Aberrant Presenilin-1 Expression Downregulates LDL Receptor-Related Protein (LRP): Is LRP Central to Alzheimer's Disease Pathogenesis?

Molecular and Cellular Neuroscience 14, 129–140 (1999) Article ID mcne.1999.0772, available online at http://www.idealibrary.com on

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Aberrant Presenilin-1 Expression Downregulates LDL Receptor-Related Protein (LRP): Is LRP Central to Alzheimer’s Disease Pathogenesis? Emily Van Uden,* George Carlson,† Peter St. George-Hyslop,‡ David Westaway,‡ Robert Orlando,§ Margaret Mallory,* Edward Rockenstein,* and Eliezer Masliah*,§ *Department of Neurosciences and §Department of Pathology, School of Medicine, University of California at San Diego, La Jolla, California 92093-0624; †McLaughlin Research Institute, Great Falls, Montana 59405; and ‡Centre for Research into Neurodegenerative Disease, Departments of Medicine and Pathology, University of Toronto, Ontario, Canada

Low density lipoprotein receptor-related protein (LRP) polymorphisms have recently been associated with an increased susceptibility of Alzheimer’s disease (AD). Furthermore, LRP has been linked to molecules that confer susceptibility to AD (apolipoprotein E, alpha-2-macroglobulin, amyloid precursor protein), previously with the exception of the presenilins. Here we report that aberrant presenilin-1 expression in vivo and in vitro downregulates LRP. Specifically, transgenic mice overexpressing the M146L or L286V presenilin-1 mutation show decreased levels of LRP expression in neuronal populations where presenilin-1 and LRP are closely colocalized or coexpressed. Moreover, cell lines transfected with presenilin-1 also expressed decreased levels of LRP. These findings suggest that LRP may be central to AD pathogenesis since all proteins genetically associated with AD can now be linked via a single pathway to LRP.

INTRODUCTION Low density lipoprotein receptor-related protein (LRP) is a member of the LDL receptor family. A remarkable feature of this receptor family is the ability of its members to bind multiple, diverse ligands (Williams et al., 1994). For this reason, it is not surprising that LRP appears to be involved in a variety of processes, including regulation of lipoprotein metabolism, proteolytic activity, hippocampal neurite outgrowth, and early developmental processes necessary for viability (Fagan et al., 1996; Herz and Willnow, 1995; Holtzman et al., 1995; Narita et al., 1997a; Okada et al., 1996; Strickland et al., 1995; Weaver et al., 1997; Wijnberg et al., 1997). 1044-7431/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

Furthermore, recent reports have shown a genetic linkage between LRP and Alzheimer’s disease (AD) (Kang et al., 1997; Lendon et al., 1997; Wavrant-DeVrieze et al., 1997). To date, in addition to LRP, five other proteins have been genetically linked to AD: amyloid precursor protein (APP), apolipoprotein E ⑀4 (apoE ⑀4), alpha-2 macroglobulin (␣-2m), presenilin-1 (PS-1), and presenilin-2 (PS-2) (Finch and Tanzi, 1997). Interestingly, APP, apoE, and ␣-2m are ligands of LRP, and these and other LRP ligands are present in senile plaques, a hallmark of AD (Blacker et al., 1998; Knauer et al., 1996; Kounnas et al., 1995; Kuchenhoff et al., 1997; Narita et al., 1997b; Rebeck et al., 1995). Genetic evidence, combined with clinical observations of increased serum LRP ligand levels [i.e., amyloid-␤ (A␤), ␣1-antichymotrypsin, plasmin and urokinase] (Aoyagi et al., 1992; Licastro et al., 1995; Martins et al., 1995), suggests that deficient LRP expression and/or function to be central to AD pathogenesis. Thus, with the exception of presenilins, the known genes that confer AD susceptibility are linked to a common pathway via LRP. Since the majority of familial AD cases are accounted for by mutations in the presenilin genes, particularly PS-1 (Clark et al., 1996; Haass, 1996), we explored the hypothesis that mutant PS-1 affects LRP expression. Support for this hypothesis is provided by studies showing that there is an increased level of A␤ protein in PS-1 transgenic mice and PS-1-transfected cell lines (Borchelt et al., 1996; Citron et al., 1997; Duff et al., 1996; Lemere et al., 1996; Mann et al., 1996; Mehta et al., 1998; Scheuner et al., 1996; Xia et al., 1997). While the mecha-

