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Originally published In Press as doi:10.1074/jbc.M604436200 on September 29, 2006

J. Biol. Chem., Vol. 281, Issue 47, 36180-36186, November 24, 2006
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Apolipoprotein E and Low Density Lipoprotein Receptor-related Protein Facilitate Intraneuronal Abeta42 Accumulation in Amyloid Model Mice*

Celina V. Zerbinatti{ddagger}, Suzanne E. Wahrle§, Hyungjin Kim{ddagger}, Judy A. Cam{ddagger}, Kelly Bales, Steven M. Paul, David M. Holtzman§||, and Guojun Bu{ddagger}**1

From the Departments of {ddagger}Pediatrics, §Neurology, ||Molecular Biology and Pharmacology, and **Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and Neuroscience Discovery Research, Lilly Research Laboratories, Indianapolis, Indiana 46285

Received for publication, May 9, 2006 , and in revised form, September 18, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The low density lipoprotein receptor-related protein (LRP) is highly expressed in the brain and has been shown to alter the metabolism of amyloid precursor protein and amyloid-beta peptide (Abeta) in vitro. Previously we developed mice that overexpress a functional LRP minireceptor (mLRP2) in their brains and crossed them to the PDAPP mouse model of Alzheimer disease. Overexpression of mLRP2 in 22-month-old PDAPP mice with amyloid plaques increased a pool of carbonate-soluble Abeta in the brain and worsened memory-related behavior. In the current study, we examined the effects of mLRP2 overexpression on 3-month-old PDAPP mice that had not yet developed amyloid plaques. We found significantly higher levels of membrane-associated Abeta42 in the hippocampus of mice that overexpressed mLRP2. Using immunohistochemical methods, we observed significant intraneuronal Abeta42 in the hippocampus and frontal cortex of PDAPP mice, which frequently co-localized with the lysosomal marker LAMP-1. Interestingly, PDAPP mice lacking apolipoprotein E (apoE) had much less intraneuronal Abeta42. We also found that PC12 cells overexpressing mLRP2 cleared Abeta42 and Abeta40 more rapidly from media than PC12 cells transfected with the vector only. Preincubation of apoE3 or apoE4 with Abeta42 increased the rate of Abeta clearance, and this effect was partially blocked by receptor-associated protein. Our results support the hypothesis that LRP binds and endocytoses Abeta42 both directly and via apoE but that endocytosed Abeta42 is not completely degraded and accumulates in intraneuronal lysosomes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The low density lipoprotein receptor-related protein (LRP)2 is a large endocytic receptor that is highly expressed in neuronal cell bodies and dendritic processes (1, 2). LRP binds and internalizes many distinct ligands, including molecules associated with Alzheimer disease (AD) such as apolipoprotein E (apoE) and {alpha}2-macroglobulin (3). LRP has also been shown to interact with amyloid precursor protein (APP) (4-8), and this interaction appears to favor the processing of APP to generate the amyloid beta-peptide or Abeta (9, 10). In turn, Abeta has been shown to directly bind LRP (11) and to form stable complexes with apoE and {alpha}2-macroglobulin (12, 13). These observations suggest that changes in LRP expression levels may affect risk for AD by altering both the processing of APP and the clearance of Abeta.

To study the role of LRP in the brain, our laboratory previously cloned a section of the extremely large human LRP receptor (mLRP2) into the MoPrP.Xho transgenic mouse vector and developed transgenic mice that overexpress mLRP2 (14). The mLRP2 section or minireceptor of LRP behaves similarly in vitro to full-length LRP with respect to ligand binding and internalization (14). Mice overexpressing mLRP2 were then bred to the well characterized PDAPP mouse model of AD, which expresses human APP with a mutation that causes familial AD (V717F) and develops amyloid plaques in the brain beginning at ~6 months of age (15). We found that 22-month-old PDAPP/mLRP2 and PDAPP/wild-type mice had similar levels of total amyloid deposition in their brains but that PDAPP/mLRP2 mice had increased levels of Abeta in carbonate-soluble brain extracts (14). Furthermore, this carbonate-soluble Abeta pool was highly correlated with memory deficits in old mice.

