Sporadic, late onset AD is the most common form of the disease and the only established genetic risk factor is possession of one or more ApoE4 alleles (Roses, 1996
; Tanzi and Bertram, 2005
). It remains unclear how ApoE participates in disease pathogenesis. ApoE is implicated as a critical regulator of the propensity of Aβ to be deposited within the brain. Examination of APP expressing mice lacking the murine Apoe
gene failed to develop compact amyloid plaques, but exhibited elevated levels of Aβ peptides within the brain (Bales et al., 1997
). Strikingly, when human ApoE isoforms were expressed in these animals there was a delay in the onset of plaque deposition and a significant reduction in plaque burden that was isoform-specific (E2>E3>E4) and gene dose-dependent (DeMattos et al., 2004
). These data suggested that ApoE influenced Aβ deposition and promoted Aβ clearance, with the ApoE4 isoform being less effective than other isoforms in facilitating removal of these peptides from the brain.
Age-related impairment of Aβ homeostatic mechanisms has been postulated to be a critical determinant of disease risk. Aβ peptides are normally generated at high levels in the brain (approximately 8%/hour) and are cleared at an equivalent rate in both humans and mice (Bateman et al., 2006
). Thus, even modest reductions in clearance of soluble Aβ could result in elevated levels of Aβ peptides and ultimately their deposition within the brain. The clearance of Aβ peptides from the brain is accomplished by their proteolytic degradation within the brain and by their efflux through the blood-brain barrier (Tanzi et al., 2004
). ApoE has been postulated to facilitate Aβ clearance across the blood—brain barrier, owing to its ability to form complexes with Aβ and to be exported to the peripheral circulation via LRP1 in vascular endothelial cells (Deane et al., 2004
). However, a recent quantitative analysis has revealed that ApoE:Aβ complexes are inefficiently cleared through this mechanism and lipidated forms of ApoE are not significantly trafficked by this process (Bell et al., 2007
). These finding suggest that ApoE promotes Aβ clearance from the brain principally through intrinsic proteolytic mechanisms. The physiological importance of intrinsic Aβ proteolysis has been demonstrated in vivo.
Mice in which Nep
have been genetically inactivated exhibit higher Aβ levels and enhanced plaque deposition in the brain (Farris et al., 2003
; Farris et al., 2007
; Iwata et al., 2001
The present study documents a novel mechanism through which ApoE stimulates the degradation of soluble Aβ peptides within the brain. We show that ApoE facilitates the proteolytic degradation of soluble Aβ, both within microglia and in the extracellular milieu, through the action of two distinct classes of proteinases. Significantly, the proteolysis of Aβ is dramatically enhanced in the presence of lipidated apolipoproteins (). The transfer of lipids to ApoE is accomplished principally by ABCA1 and several recent studies have provided direct evidence for the functional importance of this process in Aβ homeostasis. Three independent studies in different hAPP mice lacking the Abca1
gene, have demonstrated that the absence of Abca1
results in low levels of poorly-lipidated ApoE in the brain. Importantly, Abca1
inactivation did not affect APP processing or Aβ production. However, these animals exhibited much greater Aβ plaque loads and higher levels of insoluble forms of ApoE, reflective of the codeposition of these molecules (Hirsch-Reinshagen et al., 2005
; Koldamova et al., 2005a
; Wahrle et al., 2005
). Thus, the dominant effect of Abca1
inactivation in vivo
is to impair Aβ clearance, without a significant impact on Aβ synthesis.