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130 nism of this increase is unknown, it has been shown that A␤ complexes with activated ␣-2m and is internalized by LRP and degraded (Narita et al., 1997b). Thus, increased A␤ concentrations may be due to decreased surface LRP levels mediated by mutant presenilin expression. To explore this hypothesis, levels of LRP in PS-1 transgenic mice and PS-1-transfected cell lines were studied. Our experiments show that LRP is decreased in pyramidal and somatostatinergic neurons in transgenic mice expressing the PS-1 M146L and L286V mutations. These neuronal populations were found to coexpress LRP and PS-1. Furthermore, C6 cells stably transfected with PS-1 also show a significant decrease in LRP protein and transcriptional levels.

RESULTS LRP and PS-1 are closely coexpressed in cortical neurons. Since the main hypothesis of this study was that PS-1 and LRP might interact in regulating neuronal function, we wanted to determine if these two molecules were present in the same neuronal population. Consistent with previous reports, PS-1 was found to be ubiquitously expressed in neurons throughout the brain, with the strongest labeling in neurons and the neuropil (Elder et al., 1996; Huynh et al., 1996; Kim et al., 1997; Lah et al., 1997; Lee et al., 1996; Moussaoui et al., 1996; Uchihara et al., 1996). PS-1 immunoreactivity was present in diverse neuronal populations including pyramidal neurons and interneurons. Of these two neuronal populations, PS-1 immunoreactivity was more intense in interneurons in the stratum oriens of the hippocampal CA1 region and in layers 2 and 4 of the neocortex. Double-labeling studies showed that in the hippocampus, PS-1 and LRP were coexpressed by the same interneurons; however, LRP was expressed mainly at the cell surface and PS-1 in the cytoplasm (Fig. 1A). In contrast, in the neocortex, in addition to surface LRP immunoreactivity, LRP and PS-1 were colocalized to the neuronal cytoplasm of both pyramidal neurons and interneurons (Fig. 1B). LRP immunoreactivity was observed both in the neuronal cell body as well as in the initial segments of the neurites (Figs. 1A and 1B). The LRP-immunoreactive interneurons also displayed strong somatostatin (Figs. 1C and 1D) and receptor-associated protein (RAP) (Figs. 1E and 1F) immunoreactivity. Overall, the somatostatin-producing interneurons in the neocortex and hippocampus showed strong PS-1 and RAP immunoreactivity (Fig. 1). In contrast, pyramidal neurons in the neocortex and hippocampus showed only moderate PS-1, RAP, and LRP immunoreactivity and

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were somatostatin negative (not shown). In the neocortical neurons, somatostatin and RAP were colocalized with LRP in the cytoplasm (Figs. 1D and 1F), whereas in the hippocampus, LRP was more abundant at the cell surface (Figs. 1C and 1E). This might indicate that in the neocortex there might be more active recycling of this receptor. Levels of LRP are decreased in PS-1 transgenic mice. Since LRP and PS-1 were found to be coexpressed in neocortical and hippocampal interneurons and pyramidal neurons (Fig. 1), we hypothesized that altered PS-1 expression in transgenic mice might affect LRP expression in these neuronal populations. Western blot analysis showed that the antibody against human PS-1 detected a band at an approximate molecular weight of 19 kDa only in the PS-1 transgenic mice (Fig. 2A). The polyclonal antibody against LRP 1073 detected a band at approximately 85 kDa, corresponding to the C-terminal region of LRP both in the nontransgenic and PS-1 transgenic mice (Fig. 2A). Semiquantitative analysis of the Western blot using the ImageQuant software revealed that in two lines of the PS-1 transgenic mutant mice (Tg(L286V)198 and Tg(M146L)76) there was a significant decrease in LRP immunoreactivity compared to PS-1 wild-type transgenic and nontransgenic controls (Fig. 2B), indicating that overexpression of mutant PS-1 alters LRP expression in vivo. Further analysis of the patterns of LRP expression in pyramidal and interneuronal populations were performed by immunocytochemistry. Sections were immunolabeled with a polyclonal antibody against LRP and analyzed via two semiquantitative methods: (1) optical density readings on the Quantimet 570C, and (2) mean pixel intensity readings on the laser scanning confocal microscope (LSCM). Pixel intensity readings on the laser scanning confocal microscope of sections labeled with FITC showed that in comparison to the nontransgenic littermates, all three PS-1 mutant transgenic lines expressed significantly decreased LRP immunoreactivity in the pyramidal neurons (Figs. 3A and 4). Additionally, the two high PS-1 mutant overexpressor lines, Tg(M146L)76 and Tg(L286V)198, showed significantly decreased LRP immunoreactivity in the somatostatinergic interneurons (Figs. 3B and 5). Linear regression analysis between optical densitometry readings from DAB-immunoreacted tissues (not shown) and mean pixel intensity readings from FITC-immunolabeled tissues (Fig. 3) showed that the results from both methods were strongly correlated (r ⫽ 0.72, P ⬍ 0.005). Distributional analysis of LRP immunoreactivity by LSCM revealed that in the neocortical pyramidal neurons LRP immunoreactivity was observed both in non-