In the current study, we examined whether overexpression of mLRP2 also affects Abeta levels in 3-month-old mice that have not yet developed amyloid plaques. We hypothesized that changes in Abeta at this age are more likely to reflect subtle changes in Abeta metabolism resulting from mLRP2 overexpression without the confounding factor of severe pathology as in the older mice. Based on our previous work (14), we expected that overexpression of LRP would increase membrane-associated Abeta. Additionally, we investigated whether membrane-associated Abeta could represent intracellular Abeta.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—The mLRP2 transgene was expressed by the MoPrP.Xho vector as previously described (14). mLRP2 mice were crossed to PDAPP+/- mice (15) to produce PDAPP/mLRP2 and PDAPP/wild-type mice. Apoe-/- mice obtained from the Jackson Laboratory (Bar Harbor, ME) were crossed with PDAPP mice to produce PDAPP/Apoe+/+ and PDAPP/Apoe-/- mice. All the mice used for this study were on a C57Bl/6 genetic background. Mice were screened for the presence of the mLRP2 and PDAPP transgenes by PCR. Tissues were obtained following transcardial perfusion with ice-cold PBS (14). The right hemisphere was immersion fixed for 24 h in 4% paraformaldehyde and then cryoprotected in 30% sucrose in PBS for histological analysis. The left hemisphere was further dissected, and brain regions were frozen for biochemical analysis. Cerebrospinal fluid (CSF) was isolated from the cisterna magna as previously described (16).

Cells—PC12 cell were transfected with the pcDNA vector only or the pcDNA vector encoding mLRP2 cDNA tagged with the hemagglutinin (HA) epitope. Stably expressing clones were selected based on cell morphology and mLRP2 expression levels, which were evaluated by Western blotting and immunofluorescent staining with anti-HA antibody. High expressing lines were maintained in RPMI 1640 containing 10% horse serum, 5% fetal calf serum, 1% glutamine, 1% penicillin, 1% streptomycin, and 400 µg/ml G418.

Sequential Brain Extraction—Hippocampi were homogenized with 25 strokes in 15 volumes of cold Tris-buffered saline (TBS), pH 7.4, containing the protease inhibitors leupeptin (10 µg/ml) and aprotinin (20 µg/ml) using a 1-ml dounce homogenizer. Homogenates were transferred to microcentrifuge tubes and spun at 20,000 x g for 20 min at 4 °C. Supernatants (TBS extracts) were transferred to new tubes and kept on ice. After a single wash with 50 µl of cold TBS, pellets were resuspended in 15 volumes of cold TBS, pH 7.4, containing 1% Triton X-100 and protease inhibitors and incubated for 30 min at 4 °C with agitation. The homogenates were spun again at 20,000 x g for 20 min at 4 °C. Supernatants (TBS-Triton X-100 extracts) were transferred to new tubes and kept on ice. Pellets were spun once for 5 min, and all leftover supernatant was removed. 400 µl of 5 M guanidine solution containing protease inhibitors were added to the final pellet. Samples were vortexed to detach the pellet from the bottom of the tubes and incubated for 4 h at room temperature. The homogenates were spun again at 20,000 x g for 20 min at 4 °C. The supernatants (guanidine extracts) were transferred to new tubes and kept on ice.

Abeta40 and Abeta42 Determinations—Human Abeta40 and Abeta42 levels were determined in all hippocampal extracts (TBS, TBS-Triton X-100, and guanidine) and CSF by sandwich ELISA under denaturing conditions (0.5 M guanidine). The capturing antibody was 2G3 for Abeta40 and 21F12 for Abeta42, and the detection antibody was biotinylated 3D6 as previously described (14). Samples were diluted 10-fold in PBS containing 0.25% bovine serum albumin, and human Abeta40 and Abeta42 standards were also solubilized in PBS containing 0.25% bovine serum albumin as well as 10% of the buffer used to obtain each sample (TBS, TBS-Triton X-100, or 5 M guanidine).