ApoE and its lipidation status regulates the proteolytic degradation of soluble Aβ
We demonstrate that the proteolytic degradation of Aβ is stimulated by LXR agonist treatment and is reliant upon the LXR target genes, Apoe
ApoE secreted from wild type glia is fully lipidated (Hirsch-Reinshagen et al., 2004
; Wahrle et al., 2004
) and enables soluble Aβ to be efficiently degraded by proteinases. Our findings are consistent with the previous report that Aβ present within lipidated ApoE:Aβ complexes isolated from human brains was more susceptible to proteolytic degradation than with purified ApoE, which is poorly lipidated (Russo et al., 1998
). We favor a model in which ApoE interacts with Aβ acting to chaperone its proteolysis. However, we cannot exclude the possibility that enhanced intracellular proteolytic degradation of Aβ might arise from HDL-mediated changes in cellular membrane lipid composition, as Abca1
null microglia retain a partial response to the LXR agonist GW3965. Indeed, LXR has been demonstrated to induce the expression of other cholesterol transporters and apolipoproteins involved in HDL metabolism such as Abcg1
, respectively (Zelcer et al., 2007
). The ApoE-dependent soluble Aβ clearance mechanisms reported here appear to be distinct from those that occur in adult astrocytes which involve the ApoE receptor LRP1 and deposited forms of Aβ (Koistinaho et al., 2004
Much of the initial interest in LXR action centered on the roles of these receptors and ABCA1 on neuronal APP processing and Aβ generation. LXR activation in cell culture models yielded conflicting data and was shown to either increase (Fukumoto et al., 2002
) or suppress Aβ production (Burns et al., 2006
; Koldamova et al., 2003
; Sun et al., 2003
). Subsequent studies in mice have uniformly found that LXR agonist treatment of wild type (Burns et al., 2006
) or young APP-expressing mice (Koldamova et al., 2005b
; Lefterov et al., 2007
; Riddell et al., 2007
) resulted in a significant decrease in brain Aβ levels, and we report similar results in aged Tg2576 mice treated with GW3965. Moreover, we have recently reported that hAPP mice in which either Lxrα
have been knocked out exhibit a significant increase in Aβ plaque pathology (Zelcer et al., 2007
), consistent with the view that LXR activation facilitates Aβ clearance from the brain. These recent studies also found that LXRs inhibited the microglia-mediated inflammatory response and inflammatory gene expression, owing to their ability to functionally inactivate the promoters of proinflammatory genes (Zelcer and Tontonoz, 2006
). We have reproduced this finding in aged Tg2576 mice with existing plaque pathology. Thus, LXRs can act to ameliorate AD pathogenesis through their action on both Aβ clearance and suppression of the plaque-related inflammatory response.
The behavioral impairment associated with overexpression of hAPP has been postulated to be due to elevated levels of soluble forms of Aβ within the brain (Comery et al., 2005
; Riddell et al., 2007
; Walsh and Selkoe, 2004
). We found that treatment of Tg2576 mice with the LXR agonist GW3965 resulted in a dramatic improvement in contextual memory and this finding, too, is consistent with the LXR-stimulated Aβ clearance.
In summary, we provide data documenting a previously unappreciated action of ApoE in facilitating the proteolytic clearance of Aβ peptides from the brain. We postulate that ApoE acts both within microglia and in the extracellular space to affect the clearance of Aβ through promoting its proteolysis by at least two distinct classes of proteinases. Importantly, the ApoE4 isoform, which is associated with increased risk for AD, exhibits an impaired ability to promote Aβ proteolysis compared to the ApoE2 and ApoE3 isoforms. There is a remarkable correspondence in the effects of LXR activation and Abca1
inactivation (or overexpression) in animal models of AD that support an essential role for the lipidation of ApoE in removing soluble Aβ from the brain. The present study provides a mechanistic explanation of both the effects of Abca1
inactivation (or overexpression (Wahrle et al., 2008
)) and the Lxrα
knockouts (Zelcer et al., 2007
) on Aβ plaque pathology in animal models of AD. Our data suggests that therapeutic agents which increase the levels of lipidated forms of ApoE, including LXR agonists, represent a new and potentially efficacious therapy for AD.
Tg2576 mice or wild type littermates (5 animals/group), 12 months of age, were fed the AIN-76A standard rodent diet alone or containing GW3965 (120 mg/kg; 33 mg/kg/day) ad libitum for 4 months. The animals were sacrificed, and the right hemispheres were fixed and processed for immunohistochemical analysis. The left hemispheres were snap-frozen on dry ice and subject to serial extraction of total RNA, DNA and protein. Wild type mice (C57BL/6), Apoe null mice (B6129P2-Apoetm1Unc/J) and Abca1 heterozygote mice (DBA/1-Abca1tm1Jdm/J) were obtained from Jackson Laboratory (Bar Harbor, ME). Abca1+/+, +/− and −/− mice were bred from Abca1 heterozygote mice (DBA/1-Abca1tm1Jdm/J). Tg2576 mice were maintained by crossing to B6SJLF1/J animals.