FIG. 1. LRP and PS-1 are colocalized in somatostatin-expressing neurons. Double-labeled sections were imaged with the LSCM. Images in green correspond to LRP immunoreactivity, and those in red correspond to PS-1, somatostatin, or RAP immunoreactivity. Images in the top panels are from the stratum oriens of the hippocampal CA1 region, and images on the bottom panels are from layers 4 and 5 of the parietal cortex. (A,B) Neurons displaying strong LRP immunoreactivity at the cell surface also displayed intense PS-1 immunoreactivity in the cytoplasm. (C,D) Somatostatin-immunoreactive neurons displayed strong LRP immunoreactivity both on the cell surface and in the cytoplasm. (E,F) LRP-immunoreactive neurons displayed strong RAP immunoreactivity both on their surface and in the cytoplasm.

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FIG. 2. Levels of LRP immunoreactivity in PS-1 transgenic mice. (A) Western blot analysis showed that the antibody against human PS-1 detected a 19-kDa band only in the PS-1 transgenic mice. The polyclonal antibody, LRP 1073, detected a band at approximately 85 kDa, corresponding to the LRP C-terminal region both in the nontransgenic and PS-1 transgenic mice. (B) Semiquantitative analysis of the Western blot using the ImageQuant software revealed that in two lines of the mutant PS-1 transgenice mice (Tg(L286V)198 and Tg(M146L)76) there was a significant decrease in LRP immunoreactivity compared to wild-type PS-1 transgenic and nontransgenic controls.

transgenic and transgenic mice in the cytoplasm (Fig. 4). In the hippocampus of both wild-type and transgenic mice, LRP was consistently associated with the cell surface (Fig. 5). However, while in the nontransgenic mice LRP appeared as dense granular immunoreactivity, in mutant transgenic mice levels of immunoreactivity were decreased and the staining was more diffuse (Figs. 4 and 5). Since LRP transport to the membrane is dependent on the chaperone molecule RAP (Herz and Willnow, 1995), it is possible that decreased expression of LRP by PS-1 might depend on altered RAP expression. In this regard, both immunocytochemical and Western blot analysis revealed that levels of RAP were unaltered in PS-1 transgenic mice (not shown). Levels of LRP are decreased in PS-1-transfected cells. To further confirm the observations made in the transgenic mice, C6 cells were stably transfected with human wild-type or mutant PS-1 harboring the M146L missense mutation. Analysis of mRNA and protein expression showed that both transfected cell lines expressed similar levels of PS-1 (not shown). Consistent with the studies in vivo, immunocytochemical analysis of the C6 cells showed that wild-type-untransfected and vector-transfected cells displayed intense LRP-immunoreactivity, while cells transfected with mutant PS-1 showed significantly decreased LRP immunoreactivity (Fig. 6). Consistent with these observations, Western blot analysis of transfected cells showed significant decreases in LRP immunoreactivity, which was more accentuated in the mutant PS-1 than wild-type PS-1transfected cells (Fig. 7). In order to determine if alterations in LRP protein expression were due to transcrip-

tional alterations, the ribonuclease protection assay (RPA) was utilized. This assay corroborated that human PS-1 was only present in the transfected cells (identified as a 305-bp fragment), and found LRP (identified as a 225-bp fragment) to be significantly reduced in the PS-1-transfected cells (Fig. 8). Since it is possible that decreased levels of LRP immunoreactivity or message might be the result of cell death, colorimetric analysis of LDH release (Cytotox96, Promega) and DNA laddering analysis via flow cytometry was performed. These assays found no differences in cell viability between the PS-1 and vector-transfected cells (not shown).

