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A dinucleotide deletion in amyloid precursor protein (APP). mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP ...
Journal of Neurochemistry, 2001, 76, 1308±1314

A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in ef®cient secretion of truncated APP isoforms from neuroblastoma cell cultures Martin Hersberger,* Juan Santiago-Garcia,* Susannah Patarroyo-White,* Jimmy Yan² and Xiao Xu²,1 *Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California, USA ²Gladstone Institute of Neurological Disease, University of California, San Francisco, California, USA

Abstract Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38-kDa N-terminus and a novel carboxyl terminus (APP11). We investigated the subcellular localization and the processing of APP11 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP11 or fulllength APP with the enhanced green ¯uorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP11±eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full-length APP fused to eGFP, the APP11±eGFP fusion

protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts con®rmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP11±eGFP fusion protein was ef®ciently secreted from B103 cells, whereas the secreted form of full-length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP11 that is not membrane associated and is readily secreted from neurons. Keywords: amyloid precursor protein, fusion protein, green ¯uorescent protein, molecular misreading, protein processing, traf®cking. J. Neurochem. (2001) 76, 1308±1314.

Amyloid precursor protein (APP), a type-I integral membrane protein, can be processed into soluble fragments by two different proteolytic cleavage pathways. APP proteolytically cleaved at the a-secretase site is processed into a 100-kDa secreted isoform (a-APP) that acts as a trophic factor in the CNS. Alternatively, APP cleaved at b- and g-secretase sites is processed into a shorter secreted isoform (b-APP) and a 39±43 amino acid Ab peptide that appears to be neurotoxic and plays a central role in Alzheimer's disease (AD). Cell types differ in the predominant APP isoform they produce and in how much of their APP is processed to a nonmembrane-bound form and is secreted or retained within the cell (Tanzi et al. 1987; Neve et al. 1988; Sandbrink et al. 1994; LeBlanc et al. 1996; LeBlanc et al. 1997). Neurons express predominantly APP695, whereas other cell types produce mostly isoforms that contain a Kunitz-type protease

inhibitor domain, such as APP751 and APP770. In culture, brain cells and cell lines secrete only 15±30% of newly synthesized APP (Weidemann et al. 1989; Caporaso et al.

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Received May 25, 2000; revised manuscript received September 28, 2000; accepted September 29, 2000. Address correspondence and reprint requests to Martin Hersberger, Institute for Clinical Chemistry, University Hospital Zurich, Raemistr. 100, CH-8091 Zurich, Switzerland. E-mail: [email protected] 1 Current address of Xiao Xu is Nanogen Inc., 10398 Paci®c Center Court, San Diego, CA 92121, USA. Abbreviations used: a-APPs, a-secretase cleaved and secreted APP; Ab, b-amyloid; AD, Alzheimer's disease; APP, amyloid precursor protein; DMEM, Dulbecco's modi®ed Eagle's medium; ER, endoplasmic reticulum; eGFP, enhanced green ¯uorescent protein; nt, nucleotide(s); PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.

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1994; LeBlanc et al. 1996; Simons et al. 1996; LeBlanc et al. 1997), and most of the intracellular APP appears to be anchored in the endoplasmic reticulum, Golgi complex, or cell membranes. Expressing full-length APP in cultured cells or adding a-APPs (or some of its domains) to the culture medium enhances cell survival and protects cells from a variety of cytotoxic agents (Saitoh et al. 1989; Mattson et al. 1993a; Schubert and Behl 1993; Furukawa et al. 1996; Li et al. 1997; Xu et al. 1999). Furthermore, a-APPs promote neurite extension through a ®ve amino acid motif, RERMS (Fig. 1), located in the middle of the extracellular domain (Ninomiya et al. 1993; Jin et al. 1994). Low-level neuronal overexpression of human APP (hAPP) in transgenic mice or intracerebroventricular infusion of hAPP fragments in nontransgenic rodents protected neurons against ischemia and excitotoxins (Mucke et al. 1994; Roch et al. 1994; Smith-Swintosky et al. 1994; Mucke et al. 1995; Masliah et al. 1997). In contrast, deletion of the APP gene by homologous recombination in embryonic stem cells induced locomotor de®cits in mice (Zheng et al. 1995) and impaired the survival of primary hippocampal neurons in culture (Perez et al. 1997). These ®ndings suggest that APP may ful®l important neuroprotective functions. Recently, two different dinucleotide deletions were detected in the APP mRNA from cerebral cortex neurons of patients with sporadic AD and Down's syndrome (van Leeuwen et al. 1998a; van Leeuwen et al. 1998b). The deletions resulted in a translational frameshift of the APP mRNA, producing an 38-kDa APP isoform with an N-terminus that ends prematurely at amino acid 348 (Fig. 1). The two abnormally produced truncated APP isoforms (APP11) contain new C-termini of 18 and 12 amino acids, respectively, that can be recognized by a speci®c antibody. Immunohistochemical analysis with this antibody revealed that the APP11 proteins accumulate in cerebral cortex neurons of patients with AD and Down's syndrome (van Leeuwen et al. 1998b). However, the intracellular processing and pathophysiological function of APP11 in the CNS is not known. As a ®rst step toward understanding the role of the APP11 in AD development, we examined the intracellular distribution and processing of APP11 in a cell culture system using APP11 and APP fusion proteins with the enhanced green ¯uorescent protein (eGFP). Experimental procedures Generation of APP±eGFP fusion constructs The plasmid pEGFP-N3 (Clontech, Palo Alto, CA) containing the eGFP coding sequence was used to generate the APP±eGFP fusion constructs. The different APP fragments were ampli®ed by polymerase chain reaction (PCR) from plasmid NSE-hAPP (Mucke et al. 1994). The primers for the truncated APPtrunc and

