Báo cáo khóa học: Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations pptx

Eur J Biochem 271, 375–385 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03936.x Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations Janetta G Culvenor1,2,3, Nancy T Ilaya1,2,3, Michael T Ryan4, Louise Canterford1,3, David E Hoke1,3, ` Nicholas A Williamson1,3, Catriona A McLean5, Colin L Masters1,3 and Genevieve Evin1,3 Department of Pathology and 2Centre for Neuroscience, The University of Melbourne, Australia; 3Mental Health Research Institute of Victoria, Australia; 4Department of Biochemistry, La Trobe University, Bundoora, Australia; 5National Neuroscience Facility, University of Melbourne, Australia The presenilin proteins are required for intramembrane cleavage of a subset of type membrane proteins including the Alzheimer’s disease amyloid precursor protein Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N- and C-terminal fragments in association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1 proteins Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin complex mass, stability and association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1 Solubilization of mouse or human brain membranes with dodecyl-D-maltoside produced a 360-kDa species reactive with antibodies to presenilin Presenilin complex levels were high in embryonic brain Complex integrity was sensitive to Triton X-100 and SDS, but stable to reducing agent Addition of M urea caused complex dissolution and nicastrin to migrate as a subcomplex Nicastrin and presenilin enhancer-2 were detected in the presenilin complex following BN/PAGE, electroelution and second-dimension analysis Anterior pharynx defective-1 was detected as an 18-kDa form and 9-kDa C-terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36-kDa band after presenilin complex second-dimension analysis Membranes from brain cortex of Alzheimer’s disease patients, or from cases with presenilin missense mutations, indicated no change in presenilin complex mobility Higher molecular mass presenilin 1-reactive species were detected in brain containing presenilin exon deletion mutation This abnormality was confirmed using cells transfected with the same presenilin deletion mutation The presenilins are multispanning membrane proteins (presenilin 1, PS1 and presenilin 2, PS2) first identified for their genetic association with Alzheimer’s disease (AD) They are essential components of a multiprotein protease complex implicated in regulated intramembrane proteolysis of several type membrane proteins including the amyloid precursor protein (APP) and developmentally important Notch receptors [1] The variety of identified substrates indicates a critical role for PS in cell metabolism involving controlled cleavage of protein transmembrane domains and signal transduction The role of PS in c-secretase activity is compelling and includes lack of Ab amyloid peptide generation and Notch signalling in PS double knockout cells, cofractionation of activity with PS, abolition of activity with mutation of conserved aspartates in transmembrane domains six and seven, binding of c-secretase aspartyl protease inhibitors to PS, as well as identification of homologues with protease-associated domains [2–6] Following a primary cleavage event that causes ectodomain shedding, PS-dependent Ôc-secretaseÕ proteolysis results in generation of a membrane spanning stub (such as Ab peptide) and a cytoplasmic fragment such as the APP intracellular domain or the Notch intracellular domain which translocates to the nucleus for regulation of gene expression [1] The PS proteins undergo endoproteolysis between transmembrane domains six and seven to generate heterodimers of N-terminal fragments (NTF) and C-terminal fragments (CTF) [7] Many binding partners for the PS proteins have been identified including substrates and the catenins (b, d and p0071) [8] The interacting components that are required for c-secretase activity besides PS include the Correspondence to J G Culvenor, Department of Pathology, The University of Melbourne, Parkville, Victoria, 3010 Australia Fax: +61 38344 4004, Tel.: +61 38344 3990, E-mail: janetta@unimelb.edu.au Abbreviations: AD, Alzheimer’s disease; APH, anterior pharynx defective; APP, amyloid precursor protein; BACE, b-site APP-cleaving enzyme; BN/PAGE, Blue Native polyacrylamide gel electrophoresis; CTF, carboxyl-terminal