DISCUSSION The present study showed that aberrant PS-1 expression downregulates LRP in vivo and in vitro. This is of particular interest to AD pathogenesis because previous studies have shown LRP and several of its ligands confer genetic susceptibility to this disorder (Kang et al., 1997; Lendon et al., 1997; Wavrant-DeVrieze et al., 1997). Furthermore, as shown in this study, LRP and presenilin are coexpressed by the same neuronal populations in the neocortex and hippocampus. In the neocortical neurons, PS-1, somatostatin, and RAP were colocalized with LRP in the cytoplasm while, in contrast, in the hippocampus LRP was more abundant at the cell surface of somatostatin-producing neurons. This might indicate that in the neocortex there might be more active recycling of this receptor. These neuronal populations

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FIG. 3. Semiquantitative analysis of levels of LRP immunoreactivity in tissue sections of PS-1 transgenic mice. Sections immunolabeled with a polyclonal antibody against LRP and analyzed with the LSCM revealed that compared to control, nontransgenic littermates, and wild-type PS-1 transgenic mice, LRP immunoreactivity per neuron was decreased in the (A) cortical pyramidal neurons in all three mutant PS-1 tg lines as well as the (B) hippocampal interneurons of the mutant PS-1 Tg(M146L)76 and Tg(L286V)198 lines.

have previously been shown to be susceptible to degeneration in AD (Ang and Shul, 1995; Dournaud et al., 1994; Masliah et al., 1990), and downregulation of LRP via deletion of RAP results in decreased somatostatin

133 expression (Van Uden et al., 1999). Taken together, these findings suggest that LRP and PS-1 might play a role in maintenance of diverse neuronal populations of significant interest to AD pathogenesis. The mechanisms by which abnormal expression of presenilin dysregulates LRP are not completely understood. However, the present study showed that downregulation of LRP expression occurred at the protein and mRNA levels, indicating that PS-1 disrupts LRP transcription. A role for presenilin in transcriptional regulation is supported by previous studies, which have shown that overexpression of PS-1 reduces ␤-catenin levels and inhibits ␤-catenin T-cell factor-regulated transcription (Murayama et al., 1998). Furthermore, induction of DNA binding activity of the transcription factor, AP-1, by nerve growth factor, is markedly suppressed in cells expressing mutant PS-1 (Furukawa et al., 1998). Alternatively, it is possible that overexpression of presenilin might affect LRP expression via posttranscriptional alterations. Since presenilins appear to be present in the endoplasmic reticulum (Zhang et al., 1998) and colocalize with LRP in this compartment, presenilins could interfere with LRP transport to the membrane. In this regard, previous studies have shown that PS-1 binds molecules involved in AD pathogenesis such as APP, affecting its proteolytic processing (Waragai et al., 1997; Weidemann et al., 1997; Xia et al., 1997). However, recent studies in HEK 293 cells cotransfected with presenilin and APP or LDL-receptor, show that PS-1 mutants did not affect protein trafficking through the Golgi apparatus (Tan et al., 1998). Thus, it is more likely that PS-1 might affect the expression of receptors involved in AD pathogenesis via dysregulation of transcriptional pathways rather than through post-transcriptional changes. Alterations in LRP might potentially represent a common pathway in the pathogenesis of AD because genetic modifications in this molecule (Kang et al., 1997; Lendon et al., 1997; Wavrant-DeVrieze et al., 1997), as well as in its ligands are linked with increased susceptibility to AD and are found in senile plaques (Blacker et al., 1998; Knauer et al., 1996; Kounnas et al., 1995; Kuchenhoff et al., 1997; Narita et al., 1997b; Rebeck et al., 1995). Since presenilins account for the majority of familial AD cases (Clark et al., 1996), we hypothesized that presenilins might be linked with the LRP pathway. In support of this possibility, recent studies have shown that mutations in presenilins increase A␤1-42 levels (Borchelt et al., 1996; Citron et al., 1997; Duff et al., 1996; Lemere et al., 1996; Mann et al., 1996; Mehta et al., 1998; Scheuner et al., 1996; Xia et al., 1997). The mechanism by