Mouse ApoE Determination—A previously described sandwich ELISA for mouse apoE with a sensitivity of ~1 ng/ml was used (17). Brain samples were sonicated in PBS containing 0.05% Tween 20 and protease inhibitors. Samples were spun to pellet cell debris (20,000 x g for 25 min at 4 °C), and supernatants were diluted in PBS containing 0.025% Tween 20 and 0.5% bovine serum albumin. Standards were based on pooled plasma from C57/Bl6 mice containing 68 µg/ml apoE, and brain samples from apoE knock-out mice were used for background subtraction.

Western Blot Analysis—Hippocampi were dounce homogenized in 15 volumes of TBS containing 1% Triton X-100 with the protease inhibitors leupeptin (10 µg/ml) and aprotinin (20 µg/ml). Equal amounts of protein from the tissue lysates were separated using 10% Tris-Tricine gels and transferred to polyvinylidene difluoride membranes. Membranes were blotted with anti-APP antibody (Zymed Laboratories, San Francisco, CA), and immunoreactive bands were detected using ECL (Amersham Biosciences).

Immunostaining—Right hemispheres were cut in the coronal plane on a freezing sliding microtome. Floating brain sections (50 µm) were blocked with 10% normal goat serum (Vector Laboratories, Burlingame, CA) in TBS containing 0.25% Triton X-100 for 45 min. The sections were then incubated overnight at 4 °C with a polyclonal antibody to Abeta42 (Chemicon International, Temecula, CA) and a monoclonal antibody to neuron-specific nuclear protein (NeuN) (Chemicon International), both at 1:500 dilution in TBS containing 0.25% Triton X-100 and 2% normal goat serum. Secondary antibodies AlexaFluor 488 goat anti-rabbit IgG and AlexaFluor 568 goat anti-mouse IgG (Molecular Probes, Eugene, OR) were diluted 1:1000 for detection. Fluorescence was visualized by confocal laser scanning microscopy. A mouse-specific rat antibody to LAMP-1 (1D4B) was used as hybridoma tissue culture supernatant (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) at 1:4 dilution with secondary antibody Alexa-Fluor 568 goat anti-rat IgG at 1:1000 dilution (Molecular Probes) for detection. Quantitation of Abeta42 immunoreactivity associated with neurons from PDAPP/Apoe+/+ and PDAPP/Apoe-/- mice was performed using the ImageJ software from the National Institutes of Health (Bethesda, MD). Briefly, each of the images obtained under identical settings by confocal microscopy using x40 magnification was split into RGB channels (red, green, and blue) and a total of 25 neurons/section were randomly circled in the red channel (NeuN). The intensity of fluorescence signal associated with each neuron was then calculated by redirecting measurements to the green channel (Abeta42).

In Vitro Abeta Clearance—In vitro Abeta clearance experiments were performed using PC12 cells stably transfected with mLRP2 or the pcDNA vector. PC12 cells were seeded in 24-well plates at a density of ~300,000 cells/well and allowed to attach overnight. Serum-containing medium was then replaced with neurobasal medium containing N2 supplement and nerve growth factor (100 ng/ml) to stimulate differentiation overnight. Cell-secreted Abeta was obtained from PDAPP primary neuronal cultures that contained ~60% Abeta42. This high Abeta42: Abeta40 ratio is typical of Abeta produced by PDAPP mice (18, 19). The cell-secreted Abeta was diluted 10-fold in neurobasal medium plus B27 and nerve growth factor and was added to the differentiated PC12 cells (final Abeta concentration ~500 pg/ml). In some conditions, diluted Abeta-containing medium was preincubated for 2 h at 37°C with apoE particles (10 µg/ml) derived from human embryonic kidney cells stably transfected with human apoE3 or apoE4 (12, 20), and then the apoE- and Abeta-containing medium was added to the cells. In other conditions, cells were pretreated for 15 min with 0.5 µM receptor-associated protein (RAP), a LRP antagonist (21), prior to the addition of Abeta-conditioned medium preincubated with apoE3 particles. After 24 h, Abeta levels in the incubation medium were quantified by ELISA and compared with the values of the original Abeta medium, kept at 37 °C. The difference in Abeta levels was then divided by cell protein content in each well (n = 3).