fragment; DDM, n-dodecyl b-D-maltoside; ECL, enhanced chemiluminescence; FAD, familial Alzheimer’s disease; FTD, frontotemporal dementia; NCT, nicastrin; NTF, amino-terminal fragment; PEN, presenilin enhancer; PS1, presenilin 1; PS2, presenilin (Received October 2003, revised November 2003, accepted 20 November 2003) Keywords: Alzheimer’s disease; brain; Blue Native PAGE; presenilin complex Ó FEBS 2003 376 J G Culvenor et al (Eur J Biochem 271) glycoprotein, nicastrin (NCT) identified by affinity purification of PS1 complex using digitonin-solubilization of membranes from human embryonic kidney cells [9], and two further membrane proteins PEN-2 and APH-1, identified by genetic screening in Caenorhabditis elegans [10,11] NCT is a heavily glycosylated type membrane protein Maturation of NCT glycosylation is impaired in PS1deficient cells, and down-regulation of NCT inhibits c-secretase cleavage of APP and Notch [11–13] PEN-2 is a small double membrane-spanning protein with N- and C-termini facing the lumen [14] APH-1 contains seven putative transmembrane domains and has a variety of isoforms with differing C-terminal domains; in humans, APH-1a long and short forms are encoded by a gene on chromosome and APH-1b by chromosome 15 [15,16] Glycerol velocity gradient centrifugation and gel filtration of detergent-solubilized membrane extracts have characterized PS complexes of sizes ranging between 150 kDa and two million kDa [5,17,18] There is limited knowledge about the assembly and role of the PS complex components other than PS We have applied Blue Native gel electrophoresis (BN/PAGE) [19] to analyse further and characterize endogenous native complexes from mouse and human brain tissue and from the neuroblastoma line SH-SY5Y (SY5Y) Experimental procedures Antibodies Synthetic peptides derived from human sequences were conjugated to diphtheria toxoid and rabbit antibodies generated as follows: Ab 98/1, PS1 NT (1–20) [20]; Ab 00/ and Ab 00/2 PS1 loop (301–317) [21]; Ab 00/12, PS2 loop (307–336); Ab 00/19 NCT CT (691–709]) [22]; Ab 00/22 NCT ectodomain (331–346); Ab 02/45 PEN-2 NT (1–15) and Ab 02/41 APH-1b CT (244–257) Ab 00/6 was raised to human BACE CT (485–501) [23] NCT, PEN-2 and APH-1 antibodies were affinity-purified using immunizing peptide coupled to SulfoLink Coupling Gel (Pierce) Mouse mAb C1/6.1 raised to APP (676–695) was a gift from P Mathews, Nathan Kline Institute for Psychiatric Research, NY, USA [24] Cell lines Human neuroblastoma SY5Y wild-type cells were cultured as described previously and stably transfected with cDNA for PS1, PS1 with exon deletion mutation, or PS1 with D257A artificial mutation following published protocols [20] Mouse embryonic neural stem cell lines NS51 (PS1–/–) and NS52 (PS1 wt) were derived and cultured from PS1deficient mice as described [25] Membrane preparations Cells were washed in NaCl/Pi and pellets stored at )70 °C Mouse brain tissue was obtained from C57/Bl6 mice, APPC100 transgenic mice (over-expressing last 99 residues of human APP) [31], APPsw transgenic mice Tg 2576 (overexpressing APP with Swedish mutation) [32], or transgenic mice homozygous for human PS1M146 L [33] Samples were homogenized in 250 mM sucrose, 20 mM Hepes pH 7.4 with protease inhibitors (Sigma P-2714) at °C with a glass Dounce homogenizer Centrifugation at 1000 g for 10 min, °C, yielded a post-nuclear supernatant that was further centrifuged at 100 000 g for h, °C, and the resultant membrane pellets were resuspended in sucrose homogenization buffer with protease inhibitors and stored at )70 °C Protein concentration was determined with bicinchoninic acid reagent (Pierce) BN/PAGE Membrane protein (30–100 lg protein per gel lane) was resuspended in 0.2–0.5% n-dodecyl b-D-maltoside (DDM) (or alternate treatment as indicated) in buffer composed of 50 mM NaCl, mM 6-aminohexanoic acid and 50 mM imidazole pH 7.0 and then clarified after 15 at room temperature by microfuge centrifugation for before addition of 0.5% Coomassie G250 (Sigma) and 50 mM 6-aminohexanoic acid Samples were resolved on 4–16.5% gradient acrylamide gels with cooling; anode buffer contained 25 mM imidazole pH 7.0; cathode buffer contained 50 mM tricine, 7.5 mM imidazole pH 7.0 with 0.02% Coomassie G 250 [34] High molecular mass standards for native electrophoresis were from Amersham and were stained with Coomassie R250 Following