the 16 Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C. Alzheimer Society of Ontario, Medical Research Reconstitution of gamma-secretase activity. Nat Cell Biol 2003; 5: Council of Canada, Ontario Mental Health Founda- 486–488. 17 Ramabhadran TV, Gandy SE, Ghiso J, Czernik AJ, Ferris D, tion and Scottish Rite Charitable Foundation (PEF, Bhasin R et al. Proteolytic processing of human amyloid PStGH). Further support was provided by the Na- beta protein precursor in insect cells. Major carboxyl-terminal tional Health & Medical Research Council of Australia fragment is identical to its human counterpart. J Biol Chem 1993; (Unit Grant 983302, Block Grant 993050 and Fellow- 268: 2009–2012. ship 157209) and Australian Department of Veterans 18 Gandy SE, Bhasin R, Ramabhadran TV, Koo EH, Price DL, Goldgaber D et al. 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