BV2 microglia were maintained in DMEM containing 2% FBS. Primary microglia and astrocytes were prepared from P0–P3 mice and purified microglia and astrocyte cultures were obtained as previously described (Koenigsknecht and Landreth, 2004
; Zander et al., 2002
). Immortalized astrocytes derived from human ApoE knockin mice (Morikawa et al., 2005
) were maintained in DMEM containing 10% FBS, 500 µg/ml G418 and 1 mM sodium pyruvate.
Preparation of Aβ peptides
lyophilized powder (American Peptide Company, Sunnyvale, CA) was dissolved to a final concentration of 1 mg/mL in DMSO and stored at −80°C until use. Fluorescently labeled peptides were prepared by first dissolving the peptides to a concentration of 2 mmol/L in sterile H2
O. The Aβ42
was then labeled with Cy3 (Amersham Biosciences, Pittsburgh, PA) or Alexa488 fluorophores (Invitrogen, Carlsbad, CA) using manufacturer’s protocol. The Aβ42
reaction mixture was allowed to fibrillize at 37°C overnight after which unincorporated dyes were removed by ultracentrifugation at 4°C. The pellet was then resuspended in DMSO, sonicated and ultracentrifuged. This was subsequently repeated until the fAβ was solubilized in DMSO. The supernatant contains the operationally defined “soluble Aβ” which exhibits an electrophoretic mobility corresponding to 4 kDa on SDS-PAGE and is predicted to consist of primarily monomeric and small oligomeric species (Shen and Murphy, 1995
Western Blot analysis
Protein concentrations of cell lysates or brain extracts were measured using the BCA method (Pierce, Rockford, IL). For Western blot analysis of Aβ, 10–20% Tricine gels or 4–15% Bis-Tris gels (Invitrogen, Carlsbad, CA) were used. The following primary antibodies were used: anti-human Aβ, 6E10 (Covance, Dedham, MA); anti-ApoE (Calbiochem, San Diego, CA); anti-β-actin; anti-β-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA); anti-ABCA1 (Novus Biologicals, Littleton, CO); anti-APP CTF (Millipore, Billerica, MA).
Immunohistochemistry and image analysis
Post-fixed hemispheres were sectioned sagittally into 10 µm sections using a cryostat. Sections were mounted, air-dried, and then stored at 4°C until use. For Aβ immunohistochemistry, sections (3 per mouse, about 1.2 mm–1.5mm from the midline, spaced 0.1 mm apart from each other) were incubated in 70% formic acid for 3 min and the endogenous tissue peroxidase activity was quenched by incubation with 1% peroxide in methanol for 10 min. Sections were then microwaved in distilled water for 3 min, and then incubated with blocking solution (5% normal goat serum and 0.1% Triton X-100 in PBS) for 1 hour. Sections were incubated with primary antibody in the blocking solution overnight at 4°C. The antigens were detected by secondary antibodies using standard ABC-DAB methods. Sections were counterstained with hematoxylin. The 6E10 antibody against human Aβ was used to stain Aβ plaques. Images were analyzed using Image Pro-Plus software (Media Cybernetics, Silver Spring, MD).
Aβ40 and Aβ42 ELISAs were performed using commercial kits (Invitrogen, Carlsbad, California) following the manufacturer’s instructions. Alternatively, Aβ1-x, Aβ40 or Aβ42 ELISAs were performed using 6E10 as the capture antibody and monoclonal 4G8, anti-Aβ40 or anti-Aβ42 HRP-conjugated antibodies (Covance, Dedham, MA) as the detection antibodies. Synthetic Aβ40 or Aβ42 were used to generate a standard curve for each experiment. The plates were developed using TMB substrate kit (Pierce, Rockford, IL) and the reaction was stopped by addition of equal volume of 1 M HCl. The results were read using a Spectramax colorimetric plate reader (Molecular Devices, Sunnyvale, CA).
