Level 1 – LRP1 at the blood-brain barrier and in brain vascular cells
LRP1 is the main cell surface receptor mediating brain Aβ clearance at the BBB. It has been demonstrated that binding of Aβ to LRP1 at the abluminal side of the BBB in vivo
initiates a rapid Aβ clearance across the BBB into the blood (Shibata et al. 2000
; Deane et al. 2004a
; Cirrito et al. 2005
; Ito et al. 2006
; Bell et al. 2007
; Sagare et al. 2007a
). Human Aβ injected into different brain regions in mice was found intact in murine plasma confirming its elimination from the brain (Shiiki et al. 2004
; Bell et al. 2007
). It has been shown that clearance of human 125
I-labeled Aβ40 and Aβ42 injected into the caudate nucleus in mice is significantly inhibited (i.e., >70%) by RAP which blocks binding of ligands to both LRP1 and megalin, as well as by an LRP1 specific antibody, but not by antibodies against LRP2 (megalin), low density lipoprotein receptor (LDLR), very low density lipoprotein receptor (VLDLR) or apoE2R, thereby suggesting that LRP1 mediates clearance of Aβ (Shibata et al. 2000
; Deane et al. 2004a
). A significant reduction of human 125
I-Aβ40 and 125
I-Aβ42 clearance from the brain has been reported in transgenic RAP null mice exhibiting a greater than 70% decrease in the LRP1 levels in brain microvessels, but not in transgenic mice lacking LDLR or VLDLR, demonstrating again that cerebrovascular LRP1 mediates Aβ clearance across the BBB (Shibata et al. 2000
; Deane et al. 2004a
). It has been shown that LRP1 is involved in the vascular clearance of human 125
I-Aβ40 from the rat secondary somatosensory cortex (S2 region) (Ito et al. 2006
Using human specific ELISA to determine the levels of unlabeled human Aβ40 and Aβ42 in brain and plasma after their respective microinjections into the mouse caudate nucleus, it has been independently confirmed that both RAP and an anti-LRP1 antibody inhibit Aβ40 clearance from the brain and the appearance of intact Aβ in plasma, indicating LRP1 involvement (Bell et al. 2007
). Furthermore, it has been shown that inhibition of LRP1 expression in brain microvessels of CD-1 mice by a cocktail of the LRP1 antisense RNAs results in impaired Aβ clearance from the brain associated with an increase in endogenous brain Aβ levels and impaired cognition (Jaeger et al. 2009
). Studies in mouse models with genetically manipulated LRP1 expression at the BBB, such as RAP-null mice expressing substantially reduced LRP1 levels in brain microvessels and Tie-2
-LRP1-cluster-IV transgenic mice expressing the LRP1 cluster IV minigene in cerebral microvasculature, have demonstrated increased and reduced mouse endogenous Aβ40 and Aβ42 brain levels, suggesting a major role of LRP1 in regulating brain clearance of Aβ under physiological conditions (Deane et al. 2004a
; LaRue et al. 2007
; Sagare et al. 2007b
). Recently, it has been reported that the levels of LRP1 in brain microvessels can be increased by fluvastatin resulting in an enhanced Aβ clearance from the brain (Shinohara et al. 2010
It is of note, in addition to LRP1-mediated clearance of Aβ from the brain, there is also clearance of different Aβ isoforms from the CSF to the blood (Ghersi-Egea et al. 1996
; Monro et al. 2002
; Silverberg et al. 2003
). Although the preponderance of intraparenchymally-injected biologically active test-molecules in the rodent brain is likely reabsorbed across brain microvessels, the bulk flow clearance from brain interstitial fluid to CSF (Szentistvanyi et al. 1984
) and along perivascular spaces (Weller et al. 2008
) has been estimated to contribute up to 15-20%. In the case of Aβ, it has been determined that the CSF bulk flow in the normal mouse brain can mediate ~ 15% of total Aβ clearance (Shibata et al. 2000
). It is conceivable that under pathological conditions associated with amyloid accumulation and diminished overall Aβ clearance from the CNS, the CSF bulk flow component can eventually become > 15%. However, this relative increase in the CSF clearance contribution would likely reflect a diminished LRP1-mediated Aβ clearance across the BBB, rather than an increase in the clearance capacity for Aβ through the choroid plexus and via the CSF pathway.