Figure 1
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  		FIGURE 1.
mLRP2 overexpression increases Abeta42 in detergent- and guanidine-soluble extracts from the hippocampus of 3-month-old PDAPP mice. A, levels of APP in brain extracts made with carbonate and with TBS containing 1% Triton X were similar and were not affected by overexpression of mLRP2. Equal amounts of total protein from hippocampal extracts made with carbonate and TBS with 1% Triton X-100 from young 3-month-old PDAPP/wild-type (n = 2) and PDAPP/mLRP2 mice (n = 2) were separated by SDS-PAGE electrophoresis followed by Western blotting with an antibody to APP. B-D, the hippocampus underwent a three-step serial extraction with TBS, TBS with 1% Triton X-100, and 5 M guanidine. Abeta40 and Abeta42 levels were determined by ELISA. For both the PDAPP/mLRP2 and PDAPP/wild-type groups, n = 12 mice. E, CSF was obtained from the cisterna magna and assayed for Abeta40 and Abeta42 by ELISA. n = 5 PDAPP/mLRP2 and PDAPP/wild-type mice. Results are shown as mean ± S.E.; statistical analysis was by Student's two-tailed t test.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We have previously shown that overexpression of mLRP2 significantly increases a carbonate-soluble Abeta pool in the hippocampus and cortex of 22-month-old PDAPP mice with abundant plaque deposition (14). In the current study, we examined whether overexpression of mLRP2 affects Abeta levels in 3-month-old PDAPP mice that have not yet developed amyloid plaques in the brain.

To examine the different pools of Abeta, we performed sequential extraction of the brain tissue. Previously, we examined carbonate (soluble) and guanidine (insoluble) extracts from brain. For these experiments, we added an extra step into our protocol so that we could separate the soluble extract into a water-soluble extract with few membrane-associated proteins and a detergent-soluble extract enriched for membrane-associated proteins. Instead of carbonate, we used TBS buffer containing 1% Triton X-100 because this buffer is more widely used to extract membrane proteins and yields results similar to those obtained with carbonate. APP, as well as other membrane proteins such as transferrin, PS1, and calnexin, were extracted by both carbonate and TBS with 1% Triton X-100 (Fig. 1A and data not shown). The hippocampus was first homogenized in TBS and centrifuged, which provided a supernatant with water-soluble proteins that contained almost none of the intramembrane protein APP (Fig. 1A). Next, the pellet from the TBS extraction was rehomogenized in TBS with 1% Triton X-100 and centrifuged, resulting in a supernatant containing detergent-soluble, mainly membrane-associated proteins such as APP (Fig. 1A). Finally, the pellet was homogenized again in 5 M guanidine and centrifuged, allowing extraction of relatively insoluble proteins. The TBS, TBS-Triton X-100, and guanidine extracts from the brain were then assayed for Abeta40 and Abeta42 by ELISA.

We found that overexpressing mLRP2 in PDAPP mice does not significantly affect levels of Abeta40 in any of the three different extracts from the hippocampus (Fig. 1, B-D). TBS-soluble Abeta42 was also not different in PDAPP/mLRP2 and PDAPP/wild-type mice (Fig. 1B). However, detergent- and guanidine-soluble Abeta42 was significantly higher in PDAPP mice overexpressing mLRP2 compared with non-transgenic littermates. Specifically, detergent-soluble Abeta42 levels were increased by ~20% in PDAPP/mLRP2 mice, and guanidine-soluble Abeta42 levels were increased by ~12% in PDAPP/mLRP2 mice compared with PDAPP/wild-type control mice (Fig. 1, C and D). The selective increase of detergent- and guanidine-soluble Abeta42 suggests that mLRP2 affects Abeta42 that is associated with membranes and not freely soluble Abeta42. Although these effects are small, they demonstrate the involvement of LRP in regulating brain Abeta levels.