electrophoretic transfer to poly(vinylidene difluoride) membrane (Immobilon-P, Millipore), proteins were identified by immunoblotting and detection with enhanced chemiluminescence (ECL; Amersham) For second-dimension analysis, gel bands corresponding to the PS complex region derived from 2-cm wide lanes, 16-cm length, 1.5-mm thickness slab gels were excised at cm below the ferritin marker as indicated, proteins were electroeluted with BN/PAGE buffers, and samples were solubilized with 1.5% SDS, 2.3 M urea, 0.1 M dithiothreitol, 15 mM Tris/HCl pH 6.8 with heating at 50 °C for 15 before electrophoresis on 10% Tris/tricine gels Immunoblotting For analysis of PS1, NCT, PEN-2 and APH-1, membrane proteins were separated with reducing agent on Tris/glycine/ SDS or Tris/tricine/SDS polyacrylamide gels and analysed by immunoblotting as described previously [20,35] Brain bank tissue Samples of frontal cortex from human brain were obtained from the NHMRC Tissue Resource Centre (Melbourne, Australia) and were well characterized pathologically and clinically [23] Details of pathology or mutation analysis of tissue used in this study is published as follows: FTD1 and FTD2 [26], FAD PS1 L271V [27], FAD PS1 exon deletion [28], FAD PS1 S169L [29] and FAD PS1 L219P [30] Results PS1 is detected in a 360 kDa complex with nicastrin in embryonic mouse brain and SY5Y cells Preliminary investigation of optimal conditions for PS1 complex analysis from mouse brain by BN/PAGE indicated that solubilization of membranes with 0.5% DDM Ó FEBS 2003 Analysis of mouse and human brain presenilin complex (Eur J Biochem 271) 377 Fig BN/PAGE of PS1 complex reveals a 360-kDa species reactive with antibodies to PS1 and NCT (A) Protein (100 lg) from membranes of embryonic day 14 (E14) mouse brain (E14MB), or from adult mouse brain with PS1 [M146L] (AdMB), were treated with DDM or digitonin before BN/PAGE and Western blotting for PS1 or NCT detection Sharp bands of PS1 reactivity indicated that the 360-kDa PS1 complex contained both PS1 N-terminal reactivity and PS1 C-terminal reactivity in DDM A broader band of 460 kDa PS1 immunoreactivity was found after digitonin solubilization Reactivity for PS1 and NCT was stronger in E14 mouse brain than for adult mouse brain NCT was detected in a 360-kDa complex with DDM and low levels were detected as apparent monomer of  125 kDa; NCT was detected only weakly in adult brain (B) Reactivity of NCT C-terminal antibody after SDS/PAGE for PS1-deficient mouse neural stem cells, PS1 wild-type neural cells, E16 mouse brain and adult mouse brain showed three major bands of 110, 120 and 140 kDa which were absent after preabsorption with immunizing peptide Right panel shows PS1 antibody detection of PS1 29-kDa NTF after SDS/PAGE (20 lg membrane protein per lane) generated a complex of mobility  360 kDa, which migrated ahead of the ferritin marker (440 kDa) as detected by Western analysis (Fig 1A) The complex was reactive with both PS1 N-terminal (98/1) and loop antibodies (00/1) Digitonin was also compatible with maintaining the complex stability and yielded a complex of higher apparent molecular weight of about 460 kDa (Fig 1A) Chapso solubilization was also tested as it has proved useful for maintaining c-secretase activity [5,36] but did not produce a distinct band of PS1 complex immunoreactivity under these electrophoresis conditions (data not shown) Embryonic mouse brain produced stronger PS1 complex immunoreactivity (Fig 1A) compared to adult tissue Results for brain tissue from adult PS1 [M146L] transgenic mice is shown but was the same for mouse wild-type brain NCT was readily detected in association with PS1 complex in embryonic tissue as indicated by reactivity of NCT ectodomain antibody NCT was barely detectable in the PS complex from adult mouse brain as shown in the last lane of Fig 1A PS1 and NCT were also strongly detected as an apparent complex in membranes from the human neuroblastoma cell line SY5Y using PS1 Ab 98/1 and C-terminal NCT Ab 00/ 19 (Fig 2A) We investigated further these apparent changes in PS1 and NCT detection with developmental stages NCT levels in neural stem cell lines and mouse brain are compared in Fig 1B after analysis using 12% SDS/PAGE and immunodetection with NCT CT Ab 00/19 (left panel) Antibody reactivity was removed by preabsorption with immunizing peptide (middle panel) Consistent with previous reports [12,37–39] we found that cells derived from PS1 knockout mice express high levels of less mature NCT ( 110 kDa) Embryonic mouse brain contained a more intermediate glycosylated form of NCT ( 120 kDa) than adult brain where the predominant NCT species was a 140-kDa form NCT ectodomain Ab 00/22 reactivity indicated detection of only the less mature or fully deglycosylated forms of NCT (data not shown), this finding may explain the poor detection of NCT in adult mouse brain complex as high glycosylation may impair antibody access to the epitope As expected, the PS1 N-terminal antibody detected a 29-kDa immunoreactive band from these tissues corresponding to PS1 NTF which was not detectable in PS1-deficient cells Amyloid precursor protein, b-catenin, and b-secretase are not tightly associated with the PS1 complex To investigate possible association of the c-secretase substrate APP or APP C-terminal fragments with the complex, BN/PAGE blots of membranes from SY5Y, APPC100 transgenic mouse brain and APP over-expressing transgenic mouse brain (Tg2576) were probed with APP mAb C1/6.1 to the APP C terminus Fig 2A shows that APP was detected as a smear of reactivity that spanned the PS1 complex mobility Some increase in APP reactivity corresponding to PS1 complex mobility was observed for APP Tg2576 tissue when contrast of the blot was modified, indicated as Tg2576* on Fig 2A Antibody for the PS1 binding partner, b-catenin, produced a smear after BN/ PAGE of molecular mass greater than the 440 marker consistent with interaction with many alternate binding proteins Fig 2B compares immunoreactivity for b-site APPcleaving enzyme (BACE), PS2 and PS1 in wild-type mouse brain membrane extracts after BN/PAGE Immunoreactivity in a broad band of molecular mass greater than 550 kDa did not suggest association of BACE with the PS1 complex PS2 immunoreactivity indicated that the PS2 complex has a slightly higher complex mass of  420 kDa compared with  360 kDa for PS1 Complex stability to detergents and denaturing agents Samples of adult mouse brain membranes were solubilized with alternative detergents and subjected to BN/PAGE to 378 J G Culvenor et al (Eur J Biochem 271) Ó FEBS 2003 Fig PS complex does not contain b-catenin or BACE (A) Membrane protein samples (50 lg) from SY5Y, C100 adult brain, or Tg2576 adult brain in 0.4% DDM were resolved by BN/PAGE and immunoblotted with the indicated antibody PS1 detection was enriched in SY5Y as a 360kDa band APP was detected as a broad band above 140 kDa; contrast adjustment for Tg2576* indicated increased APP immunoreactivity with the PS1 complex region NCT was strongly detected in SY5Y membrane complex and weakly in adult mouse C100 brain complex b-catenin was detected as a broad smear above 440 kDa as indicated (B) Membrane protein (50 lg) from adult mouse brain were compared for immunoreactivity with antibody as indicated BACE was detected as a broad smear of apparent molecular mass greater than 440 kDa PS2 was detected as a band of  420 kDa determine PS1 complex stability Fig 3A shows that sample preparation with 1% SDS, or a combination of 1% Triton X-100/1% Nonidet P-40 abolished the 360-kDa PS1 complex signal that was observed with 0.5% DDM solubilization Treatment with 1% Triton X-100 caused incomplete complex dissociation as a weak 360-kDa signal was observed together with smearing in the 100–200-kDa range after long exposure (Fig 3A; ECL 60 compared with ECL min) Thus, we confirm that detergent type is critical for solubilization of the complex without disruption, as shown by others [5,17] The complex was stable in 0.5% DDM even after addition of reducing agent (1% b-mercaptoethanol), indicating strong interaction, independent of maintenance of disulfide bonds Stability to M urea further supports this strong interaction However, the complex breaks down with increased urea concentration of M in presence of reducing agent and 0.2% DDM (Fig 3B, left panel) At the low SDS concentration of 0.1% with 0.2% DDM, PS1 subcomplexes of 90 and 60 kDa were detected (Fig 3B) We also investigated the association of NCT with PS complex using embryonic mouse brain where we have shown optimal NCT detection Paralleling the results observed for PS1, we found that the low SDS concentration of 0.1% caused a loss of 360-kDa immunoreactivity for NCT, using either ectodomain or C-terminal directed antibodies (Fig 3B, right