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APP11 forms included an EcoRI site in the upper primer and a BamHI site in the lower primer for directional in frame subcloning into pEGFP-N3. The primers used to amplify the APP part of APPtrunc ±eGFP were Eco/APPU626 (5 0 -TTCGAATTCGCGATGCTGCCCGGTTTGG-3 0 ) and APPL1661/Bam (5 0 -GATGGATCCCAAGTTCTTTGCTTGACGTTC-3 0 ). The 1065 nucleotide (nt) APP fragment was digested with EcoRI and BamHI and was then subcloned into the EcoRI/BamHI-digested pEGFP-N3 to generate APPtrunc ±eGFP. APP12±eGFP was generated by subcloning the EcoRI/BamHI-digested 1067-nt APP12 fragment into a BamHI/ EcoRI-digested pEGFP-N3. APP12 was ampli®ed by PCR with the primers Eco/APPU626 and APP12L1661/BamHI (5 0 -GATGGATCCGGCAAGTTCTTTGCTTGACGTTC-3 0 ). The two other truncated APP constructs, APP-M1±eGFP and APP-M2±eGFP, were made from APP12±eGFP by deleting two nucleotides (GA) with the Quickchange Mutagenesis Kit (Stratagene, La Jolla, CA, USA). APP-M1±eGFP has a GA deletion in exon 9 (nt 1136±1137), whereas APP-M2±eGFP has the GA deletion in exon 10 (nt 1156±1157) (Kang et al. 1987; Lemaire et al. 1989). The primers for M1 were APP695 1 1F1 (5 0 GAGGCCAAGCACCGAGAGAATGTCCCAGGTCATG-3 0 ) and its complementary sequence APP695 1 1R1; the primers for M2 were APP695 1 1F2 (5 0 -GTCCCAGGTCATGAGAATGGGAAGAGGCAG-3 0 ) and its complementary sequence APP695 1 1R2. The full-length APP was ampli®ed with primers Hind/APPU616 (5 0 -GAGCTCAAGCTTGCGATGCTGCCCGGTTTGG-3 0 ), including a HindIII site and primer APP/SmaI 2696 (5 0 -GATGGATCCCGGGTTCTGCATCTGCTCAAAGA-3 0 ) including a SmaI site. The 2112-nt full-length APP was digested with HindIII and SmaI and was ligated into the HindIII/SmaI-digested vector pEGFP-N3, resulting in APP±eGFP. The cDNA that coded for the APP portion of the fusion proteins was sequenced. Cell culture and transient transfection The rat neuroblastoma cell line B103 was cultivated in Dulbecco's modi®ed Eagle medium (DMEM) containing 10% fetal calf serum, 5% horse serum, and 2 mm l-glutamine. Twenty-four hours after seeding into six-well plates, the semicon¯uent cells were transfected for 4 h with 2 mg of plasmid DNA/well (35 mm) using Lipofectamine (Life Technologies, Gaithersburg, MD, USA). After transfection, the cells for confocal microscopy were incubated in regular medium for 48 h and then split into chamber slides (Nalge Nunc, Milwaukee, WI, USA). Twenty-four hours later, the cells were ®xed with 3% paraformaldehyde in phosphatebuffered saline (PBS). For cellular protein extraction and analysis of the cell culture supernatant, the cells were incubated in regular medium for 68 h after transfection, washed with PBS three times, and incubated in serum-free medium. Four hours later the serumfree supernatant was collected, proteinase inhibitors (complete; Roche Molecular Biochemicals, Indianapolis, IN, USA) were added, and the supernatant was concentrated on Microcon 10 spin columns (Amicon, Beverly, MA, USA). The volume was then adjusted with PBS, Laemmli sodium dodecyl sulfate (SDS) sample buffer was added, and the supernatant was frozen until analyzed. The remaining cells were washed three times with PBS and then lyzed in RIPA buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS) containing proteinase inhibitors (complete) for 30 min at 48C. The volume was adjusted with RIPA, and the samples were frozen until analyzed.