Figure 2
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  		FIGURE 2.
Significant Abeta42 immunoreactivity was associated with neurons in 3-month-old PDAPP mice prior to plaque deposition. Brain sections (50 µm) were stained with a polyclonal anti-Abeta42 antibody and monoclonal anti-neuron-specific nuclear protein (NeuN) antibody, followed by secondary detection with goat anti-rabbit AlexaFluor 488 and goat anti-mouse AlexaFluor 568, respectively. Immunofluorescence was detected by confocal microscopy. A, Abeta42 immunoreactivity (green) was associated with neurons (red) from the CA1 hippocampal region. B, Abeta42-immunoreactive neurons were also evident within the frontal cortex region. C, neuronal-associated immunostaining for Abeta42 (green) was largely co-localized (yellow) with the lysosomal marker LAMP-1 (red). D, preincubation of anti-Abeta42 antibody with 200-fold excess molar concentration of Abeta42 peptide completely blocked neuronal-associated Abeta42 immunoreactivity.

 
Western blotting of APP showed no differences between PDAPP/mLRP2 and PDAPP/wild-type mice (Fig. 1A). We also examined the levels of CTF-beta, a fragment of APP produced by beta-secretase cleavage of APP that is cleaved by {gamma}-secretase to form Abeta. CTF-beta levels were not different in PDAPP/mLRP2 and PDAPP/wild-type mice by Western blotting (data not shown). These data suggest that APP expression and APP processing is not drastically altered by mLRP2 overexpression, although it does not rule out the possibility that subtle changes in APP expression or APP processing are occurring that affect Abeta levels.

Interestingly, although Abeta42 levels in the brain significantly increased in the PDAPP/mLRP2 overexpressing mice, total Abeta was significantly decreased in the CSF of mLRP2 versus wild-type mice, with a trend toward decreased Abeta40 and Abeta42 (Fig. 1E). The increased amount of Abeta42 in the brain tissue of the PDAPP/mLRP2 mice, coupled with the decreased amount of Abeta in the CSF of the PDAPP/mLRP2 mice, suggests that mLRP2 overexpression increases the internalization of extracellular Abeta. These findings are consistent with the hypothesis that mLRP2 binds and internalizes Abeta, either directly or via endocytosis of apoE-Abeta complexes, and that mLRP2 overexpression increases the internalization of Abeta42.

To further examine the mechanism by which Abeta42 levels are altered in young PDAPP mice, we visualized and quantified intracellular Abeta using immunohistochemical methods. We observed significant Abeta42 associated with neurons in the CA1 region of the hippocampus and frontal cortex (Fig. 2, A and B). Double labeling of Abeta42 and LAMP1 revealed that much of the Abeta42 is localized to the lysosomes within neurons (Fig. 2C). Double labeling of Abeta42 and the transferrin receptor (an endosomal marker) or BiP (an endoplasmic reticulum marker) did not show any significant co-localization (data not shown). Importantly, the anti-Abeta42 antibody showed no staining when preincubated with an excess of Abeta42 peptide (Fig. 2D), supporting the conclusion that we were staining Abeta42. Additionally, the anti-Abeta42 antibody did not stain brain sections from wild-type mice that do not express human Abeta (data not shown). Furthermore, previous studies have shown that neither APP nor CTF-beta are significantly changed in Apoe-/- mice when compared with wild-type mice, further supporting our conclusion that we were staining Abeta42 and not APP or CTF-beta (19).


Figure 3
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  		FIGURE 3.
Neuron-associated Abeta42 immunoreactivity was significantly decreased in PDAPP mice lacking apoE. A, mice overexpressing mLRP2 had decreased levels of apoE in the cortex as measured by ELISA. n = 5 mLRP2 and wild-type mice. C, brain sections (50 µm) from 6-month-old PDAPP/Apoe-/- and PDAPP/Apoe+/+ mice were stained with a polyclonal anti-Abeta42 antibody and a monoclonal anti-neuron-specific nuclear protein (NeuN) antibody followed by secondary detection with goat anti-rabbit AlexaFluor 488 and goat anti-mouse AlexaFluor 568, respectively. Immunofluorescence was detected by confocal microscopy at x40 magnification. Abeta42 immunoreactivity (green) associated with neurons (red)in the frontal cortex region was markedly reduced in PDAPP mice lacking apoE. B, quantitation of Abeta42-associated fluorescent intensity in the frontal cortical region showed a 54% significant reduction in neuronal-associated Abeta42 immunoreactivity in PDAPP/Apoe-/- (n = 4) compared with PDAPP/Apoe+/+ mice (n = 5). Results are shown as mean ± S.E.; statistical analysis was by Student's two-tailed t test.