panels) A 140-kDa signal was observed that is consistent with the apparent molecular mass of NCT The intermediate complex of 280 kDa has a size consistent with that expected for a NCT dimer, and was sensitive to the ionic detergent SDS but stable to b-mercaptoethanol and M urea (Fig 3B) It was hardly detectable with antibody 00/22, suggesting that the molecular interaction prevented antibody access to its epitope that is located within the median region of the ectodomain Detection with the C-terminal antibody 00/19 was robust, excluding interaction through the C-terminal domain This intermediate NCT complex was similar to the subcomplex described previously on BN/PAGE for transfected cells solubilized with digitonin [40,41] Second dimension denaturing electrophoretic analysis of semipurified PS complex Prior to investigation of PEN-2 and APH-1 association with the PS complex, mouse- and human-derived membrane preparations were examined for PEN-2 immunoreactivity and revealed strong detection of a 10-kDa band in PS1+/ +neural stem cells, embryonic brain, and SY5Y cells; lower levels were detected in PS1–/– cells, and in adult brain from mouse and human (Fig 4A) Down-regulation of PEN-2 with PS1-deficiency is in agreement with other reports [42] APH-1 analysis using APH-1b CT [244–257] antibody in the same samples detected APH-1b immunoreactivity at 18 kDa and kDa (Fig 4B) Levels of APH-1b were similar for PS1-deficient and wild-type neural stem cells indicating that levels are not coordinated with PS1 expression Detection was low in adult human brain as compared with SY5Y cells Detection of the APH-1b products was reduced by peptide absorption (data not shown) APH-1a expression was also investigated but was detected more weakly with the reagents currently available To analyse further the composition of the PS1 complex, samples with high expression of PS1 complex were selected for further study BN/PAGE gel bands corresponding to the region of PS1 complex were excised as indicated in Fig 4C Fig 4C shows that treatment of membranes with 0.1 M sodium carbonate, pH 11.3, for removal of nonintegral membrane proteins, enriched for PS1 immunoreactivity This treatment was previously shown to be compatible with Ó FEBS 2003 Analysis of mouse and human brain presenilin complex (Eur J Biochem 271) 379 Fig Stability of PS1 complex to detergents and urea analysed by BN/PAGE (A) Adult mouse brain samples (50 lg) were treated with 0.5% DDM or as indicated before loading Triton X-100 1%/1% NP40, or 1% SDS eliminated the PS1 complex at 360 kDa ECL exposure of h revealed low detection of PS1 complex treated with 1% Triton compared with 0.5% DDM Addition of M urea or 1% b-mercaptoethanol to 0.5% DDM did not affect complex detection by PS1 antibody (B) Treatment of 50 lg E14 mouse brain with 0.2% DDM and 0.1% SDS or M urea indicated disruption of PS1 complex With 0.1% SDS, PS1 was detected as bands of 90 and 60 kDa With 0.1% SDS, NCT was detected largely as monomer with minimal reactivity corresponding to possible dimer or subcomplex After treatment with 1% b-mercaptoethanol/5 M urea, NCT was detected primarily at 280 kDa by NCT CT antibody and only weakly as a monomer with NCT ectodomain antibody 380 J G Culvenor et al (Eur J Biochem 271) preservation of c-secretase activity [36] Samples were processed by second-dimension analysis to investigate composition and presence of complex components Ó FEBS 2003 Second-dimensional analysis of complex from carbonatewashed membranes of SY5Y and 5-day-old mouse brain were examined using 10% Tris/tricine gels with reducing Ó FEBS 2003 Analysis of mouse and human brain presenilin complex (Eur J Biochem 271) 381 Fig PEN-2 and APH-1 expression in mouse and human tissues and detection in PS complex after 2D-complex analyis (A) Membranes from tissues and cells were analysed for PEN-2 (1–15) reactivity Significant expression was detected as a 10-kDa band in PS1 wild-type neural stem cells, E15 mouse brain and SY5Y cells Low-level expression was present in PS1 deficient cells, adult mouse brain and in control and AD human brain cortex (20 lg protein per lane, 15% Tristricine gel) (B) Tissues were also examined for APH-1b CT reactivity APH-1b was detected at  18 kDa and as an apparent CTF of kDa except in adult human brain Expression was unchanged with PS1 deficiency (C) Sodium carbonate wash of membranes before BN/ PAGE analysis