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FIG. 4. Analysis of pyramidal LRP immunoreactivity by LSCM. (A) In control, nontransgenic mice strong LRP immunoreactivity was observed. (B) Wild-type PS-1 transgenic mice (Tg(PS-1wt)195) showed a slight but not statistically significant decrease in LRP immunoreactivity. (C,D) LRP immunoreactivity per neuron was significantly decreased in all three mutant PS-1 transgenic lines studied (lines Tg(M146L)76 and Tg(L286V)198 are shown).

which PS-1 mutations result in increased A␤1-42 levels is unclear; some studies suggest that presenilins might affect APP proteolytic processing via direct protein interactions (Waragai et al., 1997; Xia et al., 1997) or that presenilins might have per se ␥-secretase activity (De Strooper et al., 1998, 1999; Wolfe et al., 1999). The present study provides support for an additional explanation, namely that presenilin might dysregulate LRP, resulting in aberrant processing of APP. In effect, if LRP is downregulated, internalization of ␣2-m/A␤ complexes may be decreased resulting in increased serum A␤ levels, as is observed in PS-1 transgenic mice (Borchelt et al., 1996). In addition, inhibition of LRP with RAP, a potent LRP antagonist, results in decreased degradation of ␣-2m/A␤ complexes, suggesting LRP to be a clearance receptor for A␤ (Blacker et al., 1998). Since secreted APP is a direct ligand for LRP (Kounnas et al., 1995), downregulation of LRP may affect APP internalization and potentially its processing. In summary, the present study provides evidence that aberrant presenilin expression might alter LRP expression, further supporting the contention that LRP may be central to AD pathogenesis.

EXPERIMENTAL METHODS Transgenic mice. A total of 18 PS-1 transgenic mice (14 months old) and 5 nontransgenic littermates (14 months old) were included in the present study. Transgenic mice were generated expressing mutant or wildtype human PS-1 cDNAs using the vector cos.Tet (Scott et al., 1992). This cosmid vector contains the hamster prion gene promoter. Transgenic lines were created and maintained in the FVB/N background and back-crossed to C57BL/6J mice (Citron et al., 1997). Specifically, the lines studied were Tg(M146L)76 (n ⫽ 3, line harboring the human M146L mutation in which Leu replaces Met at codon 146), Tg(L286V)198 (n ⫽ 4, line harboring the human L286V mutation in which Leu replaces Val at codon 286), Tg(M146L)1 (n ⫽ 3, high overexpressor of the human M146L mutation), and Tg(PS-1wt)195 (n ⫽ 3, line overexpressing human wild-type PS-1). Tissue preparation. Anesthetized mice were perfused with cold saline and their brains removed. The left hemibrain was frozen in liquid nitrogen and the right hemibrain was immersion-fixed in 4% paraformaldehyde in pH 7.4 phosphate-buffered saline (PBS). The

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FIG. 5. Analysis of interneuronal LRP immunoreactivity by LSCM. (A) In control, nontransgenic mice strong LRP immunoreactivity was observed in a granular pattern. (B) Wild-type PS-1 transgenic mice (Tg(PS-1wt)195) showed a slight but not statistically significant decrease in LRP immunoreactivity. (C, D) LRP immunoreactivity per neuron was decreased and the staining was more diffuse in two of the mutant PS-1 lines: Tg(M146L)76 and Tg(L286V)198.