It is of note that LRP1 is also expressed in the choroid plexus epithelium of healthy young rats and its expression is sustained during aging (Johanson et al. 2006
). Moreover, exposure to lead (Pb) has been shown to decrease expression of LRP1 in the choroid plexus epithelium that has been associated with Aβ accumulation in the choroid plexus (Behl et al. 2009
). The exact role of LRP1 in the choroid plexus epithelium in mediating the CSF-to-blood Aβ clearance and for brain Aβ homeostasis during normal and pathological aging is an important topic deserving further research.
Studies using isolated murine cerebral microvessels have demonstrated LRP1-dependent clearance of Aβ40 and Aβ42 at the abluminal side of the BBB (Deane et al. 2004a
). It has also been shown that RAP blocks an apoE-dependent uptake of Aβ peptides by astrocytes indicating that LRP1 and/or another member of the LDLR receptor family are likely involved in the astrocyte-mediated clearance of Aβ (Koistinaho et al. 2004
). Studies using in vitro
BBB models with a conditional immortalized cell line derived from brain capillary endothelial cells of transgenic rats expressing temperature-sensitive large T antigen (Yamada et al. 2008
) and with the polarized Madin-Darby canine kidney cells expressing LRP1 mini-receptors (Nazer et al. 2008
), have also importantly demonstrated the role of LRP1 in Aβ endothelial cellular uptake and endocytosis, respectively, resulting in Aβ clearance. Moreover, our preliminary observations using a human BBB in vitro
model with primary brain endothelial cells and pericyte-conditioned media to direct LRP1 distribution mainly to the basolateral side of an endothelial monolayer have revealed LRP1-mediated transcytosis of Aβ40 and Aβ42 in the basolateral-to-apical direction corresponding to the abluminal and luminal sides of the BBB in vivo
, respectively (E. A. Winkler, Y. Sallstrom, D. Zhu, R. Deane and B. V. Zlokovic, unpublished data).
LRP1 that is expressed at the abluminal side of the BBB was shown to mediate Aβ transport from brain to blood, but others have also reported that LRP1 can be utilized for delivery of therapeutics to the brain, as for example angiopeps (Demeule et al. 2008
), implying that LRP1 might also be expressed at the luminal side of the BBB. The exact distribution of LRP1 between the luminal side of the BBB, the cytoplasmic endothelial pool and the abluminal side of the BBB is presently unknown. Our work in progress using high resolution confocal microscopy analysis indicates, however, that LRP1 is mainly confined to the abluminal side of the BBB, but a smaller portion of LRP1 is also expressed at the luminal side of the BBB (E. A. Winkler, Y. Sallstrom, D. Zhu, R. Deane and B. V. Zlokovic, unpublished data). It is possible that luminal LRP1 may participate in transport of angiopeps from blood-to-brain and that LRP1 in the cerebral vascular smooth muscle cells (Bell et al. 2009
) can be utilized as well for delivery of therapeutics to the brain and cerebral arteries.
Reduced levels of LRP1 in brain microvessels correlating with endogenous Aβ deposition have been shown in a chronic hydrocephalus model in rats (Klinge et al. 2006
). Moreover, reduced levels of LRP1 in brain microvessels associated with Aβ cerebrovascular and brain accumulation have been reported in AD patients (Shibata et al. 2000
; Donahue et al. 2006
). Several studies have indicated that LRP1 expression in the brain capillary endothelium is reduced during normal aging in rodents, non-human primates and humans, as well as in AD models and AD patients (Kang et al. 2000
; Shibata et al. 2000
; Bading et al. 2002
; Deane et al. 2004a
; Donahue et al. 2006
; Bell and Zlokovic 2009
). Similar reductions in LRP1 expression have been reported in cerebral vascular smooth muscle cells in small pial and intracerebral arteries regulating blood flow to the brain which was shown to be associated with Aβ accumulation within the wall of these brain arteries (Bell et al. 2009
). Therefore, it is likely that LRP1 downregulation in the brain endothelium and vascular cells in patients with mild cognitive impairment during the hit 1 stage and in AD patients during the hit 2 stage would lead to faulty vascular Aβ clearance promoting cerebrovascular and focal parenchymal Aβ accumulations contributing to AD pathogenesis.