increased intensity of PS1 complex reactivity (50 lg membrane protein per lane in 0.4% DDM) A region of the unstained BN/polyacrylamide gel was excised as indicated by alignment with standards (D) Carbonate washed SY5Y membranes (500 lg) were run on BN/PAGE, proteins electroeluted from the PS complex region, treated with reducing sample buffer and analysed on 10% Tris/tricine gels using mini Biorad apparatus PS1 NTF and full-length PS1 were detected with PS1 [1–20] antibody, PS1 loop antibody detected PS1 CTF, NCT was detected at  140 kDa, PEN-2 at 10 kDa and PS2 CTF detected with PS2 CTF antibody Parallel poly(vinylidene difluoride) strips were also probed with APH 1a and 1b antibodies but APH-1 was not detected (E) Carbonate washed membranes (500 lg) from mouse brain post-natal day 5, were similarly probed after PS complex isolation from BN/PAGE and second-dimension Tris/tricine gel analysis using a large gel (16 · 18 cm) format PS1 and PEN-2 were easily detected in these samples NCT was clearly detected as the mature form with very low detection of an immature form Again APH-1 was not detected with the available reagents (F) E16 membranes were analysed in the absence of carbonate treatment Second-dimension analysis after BN/PAGE of the PS complex detected full-length PS1, NTF, CTF and PEN-2 NCT was detected as the mature form APH-1b was detected primarily as a major band of 36 kDa (possible dimer) and as minor bands of 18 kDa and possible trimer at 54 kDa (after long exposure); immunoreactivity with mobility corresponding to NCT was also indicated conditions and SDS (Fig 4D and E) PS1 NTF and CTF and PS2 CTF were detected robustly in these samples Low levels of full-length PS1 were also detected with N-terminal antibody NCT and PEN-2 were also detected in this semipurified complex APH-1 was not detectable with the reagents tested which included affinity-purified antibodies to the C-terminal regions of APH-1a (short form) and APH1b, and to the loop four region of APH-1b (data not shown) Investigation of noncarbonate washed embryonic mouse brain PS complex revealed similar detection of PS1 NTF, PS1 CTF, low level of full-length PS1, mature NCT, and PEN-2 (Fig 4F) PS1 CTF was detected with PS1 loop antibody as well as low amounts of full-length PS1 The epitope for this antibody is largely buried in full-length PS1 as shown previously by us and others [26,43] APH-1b was detected for this complex as immunoreactivity at about 36 kDa with low level detection also at about 18 kDa and 54 kDa suggestive of self-association as dimer and trimer; mobility corresponding to putative association with mature NCT (140 kDa) was also detected Evidence for selfassociation of APH-1a with itself and with APH-1b was also reported previously [16] Morais et al demonstrated that NCT and APH-1 were able to interact in vitro in the absence of PS [44] Our results suggest that all four putative complex components could be detected in the PS complex for embryonic mouse brain after BN/PAGE, and that APH-1 association may be diminished by carbonate wash Analysis of complex components by second-dimension analysis for control and AD human brain samples without carbonate wash revealed detection of PS1 NTF, PS1 CTF, full-length PS1, NCT and PEN-2 (data not shown) but not APH-1 which may be below detection levels Analysis of PS1 complex from sporadic AD and tissue with early onset familial AD mutations Membrane fractions were prepared from frontal cortex of cases with sporadic AD and compared with age-matched controls BN/PAGE analysis indicated no major differences in PS1 complex mobility with AD (Fig 5A) Human brain cortex had lower detectable levels of PS1 complex compared with preparations from SY5Y No major differences in expression of PS1 and NCT levels were found in these tissues as shown in Fig 5C using Western analysis after standard SDS gel electrophoresis We next examined cases of early onset familial AD with pathogenic PS1 missense mutations and found no alteration in complex mobility by BN/PAGE analysis for tissue containing PS1 [L271V], PS1 [S169L] or PS1 [L219P] (Fig 5B) No change in PS1 complex was found for two fronto-temporal dementia cases associated with altered PS1 transcript expression [26] However tissue from a case with pathogenic PS1 exon deletion showed an additional oligomeric species of about 600 kDa (demonstrated twice) PS1 expression