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Fig. 1 APP±eGFP fusion constructs. The coding sequences for the full-length APP protein, the truncated APP (APPtrunc) and the truncated APP 1 1 proteins were subcloned in frame to the eGFP coding sequence. APP±eGFP translates the fulllength hAPP695 (695 amino acid; white boxes) fused to the eGFP protein. APPtrunc ±eGFP translates the N-terminal 348 amino acid of APP fused to eGFP. APP-M1±eGFP translates 330 amino acid of APP and 18 amino acid of a new C-terminus (black box) fused to eGFP. APP-M2±eGFP translates 336 amino acid of APP and 12 amino acid of the new C-terminus (black box) fused to eGFP.

Confocal microscopy The subcellular localization of the eGFP fusion proteins was investigated with the Bio-Rad MRC-600 laser scanning confocal imaging system. A single channel ®lter set [blue excitation (BHS) for ¯uorescein isothiocyanate (FITC)] was used on a Nikon Optiphot upright microscope with a 60  objective [numerical aperture (N.A.) ˆ 1.4]. Immunoblot analysis Cell extracts (maximum of 75 mg of cellular protein) were separated on 10% or 7.5% polyacrylamide±SDS gels, electrotransfered onto nitrocellulose membranes and blocked with 5% milk powder in PBS-Tween (0.1% Tween 20 in PBS). The primary antibody to detect the fusion proteins in the cell extract was a GFP polyclonal antibody (1 : 1000) (Clontech, Palo Alto, CA, USA) and detection was performed with the Super Signal Ultra (Pierce, Rockford, IL, USA) detection system. To correct for differences in transfection ef®ciency between the vectors, different amounts of cell extract were loaded for SDS±PAGE (i.e. 3 mL of EGFP, 25 mL of APPtrunc ±eGFP, 9 mL of APP-M1±eGFP, 12 mL of APP-M2±eGFP, and 25 mL of APP±eGFP). For the subsequent detection of APP in the supernatant, the same ratio between samples was loaded (i.e. 2 mL of EGFP, 17 mL of APPtrunc ±eGFP, 6 mL of APP-M1±eGFP, 8 mL of APP-M2±eGFP, and 17 mL of APP±eGFP). The primary antibody used to analyze the supernatant was the monoclonal APP antibody 22C11 (Roche) that binds to the amino terminus of the APP protein (amino acids 60±100) which is present in all the fusion proteins and in the secreted form of the fulllength APP. Secretion of the truncated APP±eGFP proteins in CHO cells was detected with the polyclonal GFP antibody to avoid detection of endogenous hamster sAPP by 22C11 (Xia et al. 1997). Therefore, secreted sAPP from full length APP±eGFP was not detected.

Results and discussion To investigate the subcellular localization and traf®cking of the truncated APP (APP11) resulting from the dinucleotide deletions in the APP mRNA (van Leeuwen et al. 1998b), we made fusion constructs of APP and eGFP. These fusion

