In the current study, we hypothesized that overexpression of LDLR in the brain would decrease brain apoE protein levels, subsequently decreasing amyloid deposition. To test this hypothesis, we created several transgenic mouse lines that overexpress LDLR in the brain and then bred them with APP/PS1 transgenic mice. Brain apoE levels in LDLR transgenic mice were decreased by 50–90% in a dose-dependent manner. Most importantly, LDLR overexpression led to dramatic reductions in Aβ aggregation and neuroinflammatory responses. In addition, increasing expression of LDLR facilitated the elimination of soluble Aβ from the ISF, leading to lower levels of Aβ in the hippocampal extracellular space. This result strongly suggests that LDLR enhances brain Aβ clearance, serving as an important pathway that modulates Aβ metabolism. Overall, the results suggest that LDLR may be an attractive therapeutic target for AD.
Although numerous putative susceptibility genes for AD have been reported so far, the strongest genetic risk factor is APOE
genotype; the ε4 allele is an AD risk factor and the ε2 allele appears to be protective (Bertram et al., 2007b
). Given the considerable genetic evidence and the immunoreactivity of apoE in amyloid plaques, the effect of apoE isoforms on Aβ aggregation has been investigated extensively in vitro (Kim et al., 2009
). Later, in vivo studies demonstrated that the lack of apoE led to a dramatic reduction of fibrillar Aβ deposition in APP transgenic mouse models (Bales et al., 1999
; Bales et al., 1997
; Holtzman et al., 2000a
; Holtzman et al., 2000b
). Furthermore, apoE has been shown to regulate Aβ clearance in the brain (Bell et al., 2007
; Deane et al., 2008
; DeMattos et al., 2004
; Jiang et al., 2008
). These and other findings strongly suggest that the effects of apoE on Aβ aggregation and clearance play a major role in AD pathogenesis (Kim et al., 2009
). Consequently, modulating the function or levels of proteins that affect apoE metabolism in the brain seems to be a logical therapeutic strategy to alter Aβ-dependent pathogenic processes in AD. Results presented in the current study corroborate the feasibility and efficacy of apoE targeting therapeutics.
ApoE in the periphery is known to bind to several LDL receptor family members. Since the lipid composition and lipidation state of apoE-containing lipoprotein particles are different between brain and peripheral tissues, it would be important to know which LDL receptor members can regulate apoE protein levels in the brain (Kim et al., 2009
). Knockout mouse studies have provided direct evidence for LDLR and LRP1 as major apoE receptors in the brain (Elder et al., 2007
; Fryer et al., 2005
; Liu et al., 2007
). Fryer et al. also demonstrated that LDLR differentially regulates the levels of human apoE isoforms in the brain through its binding specificity. Zerbinatti et al. generated a LRP1 mini-receptor transgenic mouse model with 3.7-fold increased LRP1 levels in the brain (Zerbinatti et al., 2004
). Although an ~25% reduction in brain apoE levels was observed in LRP1 transgenic mice, there was an increase in soluble and insoluble Aβ in old mice (Zerbinatti et al., 2006
; Zerbinatti et al., 2004
). The reason for the LRP1 mini-receptor overexpression causing an increase in Aβ levels is not entirely clear but is likely due to the effects of LRP1 on APP and not due to its effects on apoE. For example, unlike LDLR, LRP1 is an APP binding protein that influences APP endocytic trafficking and cellular distribution such that processing to Aβ and its extracellular release is enhanced (Pietrzik et al., 2002
; Ulery et al., 2000
). This effect of LRP1 on APP and Aβ may supersede the effects of the LRP1 minireceptor on decreasing apoE levels by 25% and its effects on Aβ in the brain. In the current study, only 2-fold overexpression of LDLR protein was sufficient to decrease brain apoE levels and Aβ accumulation by more than 50%. Taken together, these data clearly demonstrate both LDLR and LRP1 regulate apoE protein levels in the brain. However, it is unclear whether other LDL receptor family members, such as LR11, ApoER2, and VLDLR, also efficiently mediate the endocytosis of apoE in the brain. Given the known apoE isoform-specific interactions with LDLR (Kim et al., 2009
), it would be interesting to determine whether the effect of LDLR overexpression differs in APP transgenic mouse models with humanized apoE isoforms. In addition, it will be important to determine the effects of LDLR overexpression on cognitive abnormalities observed in APP/PS1 mice.
Although the effects of LRP1 on Aβ clearance and APP processing have been extensively studied (Cam and Bu, 2006
), the potential role of LDLR on AD pathogenesis has been unclear. Several studies reported that a few single-nucleotide polymorphisms (SNPs) in LDLR
gene are associated with the risk of developing AD in case-control studies (Cheng et al., 2005
; Gopalraj et al., 2005
; Retz et al., 2001
). However, others could not replicate the earlier studies and a meta-analysis of the previously reported case-control data failed to detect any significant summary odds ratios (Bertram et al., 2007a
; Rodriguez et al., 2006
). More recent findings suggest that other SNPs may be associated with a risk of AD in a sex-specific manner. SNP rs688 and haplotype GTT were significantly associated with an increased risk of AD in males and females, respectively (Lämsä et al., 2008
; Zou et al., 2008
). Unlike other studies, both studies also demonstrated functional effects of SNPs on LDLR splicing and Aβ42 levels.