and NCT levels for some of these early onset cases is shown in Fig 5D SY5Y cells stably transfected with PS1 cDNA constructs were used to examine effects of over-expression of PS1 mutations on complex formation Abnormal accumulation of PS1 at about 600 kDa was detected in SY5Y cells overexpressing the PS1 delta construct Increased PS1 expression or expression of the artificial loss of function D257A mutation did not alter complex mobility Interestingly for all cell lines over-expression of PS1 did cause increased complex detection by PS1 antibody (Fig 5E) but significant amounts of PS1 were not incorporated in the complex, as indicated by a smear of reactivity between 50 and 100 kDa Discussion The PS complex is critical for normal biological processes such as Notch signalling in development in addition to the generation of Ab peptides in pathological processes Study of the characteristics of the PS complex will facilitate elaboration of the molecular processes involved in the production of Ab Analysis of these complexes requires detergent solubilization in conditions that not disrupt protein interactions This can lead to loss of weakly interacting proteins or generation of artefact from nonspecific associations BN/PAGE utilizes Coomassie Blue G250 rather than SDS in the cathode buffer and sample buffer to assist with protein solubilization and addition of charge for electrophoretic field migration of proteins according to mass [19] It is ideal for analysis and partial isolation of membrane 382 J G Culvenor et al (Eur J Biochem 271) Ó FEBS 2003 Fig BN/PAGE analysis of PS1 complex in membranes from human brain control and AD brains (A) After BN/PAGE, PS1 complex from control (CT) or AD cortex was detected in 50 lg membrane protein at 360 kDa (B) Analysis of samples from AD cases with PS1 missense mutations and fronto-temporal dementia (FTD) indicated no change in PS1 complex mobility by BN/PAGE In familial AD (FAD) with exon deletion, a higher band of  600 kDa was detected as well as the major band for PS1 at 360 kDa (shown twice for confirmation) (C) Detection of PS1 and NCT after 12% Tris-glycine SDS/PAGE of 20 lg membrane samples of human brain analysed in (A) indicated no major difference in PS1 or NCT expression in AD (D) Analysis of samples used in (B) for PS1 and NCT expression with 12% Tris-glycine SDS/PAGE, indicated additional full-length PS1 detection with exon deletion and no alteration in NCT expression (E) Detection of PS1 in SY5Y stably transfected with PS1, PS1 exon deletion (delta E9), and PS1-D257A following BN/PAGE, indicated increased levels of PS1 complex with PS1 over-expression Increased immunoreactivity was detected at  600 kDa with PS1 delta E9 (30 lg protein per lane from 18 000 g · 20-min post-nuclear pellets) Ó FEBS 2003 Analysis of mouse and human brain presenilin complex (Eur J Biochem 271) 383 protein complexes under nondenaturing conditions This technique complements immunoprecipitation and velocity gradient analyses that have been applied more extensively to examine PS interactions with binding partners and complex size The molecular mass of PS1 complex determined here with BN/PAGE corresponds to the size of  400 kDa recently determined by velocity gradient centrifugation of digitonin-solubilized membranes from HEK293 cells [45] It is also consistent with the size of Chapso-solubilized PS complex obtained after over-expression of Drosophila PS in Drosophila cells or N2a neuroblastoma cells (between 232 and 443 kDa) [46] The detection of some full-length PS1 after second-dimension analysis of complex components suggests assembly with full-length PS before endoproteolysis to generate heterodimers of NTF and CTF components consistent with previous reports [47] Sensitivity of the PS complex to SDS and Triton X-100 and stability to reducing agent is consistent with previous studies [17,48] Conditions causing partial complex disruption can contribute to knowledge of complex assembly With 0.1% SDS, PS1 high molecular mass complex was disrupted, and some PS1 with mobility of about 90 kDa was detected, which may correspond to a subcomplex or ´ PS1 dimer A recent report by Hebert et al showed evidence for PS1 dimerization using 0.1% SDS treatment and alternative native electrophoresis conditions as well as by yeast two-hybrid analysis [49] We also document that NCT migrates in BN/PAGE as a subcomplex or possible