 
Because overexpression of mLRP2 is likely to affect levels of ligands such as apoE, and apoE levels are known to affect Abeta metabolism, we determined the apoE levels in mLRP2 and wild-type mice. We found that apoE levels were 25% lower in mice overexpressing mLRP2 (Fig. 3A). This is consistent with the known role of LRP as an apoE receptor that facilitates internalization and degradation of apoE. Furthermore, we examined whether the presence of the intraneuronal Abeta42 we previously observed (Fig. 2) depended on apoE. We found that PDAPP/Apoe-/- mice had ~50% less intraneuronal Abeta42 (Fig. 3, B and C). This demonstrates that apoE plays a major role in the uptake of Abeta42 by neurons, possibly by forming apoE-Abeta complexes that bind to LRP and other receptors and are endocytosed.


Figure 4
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  		FIGURE 4.
Overexpression of mLRP2 in PC12 cells increased the clearance of Abeta-apoE complexes in vitro. A, clearance of naturally secreted Abeta42 (~300 pg/ml) by PC12 cells stably transfected with mLRP2 was significantly greater than by PC12 cells transfected with the pcDNA vector only. Abeta42 clearance was enhanced by preincubation of the Abeta-containing medium with apoE3 or apoE4 particles (10 µg/ml). Preincubation of cells with LRP antagonist RAP (0.5 µM) significantly reduced Abeta42 clearance mediated by apoE3 particles. B, clearance of naturally secreted Abeta40 (~200 pg/ml) by PC12 cells was slower than clearance of Abeta42 and was moderately increased by overexpression of mLRP2. Preincubation of Abeta-containing medium with apoE3 did not affect clearance of Abeta40. For each group, n = 3. Results are shown as mean ± S.E.; statistical analysis was by one-way analysis of variance with Newman-Keuls multiple comparison post-hoc testing.

 
To directly address the question of whether mLRP2 overexpression affects intraneuronal Abeta42 levels, we examined whether PDAPP/mLRP2 and PDAPP/wild-type mice had different levels of intraneuronal Abeta42 staining. We did not see any significant differences in staining (data not shown). However, the sensitivity of this technique is relatively low and we were expecting only a small difference, so we performed another experiment to answer this question. We stably transfected PC12 cells with mLRP2 or the vector only and induced differentiation of the PC12 cells with nerve growth factor. We then added Abeta secreted by primary neurons derived from PDAPP mice. PC12 cells overexpressing mLRP2 cleared significantly more Abeta42 than cells expressing the vector only (Fig. 4A). Similar results were obtained when preparations of oligomeric Abeta42 were added to the cells (data not shown). Clearance of Abeta42 by mLRP2-overexpressing cells was increased by preincubation of Abeta with apoE3 or apoE4 particles (Fig. 4A). It is not surprising that Abeta42 preincubated with apoE3 or apoE4 had a similar rate of clearance because apoE3 and apoE4 have similar binding properties to LRP (22, 23). The addition of RAP partially blocked clearance of Abeta42 preincubated with apoE3 (Fig. 4A). We also examined the clearance of Abeta40 and found that it was much slower than Abeta42 clearance (Fig. 4B). Abeta40 clearance was increased by the overexpression of mLRP2, but to a lesser extent than Abeta42 (54% increase in clearance for Abeta40, 80% increase for Abeta42). Additionally, clearance of Abeta40 was not affected by preincubation with apoE3 particles (Fig. 4B). This suggests that both Abeta42 and Abeta40 are cleared by an LRP-mediated process but that apoE plays a larger role in clearance of Abeta42. Overall, these data support two conclusions. 1) mLRP2 overexpression increases the internalization of Abeta42, even in the absence of apoE. This suggests that Abeta42 binds directly to mLRP2 and undergoes endocytosis. 2) ApoE enhances the clearance of Abeta42 via mLRP2. We hypothesize that Abeta binds to apoE and forms an apoE-Abeta complex, which then binds to mLRP2 and undergoes endocytosis. However, while the endocytosed apoE is rapidly degraded, a portion of the Abeta42 accumulates and possibly aggregates inside lysosomes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our current study demonstrates that overexpression of mLRP2 increases detergent- and guanidine-soluble Abeta42 in the hippocampus of 3-month-old PDAPP mice that have not yet developed amyloid plaques. We also found significantly decreased total Abeta in the CSF of PDAPP/mLRP2 versus PDAPP/wild-type mice, with a trend toward decreases in both Abeta40 and Abeta42. The increase in cellular Abeta42 and decrease in extracellular Abeta suggest that Abeta42 may be endocytosed more rapidly by mLRP2 overexpressing cells and may accumulate intracellularly. To test this hypothesis, we developed a protocol for staining Abeta42 associated with neurons. Using this protocol, we found significant amounts of intraneuronal Abeta42 in 3-month-old PDAPP mice, most of which was localized inside lysosomes. Interestingly, we found that the levels of intraneuronal Abeta42 were dramatically lower in PDAPP mice lacking apoE. Because mLRP2 mice have slightly higher levels of membrane-associated Abeta42, we expected PDAPP/mLRP2 mice to have higher levels of intraneuronal Abeta42 than PDAPP/wild-type mice. However, we were not able to detect a significant difference in intraneuronal Abeta42 between PDAPP/mLRP2 and PDAPP/wild-type animals, probably because the protocol was not sensitive enough to detect a small effect. To study whether mLRP2 affects Abeta42 clearance in another system, we made PC12 cell lines that were stably transfected with mLRP2 or vector only. We found that the mLRP2-overexpressing cells cleared significantly more Abeta42 from the medium than cells expressing the vector only. In addition, preincubation of the Abeta-containing medium with apoE3 or apoE4 particles increased the clearance of Abeta42 by the cells, and this effect was partially blocked by addition of RAP.