proteins can be detected directly by ¯uorescence microscopy in living cells. To determine if the carboxyl terminus of APP11 in¯uences the subcellular localization of the truncated proteins, we made fusion constructs with three truncated APP sequences that were cloned in-frame with eGFP (Fig. 1). The ®rst two truncation constructs, APP-M1± eGFP and APP-M2±eGFP, contain the described dinucleotide deletion in exons 9 and 10, respectively, of the APP cDNA (van Leeuwen et al. 1998b). In vivo, the dinucleotide deletions cause one APP11 protein to acquire a new carboxyl terminus of 18 amino acids (M1) and the other to acquire a novel carboxyl terminus of 12 amino acids (M2) before translation is terminated prematurely (van Leeuwen et al. 1998b). The constructs APP-M1±eGFP and APP-M2± eGFP contain the entire predicted APP11 coding sequence but lack the premature stop codon because the cDNA sequences were subcloned in-frame to the eGFP sequence. The third truncated fusion construct, APPtrunc ±eGFP, translates 348 amino acid of the wild-type N-terminus. Despite their different carboxyl-terminal sequences, all three fusion proteins with truncated APP showed a similar localization after transient transfection into B103 cells (Fig. 2). APP-M1±eGFP, APP-M2±eGFP and APPtrunc ± eGFP showed a reticular staining and the most intense unipolar staining, presumably within the Golgi complex. Therefore, the different carboxyl termini produced by the dinucleotide deletion appears to have no effect on the subcellular localization of the two APP11±eGFP proteins in our system. Since the intracellular distribution patterns of the APP-M1±eGFP, APP-M2±eGFP and APPtrunc ±eGFP differ from that of eGFP (Fig. 2), we conclude that the unique distribution pattern seen with APP-M1±eGFP, APP-M2±eGFP, and APPtrunc ±eGFP transfected B103 cells results from the APP portion. To compare further the distribution pattern of the two truncated APP11 proteins to the full-length APP protein, we

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Fig. 2 Subcellular localization of the APP11±eGFP fusion proteins and the full-length APP±eGFP fusion protein in neuroblastoma B103 cells. B103 cells were transiently transfected with the constructs shown in Fig. 1 and the eGFP-containing fusion proteins were directly detected by confocal ¯uorescence microscopy with a single channel ®lter set for FITCI. Bars are 5 mm in the pictures and 25 mm in the inlets. (a) eGFP alone. (b) APPtrunc ±eGFP. (c) APP-M1±eGFP. (d) APP-M2±eGFP. (e) APP±eGFP.

transiently transfected APP±eGFP into B103 cells (Fig. 2e). The full-length APP±eGFP fusion protein was localized to the perinuclear area and along the plasma membrane. Furthermore, APP±eGFP showed a faint reticular distribution with an intense polarized staining similar to the reticular distribution of the truncated APP11±eGFP forms.

According to the O-glycosylation of the full-length APP protein (Weidemann et al. 1989; Oltersdorf et al. 1990), we assume that the intense polarized staining localizes within the Golgi complex. This distribution pattern of APP±eGFP fusion protein in transiently transfected B103 cells is similar to the intracellular localization of endogenous APP protein

Fig. 3 The truncated APP11±eGFP fusion proteins are secreted from B103 cells. Western blot analysis with cell extracts and supernatant from B103 cells transiently transfected with the constructs shown in Fig. 1. The supernatant was collected for 4 h in serum free medium, and the cell extracts were harvested afterwards. (a) Western detection was performed on cell extracts with polyclonal GFP antibody (mini-gel, 10% SDS±PAGE). (b) Western detection was performed on cell extracts with the monoclonal APP antibody 22C11 detecting amino acid 60±100 of APP (15  15 cm, 7.5% SDS±PAGE). (c) short and (d) long exposure of western detection performed on supernatant with 22C11 (mini-gel, 10% SDS±PAGE). Numbers indicate molecular mass (in kDa).