dimer stable to reducing agent and high urea concentration This finding supports previous evidence, that NCT and APH-1 may associate independently of PS1 complexes [40,41,44] and that NCT may form a subcomplex in the absence of PS1 [13] Low or no detection of APH-1 in the partially purified endogenous PS complex by second-dimension analysis, and minimal effect on APH-1 expression level with PS1 deficiency supports the proposal that APH-1 may be important for early assembly and stabilization of the complex [50–52] High levels of PS complex were detected in mouse embryonic brain consistent with high expression of PS1 in embryogenesis and supporting an important role for PS in development, presumably due to its involvement in the Notch signalling system [53] Indeed studies with PS1 knockout mouse embryos demonstrated severe neural and skeletal defects and PS1/PS2 double knockouts display a severe phenotype resembling Notch gene knockout [3] Alternative approaches have provided evidence that PS and cofactors interact directly: affinity isolation of the complexes [9], coimmunoprecipitation studies [12,37–39], and complex isolation with c-secretase inhibitor [4,22,54] Other recent studies have also utilized BN/PAGE for analysis of PS complexes DDM at 0.5% has been recently reported independently to preserve PS complex integrity in BN/PAGE with reports of complex size from 440 to 600 kDa [12,13,42,55,56] Digitonin (1%) and BN/ PAGE produced PS1 complex mobility of  250–270 kDa in cells over-expressing the four main constituents of the c-secretase complex [40,41] Estimates of complex mobility may vary depending on sample preparation, detergent type and concentration, as well as buffer and gel composition Detection of APH-1b as an 18-kDa and CTF 9-kDa form indicates that native APH-1 undergoes endoproteolysis Lack of detection of a free 9-kDa form after seconddimension complex analysis indicates that this fragment is unlikely to be associated with the mature complex This is in agreement with the report by Kimberley et al that APH-1a also generated a small CTF which did not associate with other PS complex components after glycerol velocity gradient fractionation for tagged APH-1 in transfected cells [40] This is the first report of native PS1 complex analysis from human brain of late onset sporadic AD cases and from cases with early onset PS1 mutations We found that PS1 complex from cortex of sporadic late-onset AD cases or from pathogenic PS1 point mutations did not have altered apparent mobility in BN/PAGE This was consistent with study of PS1 complex from transfected SY5Y cells reported here for the artificial PS1 Asp257Ala mutation, and for Asp257 or Asp385 mutations in transfected mouse embryonic fibroblasts [56] Additional high molecular weight PS1 species of  600 kDa were detected for brain carrying the severe PS1 exon deletion and for SY5Y cells over-expressing this mutation, indicating impairment of normal complex formation in the presence of the mutation In vitro co-expression studies in transfected cell systems indicates PS1 or PS2 and NCT, APH-1, and PEN-2 are all required for generation of active c-secretase activity [40,50,52,57] The current study of endogenous tissue levels and detection within native endogenous complex for these membrane proteins from mouse and human brain contributes to further understanding of the nature of the native mature presenilin/c-secretase complex complementing in vitro cell-based studies Acknowledgements We thank P M Mathews and R.M.D Holsinger for antibodies, F B Reinhard for SY5Y-PS1 mutant cell lines, and Q.-X Li for transgenic mouse tissue The work was supported by the Australian NHMRC (Grants 114132 and 208978) and ANZ Charitable Trusts D H was supported by an NIH post 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carbonatewashed membranes of SY5Y and 5-day-old mouse brain were examined using 10% Tris/tricine gels with reducing Ó FEBS 2003 Analysis of mouse and human brain presenilin complex. .. assembly and stabilization of the complex [50–52] High levels of PS complex were detected in mouse embryonic brain consistent with high expression of PS1 in embryogenesis and supporting an important... analysis from mouse brain by BN/PAGE indicated that solubilization of membranes with 0.5% DDM Ó FEBS 2003 Analysis of mouse and human brain presenilin complex (Eur J Biochem 271) 377 Fig BN/PAGE of