Conditioned media harvesting
Confluent primary astrocytes were washed twice with PBS (pH7.4) and incubated with fresh serum-free F12-DMEM media for 24 hours. Immortalized astrocytes were washed twice with PBS (pH7.4) and incubated with fresh serum-free DMEM supplemented with 1 mM sodium pyruvate. Conditioned media were collected and sterilized by filtering through 0.22 µm filters.
BV2 or primary microglia were incubated with 2 µg/ml Alexa488 labeled Aβ42 in serum-free DMEM or F12 DMEM, respectively, in the presence of 10 µg/ml BSA for 3 hours, washed extensively with PBS and fixed with 4% paraformaldehyde for 5 min. Cells were washed again with PBS and collected for flow cytometry using Beckman-Coulter XL flow cytometer. The total amount of Aβ internalized was determined by fluorescent intensity of Alexa488 as measured by flow cytometry.
Live cell imaging
For live cell imaging, microglial BV-2 cells were plated overnight in DMEM containing 2% FBS on Delta T tissue culture plates. Cells were incubated with 2 µg/ml Cy3-Aβ and 100 nmol/L Lysotracker Green DND-26 (Invitrogen, Carlsbad, CA) in serum-free DMEM containing 10 µg/ml BSA and imaged using a Zeiss LSM 510 confocal microscope.
Intracellular Aβ clearance
BV2 or primary mouse microglia cells were incubated with DMSO or 1 µmol/L GW3965 for 18 hours at 37°C. Cells were then treated with 2 µg/ml soluble Aβ42 in serum-free medium containing 10 µg/ml BSA for 3 hours or 24 hours respectively in the presence or absence of drug. For experiments using apolipoproteins, purified human HDL ApoA-I (Sigma, Saint Louis, MO), purified human plasma ApoE (rPeptide, Athens, GA), native ApoE2, ApoE3, or ApoE4 particles were applied at the same time as soluble Aβ42. Aβ42 levels in the cell lysates were determined by immunoblotting with the anti-Aβ antibody 6E10. Briefly, cells were extensively washed with PBS to ensure removing Aβ which is attached to the cell surface. Cells were then lysed in ice-cold RIPA buffer (Upstate Biotechnology, Lake Placid, NY), sonicated briefly, then collected by centrifugation at 13,000 rpm at 4 °C for 15 min. The samples were resolved by 4–15% Bis-Tris SDS-PAGE. ABCA1, ApoE (for positive controls) and β-actin or β-tubulin levels (for loading controls) in the cell lysates were measured by Western blot. For ELISA measurements, cells were treated similarly, washed with PBS and lysed in 1% SDS. Aβ42 levels were measured using ELISA and normalized to total protein.
Aβ degradation in astrocyte-conditioned medium
Conditioned media from Abca1+/+, +/− and −/− astrocyte cultures were incubated with 1 µg/ml Aβ42 for 0 or 24 hours. Aβ42 in the media was assessed using Western analysis or ELISA.
In vitro IDE assay
ApoE-containing HDL particles were immunprecipitated from the conditioned medium using the anti-ApoE antibody. Recombinant (500 ng/ml) IDE (R&D Systems, Minneapolis, MN) was incubated with 2 µg/ml Aβ42 for 1 hour in the presence of ApoE containing HDL particles. The samples were resolved by 4–15% Bis-Tris SDS-PAGE. The levels of IDE, ApoE and Aβ were monitored using Western analysis.
Contextual Fear Conditioning Studies
Following six days of treatment by oral gavage with 50 mg/kg/day GW3965 or vehicle, 20 week old Tg2576 mice (n=11/genotype/treatment) were trained and tested on two consecutive days as described previously (Comery et al., 2005
To compare differences between the experimental groups, two-tailed t-test or one-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test was performed using GraphPad Prizm software (GraphPad Software, San Diego, CA). Contextual memory was analyzed using a two-way ANOVA and post hoc pairwise comparison made using SAS Statistical Software (SAS Institute, Cary, NC).