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in primary neuronal cultures and cell lines (Weidemann et al. 1989; Haass et al. 1992; Nordstedt et al. 1993; Sahasrabudhe et al. 1993; Caporaso et al. 1994; Koo and Squazzo 1994; Tomita et al. 1998). Hence, the APP±eGFP fusion protein faithfully mimics the subcellular localization of wild-type APP, indicating that the absence of membrane association of the truncated APP11±eGFP proteins results from the deletion of the APP transmembrane domain (Figs 1 and 2). To investigate intracellular traf®cking of the fusion proteins (Fig. 2), we analyzed cellular extracts of transfected B103 cells by Western blot with a polyclonal GFP antibody (Fig. 3a). The antibody detected proteins of the expected sizes: 27 kDa for eGFP, and 70±75 kDa for the truncated APP-M1±eGFP, APP-M2±eGFP, and APPtrunc ±eGFP proteins. However, the cellular extract of the B103 cells transfected with full-length APP±eGFP showed two proteins of approximately 118 kDa and 137 kDa. These two proteins apparently correspond to the N- and O-glycosylated forms of hAPP695 that have molecular masses of 91 kDa and 110 kDa, respectively (Dyrks et al. 1988; Weidemann et al. 1989). These results demonstrate that the full-length APP±eGFP protein is N- and O-glycosylated and suggest that the intense polarized staining re¯ects accumulation in the Golgi complex (Caporaso et al. 1994; Tomita et al. 1998). The truncated APP11 proteins lack the N-glycosylation site but contain potential O-glycosylation sites located in the APP N-terminus (Weidemann et al. 1989; Tomita et al. 1998). To investigate if APP-M1±eGFP, APP-M2±eGFP and APPtrunc ±eGFP were O-glycosylated, cell extracts were separated on longer polyacrylamide SDS gels and analyzed by Western blot with the antibody 22C11 (Fig. 3b). Detection of two truncated APP proteins of approximately 70 and 75 kDa suggests that APP-M1±eGFP, APP-M2± eGFP and APPtrunc ±eGFP are O-glycosylated. Since the APP-M1±eGFP, APP-M2±eGFP and APPtrunc ± eGFP proteins lack the transmembrane domain, we asked if they accumulate within the cells, or are secreted. Transiently transfected B103 cells were incubated with serum-free medium for 4 h, and the supernatant was analyzed for the presence of the amino terminus of APP by western blot analysis (Fig. 3c). The 22C11 antibody recognizes amino acid 60±100 of APP and thus recognizes APP-M1±eGFP, APP-M2±eGFP, APPtrunc ±eGFP, and the secreted fulllength APP cleaved either by a- or b-secretase. All three truncated fusion proteins were detected as approximately 75 kDa single proteins in the supernatant (Fig. 3c) which corresponds to the larger protein in cell extracts (Fig. 3b). Identical results were obtained on longer polyacrylamide SDS gels (data not shown). In contrast, no sAPP was detected in the supernatant of cells transfected with the fulllength APP±eGFP fusion construct after short exposure of western blots. Overexposure of western detection con®rmed that the B103 cells have secretase activity (Xu et al. 1999)

and that the full length APP±eGFP fusion-protein can be cleaved by secretases. A faint band representing a ,100-kDa s-APP isoform was detected in the supernatant from B103 cells transfected with full length APP±eGFP (Fig. 3d). However, little sAPP was secreted from fulllength APP±eGFP and from hAPP695 (data not shown) compared to the robust secretion of the truncated APP11± eGFP proteins (Fig. 3c). This abundance of APP11±eGFP proteins in the supernatant indicates that the truncated APP11±eGFP proteins are readily secreted from B103 neuroblastoma cells. We roughly estimate that about 50% of each truncated protein was secreted within 4 h. Because the B103 neuroblastoma cell line is APPde®cient, the question was addressed of whether the differential intracellular distribution and secretion pattern of the truncated APP fusion proteins and the full-length APP fusion protein is cell-type speci®c. Therefore, CHO cells were transiently transfected with the fusion constructs. The localizations of the APP-M1±eGFP, APP-M2±eGFP, APPtrunc ±eGFP, and full-length APP±eGFP fusion proteins were similar to that in B103 cells (data not shown). Furthermore, CHO cells ef®ciently secreted all three truncated APP±eGFP fusion proteins (data not shown). The implications of these APP11 proteins on the development of AD are unclear. The dinucleotide deletion in the APP mRNA results in loss of membrane association and in ef®cient secretion of truncated APP11 isoforms from neuroblastoma cell cultures. Such secreted APP11 isoforms lack several protein domains that were shown to be neuroprotective in physiologically secreted a-APPs. For example, M2 contains the mutated growth-promoting region (Fig. 1) (Saitoh et al. 1989; Ninomiya et al. 1993; Yamamoto et al. 1994) and lacks the neuroprotective region (Mattson et al. 1993b) but still contains the heparin-binding domain (Small et al. 1994). Considering the lack of several functional domains in APP11 and the potential abundance of the secreted APP11 proteins, they may ef®ciently interfere with physiological functions of a-APPs, like neurite outgrowth, neuroprotection, and mediation of cell adhesion (Schubert et al. 1989; Klier et al. 1990; Breen et al. 1991; Chen and Yankner 1991; Milward et al. 1992). Hence, APP11 may competitively inhibit the neuroprotective function of a-APPs which could explain its association with the pathogenesis of AD and Down's syndrome.

Acknowledgements This work was supported by the PPG2 Project 3 grant (#PO1 HL47660-09) from the NIH/NHLBI. We are indebted to David Sanan and Lara Jensen for their help with the confocal microscopy and for superb technical support, Robert Pitas, Lennart Mucke and Thomas Innerarity for critical reading of the manuscript, Gary Howard and Stephen Ordway for editorial support, and September Plumlee for manuscript preparation.

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q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1308±1314