frozen hemibrain was divided in two and one portion was homogenized and fractionated as previously described for subsequent Western blot analysis (Masliah et al., 1995b) and from the other portion RNA was extracted for subsequent RPA analysis. Fixed hemibrains were serially sectioned at 40 µm with a vibratome (Leica VT1000E, Deerfield, IL) for subsequent immunocytochemical/computer-aided image analysis. Stable transfection of PS-1. For these experiments, rat glioma (C6) cells were utilized because after preliminary screening of both neuronal and glial cell lines it was found that this line expressed the highest levels of LRP. Furthermore, previous studies have shown that after injury, glial cells express increased LRP levels under in vivo conditions (Lopes et al., 1994). Rat glioma (C6) cells were maintained at 37°C (5% CO2 atmosphere) in DMEM media supplemented with 10% heat-inactivated fetal bovine serum. Wild-type and mutant PS-1

cDNAs subcloned into the CMV-based expression vector pCI-neo (Promega) were kindly provided by David Kang and Dr. E. Koo. The mutant PS-1 vector contained the M146L missense mutation. C6 cells were transfected with Lipofectamine (GIBCO Life Technologies) and maintained under selection with 600 µg/ml G418 for 2–3 weeks until stable clones were isolated. These clones were screened for PS-1 expression by ribonuclease protection assay, as described below. Ribonuclease protection assay. Riboprobe templates were amplified by PCR from human, rat, and mouse cDNA libraries. For the PS-1 template, a 305-bp fragment corresponding to bases 1133 to 1437 of human PS-1 (GenBank Accession No. L76517) was obtained and subcloned into pCR II (Invitrogen). For the mouse and rat LRP templates, a 225-bp fragment corresponding to bases 2378 to 2601 of mouse LRP (GenBank accession No. X67469) were similarly isolated and subcloned. An

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FIG. 6. Patterns of LRP and PS-1 immunoreactivity in PS-1transfected cells. (A) Immunocytochemical analysis of the C6 cells showed that vector-transfected cells displayed intense LRP immunoreactivity, while (B) cells transfected with PS-1(M146L) showed decreased LRP immunoreactivity. (C) Vector-transfected cells showed no immunoreactivity with an antibody against human PS-1, while (D) cells transfected with PS-1 (M146L) showed intense PS-1 immunoreactivity.

FIG. 7. Levels of LRP immunoreactivity in PS-1-transfected cells. Western blot analysis of PS-1-transfected cells showed significant decreases in LRP immunoreactivity, which was more accentuated in the mutant PS-1 (M146L) than wild-type PS-1-transfected cells.

actin riboprobe was used that is complementary to nt 480–559 of mouse ␤-actin (Genbank Accession No. M18194). RPAs were carried out with 32P-labeled antisense riboprobes, and signals were quantified with a PhosphorImager and the ImageQuant software, as previously described (Rockenstein et al., 1995). Actin signals were used to correct for variations in mRNA content and loading. Western blot analysis. Levels of LRP and PS-1 expression in the transgenic mice and PS-1-transfected cells were assessed by Western blot, as previously described (Masliah et al., 1995b). Briefly, samples were loaded onto 10% SDS–PAGE gels (15 µg per lane) and then blotted onto nitrocellulose paper. Blots were incubated with rabbit polyclonal antibody LRP 1073 (1:5000) against the LRP C-terminus (Czekay et al., 1995) or goat polyclonal antibody against PS-1 (1:1,000, Cortex Biochem, San Leandro, CA). For the LRP 1073 antibody, blots were incubated with [ 125I]Protein A and for the PS-1 antibody, blots were first incubated in the rabbit anti-goat secondary antibody followed by [ 125I]Protein

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FIG. 8. Levels of LRP mRNA by RPA in PS-1-transfected cells. (A) LRP was identified as a 225-bp protected fragment, which (B) was significantly reduced in the PS-1-transfected cells.