Our initial hypothesis based on previous studies was that Abeta in detergent-soluble extracts from the hippocampus of 3-month-old PDAPP/mLRP2 mice would be higher than in PDAPP/wild-type mice. This was essentially correct, but the increase in Abeta was only in the more amyloidogenic Abeta42 species and only in the detergent- and guanidine-soluble extracts. We speculate that endocytosed Abeta42 may not be degraded as effectively as Abeta40 in lysosomes. The finding that only detergent- and guanidine-soluble Abeta42 was increased in the PDAPP/mLRP2 mice suggests that mLRP2 is specifically affecting a pool of Abeta42 that is membrane bound or insoluble. We did not specifically test whether the Abeta42 that required guanidine for extraction was in an insoluble, oligomeric state or bound to membrane domains that are poorly soluble in 1% Triton X-100. However, other investigators have found that intraneuronal Abeta42 deposits are reactive to antibodies specific for Abeta42 oligomers (24).

The increase of Abeta42 in brain tissue and decrease in Abeta in CSF suggested that the ratio of intracellular to extracellular Abeta was increasing, perhaps as a result of intracellular Abeta42 accumulation. We investigated this possibility using immunohistochemistry to visualize intracellular Abeta42. The accumulation of Abeta inside neurons has been observed in both AD patients and mice with brain amyloid deposition (25-29). Intraneuronal Abeta accumulation appears to occur prior to extracellular amyloid deposition and is a prominent neuropathological feature in brain regions that are vulnerable in AD, such as the frontal cortex and the hippocampus. In a triple transgenic AD mouse model with the PS1 (M146V), APP (Swe), and tau (P301L) transgenes, deficits in synaptic transmission and long term potentiation were observed before plaque and tau pathology, but in the presence of early intraneuronal Abeta accumulation (29). It has been suggested that intraneuronal Abeta pathology causes the onset of early cognitive deficits in this triple transgenic AD mouse model (30). In other experiments, it has been shown that intracellular Abeta accumulation directly affects cell viability, ultimately resulting in neuronal death (31-34). These observations raise the hypothesis that intraneuronal Abeta accumulation may be one of the initial steps in a cascade of events leading to AD (35).