In order to investigate the effect of LDLR deficiency on cholesterol and Aβ in the brain, several groups have analyzed LDLR knockout mice. Although LDLR deficiency significantly increased murine brain apoE levels by ~50%, it did not alter brain cholesterol levels (Elder et al., 2007
; Fryer et al., 2005
; Quan et al., 2003
; Taha et al., 2008
). Previously, we demonstrated that there was no significant change in brain Aβ levels both before and after the onset of amyloid deposition in PDAPP transgenic mice on a LDLR-deficient background (Fryer et al., 2005
). However, there was a trend for an increase in Aβ accumulation in PDAPP/LDLR knockout mice. Recently, Buxbaum and colleagues also reported that LDLR deficiency did not affect endogenous murine Aβ levels in the brain (Elder et al., 2007
). In contrast, lack of LDLR was associated with increased amyloid deposition in Tg2576 mice (Cao et al., 2006
Prior to our current study, it was unknown whether increased levels of LDLR in the brain would affect Aβ accumulation in vivo, and if so, via what mechanism. Given the role of apoE in Aβ clearance and aggregation, we hypothesized that the reduction of apoE levels by LDLR overexpression would promote the elimination of soluble Aβ from the brain ISF, i.e., via transcytosis across the blood-brain barrier into the plasma or by local cellular uptake and degradation within the brain. We predicted that increased elimination of soluble Aβ through either of these elimination routes would result in decreased Aβ accumulation. Our in vivo microdialysis results suggest that the mechanism by which LDLR overexpression alters Aβ metabolism is to enhance the extracellular clearance of Aβ peptide. It is possible that receptor-mediated clearance of Aβ-ApoE complex or Aβ alone from the brain ISF might be enhanced by LDLR overexpression. Interestingly, other LDL receptor family members, such as LRP1, LR11, and ApoER2, are known to directly or indirectly bind to APP and affect its amyloidogenic processing (Kim et al., 2009
). Since levels of carboxyl-terminal fragments of APP, generated by APP processing, were not different between genotypes, it is unlikely that LDLR overexpression alters APP processing. Though it is likely that the reduction of apoE protein levels by LDLR overexpression enhanced Aβ clearance (DeMattos et al., 2004
), we cannot exclude the possibility that LDLR may directly affect Aβ clearance independent of apoE.
Transgenic mouse models of amyloidosis have been invaluable for investigating AD pathogenic mechanisms and evaluating the efficacy of novel therapeutic targets. Interestingly, female APP/PS1 transgenic mice used in the current study had a more than 2-fold increase in plaque load and insoluble Aβ accumulation, compared with male littermates ( and ). Our finding is consistent with a recent study that used APP/PS1 mice on a different genetic background (Halford and Russell, 2009
). A similar sex-specific amyloid deposition phenotype has been previously reported with other APP transgenic mouse models (Callahan et al., 2001
; Wang et al., 2003
). The APP/PS1 transgenic mouse used in our study is one of the most commonly used Aβ amyloidosis models. Effects of genetic and pharmacological manipulations on Aβ accumulation and Aβ-related pathological changes have been tested using this model. However, most previous studies did not analyze the extent of Aβ accumulation by sex. It is possible that sex differences were not obviously recognized in other studies due to the limited sample size for each sex. Given the dramatic effect of sex on Aβ aggregation, the sex of APP/PS1 transgenic mice should be carefully considered for the proper interpretation of results. Since the prevalence of AD is higher in women even after adjusting for age and education levels (Andersen et al., 1999
), it is intriguing that several mouse models of amyloidosis have similar sex-dependent phenotypes. Several studies suggest that female hormones may, in part, contribute to sex differences in AD (Carroll et al., 2007
; Yue et al., 2005
). Given the inconsistent findings among studies, the exact mechanism underlying sex differences in AD pathogenesis requires further investigation. It is possible that the elevated apoE levels in the females APP/PS1 mice is related to why females develop more amyloid deposition (). Interestingly, while the clearance of soluble Aβ in APP/PS1 males trended towards being faster than that for APP/PS1 females (), we cannot rule out that an Aβ clearance-independent mechanism may account for the sex differences in plaque load and insoluble Aβ accumulation in older mice. Understanding the factors that regulate sex-dependent phenotypes may provide additional insight into new therapeutic targets.
Notably, an increase of LDLR protein levels by only ~2-fold was sufficient to decrease Aβ accumulation by ~50% in APP/PS1 female transgenic mice. Our findings suggest that even a small increase in LDLR levels or function in the brain may be exploited as a novel approach for developing AD therapeutics. Due to the critical role of LDLR in the metabolism of apoB-containing LDL particles in the circulation, strategies increasing the function and amount of LDLR protein in the liver have been extensively pursued as promising therapies for atherosclerosis and premature coronary heart disease (Soutar and Naoumova, 2007
). Overexpression of LDLR in the liver facilitated LDL elimination by receptor-mediated endocytosis and prevented diet-induced hypercholesterolemia (Hofmann et al., 1988
; Yokode et al., 1990
). However, the modulation of LDLR function in the brain as a treatment modality for AD has not been previously investigated. Our study clearly demonstrates the beneficial effects of LDLR overexpression in the brain on pathogenic Aβ aggregation and subsequent neuroinflammatory responses. Although other LDL receptor family members bind to multiple ligands (i.e. LRP1 having more than 20 ligands), there are only two known ligands, apoB and apoE, for LDLR. Since apoB is not expressed in the brain, modulating LDLR function in the brain is likely to target apoE specifically. A couple of recently identified genes are known to regulate LDLR protein levels by affecting the trafficking and degradation of LDLR in peripheral tissues (Soutar and Naoumova, 2007
). Since these proteins are also expressed in the brain, their potential roles in the clearance and accumulation of Aβ warrant further investigations. In addition, several compounds have been identified to increase hepatic LDLR protein levels by modulating synthesis or degradation of LDLR and LDLR-regulating proteins. Given our results from transgenic mice overexpressing LDLR in the brain, the therapeutic potential of these lead compounds merit additional testing in animal models of Aβ amyloidosis.