A. Blots were then exposed to a PhosphorImager screen and analyzed with ImageQuant software (Molecular Dynamics). Immunocytochemistry, laser scanning confocal microscopy (LSCM), and image analysis. In order to determine the levels of LRP immunoreactivity in the brains of PS-1 transgenic mice and control nontransgenic littermates, free-floating vibratome sections were first washed in PBS (pH 7.4), blocked with 10% normal serum, and incubated overnight at 4°C with the rabbit polyclonal antibody LRP 456, against full-length LRP (1:1000) (Czekay et al., 1995), as previously described (Masliah et al., 1995a; Van Uden et al., 1999). Sections were then washed in PBS and incubated with the biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA), followed by Avidin D-HRP (ABC Elite, Vector) and diaminobenzidine (DAB) (0.2 mg/ml) in 50 mM Tris buffer (pH 7.4) with 0.001% hydrogen peroxide for Quantimet analysis or FITC-goat anti-rabbit secondary antibody (Vector Laboratories) for LSCM analysis. All sections were processed simultaneously and under identical conditions. Reproducibility was assessed by repeating the experiments twice for each method. For semiquantitative assessment of levels of LRP 456 DAB-immunoreactivity, sections were analyzed with the Quantimet 570C densitometer as previously described (Masliah et al., 1995a). Briefly, the system was calibrated with a set of intermediate and high density filters, then for each section a total of 15–20 neurons were analyzed in the hippocampus and neocortex. The mean optical density per neuron was averaged

to obtain the average corrected optical density per case. For each case a total of two serial sections were analyzed, and the average per case was used for subsequent statistical analysis. For semiquantitative assessment of levels of LRP 456 FITC-immunoreactivity, sections were analyzed with the LSCM as previously described (Masliah et al., 1990). Briefly, the system was set for detection within a linear range, then for each section a total of 10–15 neurons were analyzed in the hippocampus and neocortex. The mean optical density per neuron was averaged to obtain the average corrected pixel intensity per case. For each case a total of two serial sections were analyzed, and the average per case was used for subsequent statistical analysis. To further determine the colocalization of PS-1, LRP, RAP, and somatostatin in the murine nervous system, sections from wild-type mice were double-immunolabeled, as previously described (Van Uden et al., 1999). For studies using the rabbit polyclonal antibody against LRP 456 (1:500), and either goat polyclonal anti-PS-1 antibody (1:100) or rat monoclonal antibody against somatostatin (1:50) (Chemicon, Temecula, CA), overnight incubation at 4°C in the primary antibodies was followed by FITC-conjugated goat anti-rabbit IgG (for LRP) and Texas Red-conjugated rabbit anti-goat (for PS-1) or Texas Red-conjugated goat anti-rat (for somatostatin). For double-immunolabeling studies using antiLRP456 and anti-RAP antibodies, vibratome sections were first incubated overnight at 4°C with the anti-RAP antibody (1:40,000) (Orlando and Farquhar, 1993), followed by detection with the Tyramide Signal Amplifica-

138 tion-Direct (Red) system (dilution 1:100, NEN Life Sciences, Boston, MA). Next, sections were incubated overnight at 4°C with the anti-LRP 456 (1:500), followed by incubation with FITC-conjugated anti-rabbit IgG secondary antibody (dilution 1:75, Vector). This approach allows the identification of two different antigens using secondary antibodies generated in the same species. Double-labeled sections were imaged with the LSCM, as previously described (Masliah et al., 1995a). Sections were also single-labeled with the LRP 456 antibody, developed using the FITC-conjugated goat anti-rabbit secondary antibody, and imaged with the LSCM. Further immunocytochemical experiments were carried out in PS-1-transfected and control cells plated onto poly-L-lysine-coated 35-mm glass coverslips. Briefly, 1 ⫻ 104 cells were seeded onto coverslips and after 24 h cells were fixed for 20 min in 4% paraformaldehyde. The coverslips were immunostained with either PS-1 or LRP 456 antibodies, followed by development with DAB (as described above). Statistical analysis. After the results were obtained, the code was broken and sets of data were assigned to their corresponding groups. Statistical analyses were performed using the STAT VIEW II software package (Abacus Concepts). Statistical comparisons between the control and PS-1 transgenic mice or between the control and PS-1-transfected cells were done by factorial ANOVA and Fisher’s post-hoc tests. All values are expressed as mean ⫾ SEM.

ACKNOWLEDGMENTS This work was supported by the Alzheimer’s Disease and Related Disorders Association, the Ruth K. Broad Foundation, the Sam and Rose Stein Foundation, The Medical Research Council of Canada, the Alzheimer Assciation of Ontario, the Howard Hughes Medical Research Foundation, and NIH Grants AG10869 and AG5131.

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