We found that intraneuronal Abeta42 co-localized with the lysosomal marker LAMP-1. Previous studies have shown that intraneuronal Abeta is present within endosomes, multivesicular bodies, and lysosomes (24, 28, 36-38). If Abeta42 is concentrated within small organelles such as lysosomes, this could favor the aggregation of Abeta42. Additionally, the acidic pH characteristic of endocytic compartments may provide a favorable environment for Abeta oligomerization (39, 40). Interestingly, early abnormalities in the endosomal-lysosomal system that precede amyloid deposition have been reported in sporadic AD and Down syndrome (36, 41). Whether the Abeta42 that accumulates within the endocytic compartments of neurons results from extracellular uptake of Abeta42 or intracellular generation of Abeta42 is not yet clear. However, chronic infusion of synthetic Abeta42 oligomers with transforming growth factor beta-2 into the ventricles of wild-type mice results in intraneuronal Abeta42 accumulation, suggesting that extracellular uptake of Abeta42 may result in intraneuronal Abeta42 accumulation. RAP markedly reduces this accumulation, which indicates that LRP plays a role in this process (42).

The finding that PDAPP mice lacking apoE have markedly reduced levels of intraneuronal Abeta42 demonstrates that apoE plays an important role in the neuronal uptake and/or accumulation of Abeta42. Although it has previously been shown that PDAPP/Apoe-/- mice have reductions in both total Abeta and fibrillar Abeta, these studies were performed with older mice with prominent Abeta deposition (43-45). In contrast, our studies were performed in 3-month-old mice prior to plaque deposition and therefore suggest that Abeta42 begins to accumulate inside neurons even before plaques are deposited in tissues. Additionally, our in vitro results demonstrate that apoE can increase the uptake of Abeta42 via binding to LRP and perhaps other members of the low density lipoprotein receptor family. This supports the hypothesis that extracellular uptake of apoE-Abeta complexes can lead to intraneuronal Abeta42 accumulation. However, although increased endocytosis of apoE-Abeta complexes likely leads to lower brain apoE levels, such as we found in mLRP2 mice, Abeta42 is not degraded completely following endocytosis and accumulates.

Although our current results support a role of LRP in intraneuronal Abeta accumulation, our studies were largely performed with a LRP minireceptor, mLRP2. The extremely large size of LRP (~600 kDa) makes it technically challenging to overexpress the full-length receptor either in transgenic animals or stable cell lines. Because our previous studies have confirmed the functionality of mLRP2 (46), we believe that mLRP2 is an appropriate tool for studying ligand binding to full-length LRP. Future studies should be directed to studying the function of endogenous LRP in intraneuronal Abeta accumulation by conditional knock out or virally mediated gene knock-down approaches.

In summary, the hypothesis that Abeta clearance by neurons is a protective mechanism against AD needs to be reconsidered. Provided that all internalized Abeta is efficiently degraded within lysosomes, this pathway may indeed be protective. However, our present and previous published studies suggest that this may not be the case and that intraneuronal Abeta accumulation may be pathogenic. Furthermore, reducing the endocytosis of Abeta by neurons may have a therapeutic effect on AD.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants F32-NS41872 (to C. V. Z.) and R01-AG027924 (to G. B.) and a grant from the Alzheimer Association (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Campus Box 8208, 660 S. Euclid Ave., Saint Louis, MO 63110. Tel.: 314-286-2871; Fax: 314-286-2894; E-mail: bu{at}wustl.edu.

2 The abbreviations used are: LRP, low density lipoprotein receptor-related protein; mLRP2, functional LRP minireceptor; Abeta, amyloid-beta peptide; AD, Alzheimer disease; APP, amyloid precursor protein; apoE, apolipoprotein E; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RAP, receptor-associated protein; TBS, Tris-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Hong Jiang and Wenyan Lu for technical assistance, Dr. Mary Jo LaDu (University of Illinois at Chicago) for providing the human embryonic kidney-apoE3 particles, Dr. David Borchelt (Johns Hopkins University) for providing the MoPrP.Xho vector, and Dr. David Sibley (Washington University) for providing the anti-LAMP-1 antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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