Technical Reference #294

294.

Presenilin-1 Affects Trafficking and Processing of BetaAPP and is Targeted in a Complex with Nicastrin to the Plasma Membrane Christoph Kaether; Sven Lammich; Dieter Edbauer; Michaela Ertl; Jens Rietdorf; Anja Capell; Harald Steiner; Christian Haass, Ludwig-Maximilians University, The Journal of Biological Chemistry, 158(294), (2002)
Link To Paper

Abstract:
Amyloid B-peptide (AB) is generated by the consecutive cleavages of B- and B-secretase. The intramembraneous r-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases w

Keywords:
Alzheimer's disease; presenilin; GFP; nicastrin; amyloid precursor protein

Materials & Methods:
(Capell et al. 2000b). A APP and APP CTFs were detected using antibodies 3926/6E10 (Sastre et al. 2001) 5313 and 6687 (Steiner et al. 2000) respectively. The PS1 CTF was immunoprecipitated with antiserum 3027 and detected with monoclonal BI.3D7 or 3027 as previously described (Steiner et al. 1999b). The PS1 NTF was detected using the monoclonal PS1 N antibody (provided by R. Nixon Nathan Kline Institute Orangeburg NY). The PS2 CTF was immunoprecipitated with antiserum 3711 and detected with monoclonal BI.HF5c as previously described (Capell et al. 1998). EEA1 was detected using the monoclonal EEA1 antibody (Sigma-Aldrich). Notch E (Schroeter et al. 1998) and NICD were detected using monoclonal 9E10 antibody (Santa Cruz Biotechnology Inc.). Nct was detected using polyclonal anti-Nct COOHterminal antibody (Sigma-Aldrich). GSK-3 was detected using monoclonal antibody against GSK-3 (Santa Cruz Biotechnology Inc.). HEK293 cells stably expressing Swedish mutant APP (Citron et al. 1992) and PS1 wt PS1 D385N PS2 wt or PS2 D266A were described before (Steiner et al. 1999ac). HEK293 cells stably expressing Swedish mutant APP and PS1 wt or PS1 D385N were transfected with BACE cDNA in pcDNA3.1/Hygro (Invitrogen) and pools of stably expressing cells were selected. cDNA constructs transfections and screening of stably transfected cell lines To generate an EGFP-tagged PS a NotI restriction site was introduced between codon 351 and 352 of the cytoplasmic loop of human PS1 resulting in PS1not. EGFP cDNA was then fused in-frame into the PS sequence using the NotI site. Introduction of the NotI site results in two additional glycine codons at NH2 and COOH termini of EGFP respectively. Proper orientation of EGFP was checked by transfecting miniprep DNA into COS7 cells and analyzing GFP fluorescence. One clone PE was subcloned in pCDNA3.1Zeo (Invitrogen). PS1 D385N–EGFP and PS1 L166P–EGFP were generated by mutagenizing PE with appropriate oligonucleotides using QuikChange Site-Directed Mutagenesis Kit (Stratagene). The former construct was called PEASP the latter PE (L166P). For transient or stable transfection Fugene (Roche) was used. HEK293 cells stably expressing Swedish APP were transfected with PE or PEASP or PE (L166P) and clones were selected for stable integration. Several clones expressing each construct were analyzed and one representative clone was chosen for further analysis. PE17 was the clone used for analysis of PE and PEASP1 was the one used for PEASP. For analysis of Notch processing PE 17 cells were transfected with Notch E/pcDNA3.1/Hygro and a pool of stably expressing cells was obtained by selection with hygromycin. Alternatively PE 17 cells were transfected and processed for immunofluorescence the following day. As a control for microscopy HEK293 cells were transiently transfected with pEF/myc/ER/GFP (Invitrogen) coding for a GFP–KDEL. For APP uptake experiments COS7 cells were transiently transfected with APP695. Quantitation of expression levels of exogenous PS For quantitating membrane lysates (Capell et al. 1998) and total lysates of HEK293 cells expressing endogenous PSs or stably expressing PS1 wt or PE were separated on 12% urea gels blotted and probed with antiserum 3027. Expression levels were quantitated using 125I secondary antibodies and a phosphoimager system. Total exogenous PS (holoprotein and CTF) was overexpressed 10-fold compared with endogenous PS1 levels. Only a twofold overexpression of exogenous PS CTF was observed. Because the biologically active PS complex contains the PS fragments (Capell et al. 1998; Li et al. 2000a) this demonstrates a rather low level of overexpression. Surface biotinylation HEK293 cells grown on poly-L-lysine–coated 10-cm (for PS and Nct detection) or 6-cm dishes (for APP detection) were washed in ice cold PCM (PBS supplemented with 1 mM CaCl2 0.5 mM MgCl) and incubated for 30 min on ice in PCM containing 1 mg/ml (for PS and Nct) or 0.5 mg/ml (for APP) sulfo-succinimidyl-6-([ ]-biotinamido)-hexanoate (Molecular Biosciences). Thereafter biotinylation was quenched by washing two times in 50 mM NH4Cl–PBS on ice followed by a 10-min incubation on ice in 50 mM NH4Cl–PBS. In some experiments 20 mM glycine–PBS was used for quenching. After two additional washes cells were lysed in STEN lysis buffer (50 mM Tris pH 7.6 150 mM NaCl 2 mM EDTA 1% NP-40) and biotinylated proteins were precipitated with streptavidin-Sepharose. Biotinylated proteins and 1/60 of total cell lysates were separated on 12% SDSurea gels (PS) or 8% SDS gels (APP and Nct) and blotted onto PVDF membranes. PS1 was detected using antibodies 3027 or PS1 N Nct using anti-Nct antibody and APP using antiserum 5313. As a control blots were stripped and reprobed with EEA1 or GSK-3 antibody. Similar blots to those shown in Fig. 5 were incubated with 125I secondary antibodies and the radioactive signal was detected using a phosphoimager system. The ratio of biotinylated versus total PS was then calculated. Quantitation of cell surface APP For quantitation of surface APP cells were biotinylated as described above. Biotinylated APP was detected using antiserum 5313 and 125I secondary antibody and quantified by phosphoimaging. In each experiment the values of biotinylated APP divided by total APP of PS1 wt–expressing cells were set to 1 and the values of biotinylated APP divided by total APP of PS1 D385N–expressing cells was related to 1.

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
HEK-293