Pitfalls in Aβ clearance measurements in animal models
Reproducible and accurate measurements of Aβ clearance from the brain are challenging because of the hydrophobic nature of the full length peptide, possible conformational and structural changes of Aβ and heterogeneity of truncated Aβ fragments. Studies with 125
I-labeled Aβ have generated critical data for the field. However, work with 125
I-Aβ preparations also requires special precautions due to rapid radiolysis of the labeled peptide. We have recommended that the radiolabeled Aβ should be used either immediately after labeling within 24 h or alternatively can be stored in ethanol over a short period of time (i.e., 3-4 days), and re-purified before use on the day of the experiment by HPLC to eliminate free iodine and possible Aβ degradation products, and separate mono-iodinated from di-iodinated Aβ species and oxidized from reduced Aβ (LaRue et al. 2004
). It is of note, Aβ radioiodination by a mild lactoperoxidase method typically provides less damage to the peptide than a more robust chloramine-T method (LaRue et al. 2004
). In brain clearance studies, the integrity of 125
I-Aβ in the brain should be additionally confirmed by different analytical methods such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and HPLC (Deane et al. 2004a
It has also been raised that iodination can potentially introduce a conformational change in the peptide. Therefore, more recently some important control measurements have been performed by different groups (as discussed above) to complement the radiotracer studies. This includes, but is not limited to the use of unlabeled Aβ and measurements of its concentrations in brain and blood by ELISA, as well as measurements of endogenous Aβ levels in transgenic models with manipulated LRP1 expression, or LRP1 silencing and/or by pharmacologically manipulating LRP1 expression in brain microvessels.
In a recent paper, Ito et al. (2010)
have confirmed 125
I-Aβ40 clearance from the mouse S2 region using an established brain clearance technique (Kakee et al. 1996
), but failed to demonstrate inhibition of Aβ clearance by RAP. Based on this single negative result with RAP, Ito et al.
have suggested that the members of the LDLR receptor family including LRP1 do not participate in Aβ40 clearance, challenging findings from several different groups that have demonstrated a major role of LRP1 in Aβ clearance, as discussed above. However, Ito et al.
have not performed any control experiments to determine 125
I-Aβ integrity immediately prior to its use on the day of the experiment and/or at the end of the experiment in brain extracts. They also did not use unlabeled Aβ as a control for radiolabeled Aβ. Moreover, they have not employed any complementary approach to determine the role of LRP1 in Aβ clearance, such as blockade of LRP1 by an LRP1-specific antibody, inhibition of LRP1 expression by LRP1 silencing by siRNA and/or antisense RNAs strategies, or use of transgenic models with genetically manipulated LRP1 expression.
By analyzing the experimental design in Ito et al.'s (2010)
study, one can find out that Aβ40 was radiolabeled with chloramine-T and after labeling it was lyophilized and mixed with different excipients including aprotinin, as per the PerkinElmer Technical Data Certificate of Analysis, NEX361 sheet and Dr. Terasaki's (senior author in Ito et al. 2010
) personal communication to us. Aprotinin is a protease inhibitor, but it unfortunately also binds to LRP1 and prevents an LRP1-dependent uptake of RAP (Demeule et al. 2008
), which therefore could potentially interfere with Aβ clearance measurements in vivo
as performed by Ito et al. (2010)
. According to table 1 [from Ito et al. 2010
], one can calculate the final concentration of aprotinin in the injectate in their experiments which was about 400 KIU (Kallikrein Inhibitor Unit)/mL of aprotinin or about 8.6 μM.
To test whether co-administered aprotinin interferes with the RAP-mediated blockade of Aβ clearance, we have determined clearance of unlabeled Aβ40 from the mouse caudate nucleus in the presence or absence of RAP and with or without aprotinin by using human specific Aβ40 ELISA over a 30 min period of time. In all experiments, 14
C-inulin (an inert polar molecule) was administered into the brain simultaneously with Aβ as a reference standard (Shibata et al. 2000
). As we have reported previously by using unlabeled Aβ (Bell et al. 2007
), RAP (5 μM) decreased human Aβ40 clearance from the brain by ~50% (; for details of calculations please see Bell et al. 2007
). However, in the presence of aprotinin (8.6 μM), RAP did not have any significant effect on Aβ40 clearance () corroborating data by Ito et al. (2010)
. The appearance of an intact human Aβ40 in plasma was abolished by RAP in the absence of aprotinin (). However, the presence of aprotinin completely inhibited RAP-mediated blockade of Aβ brain-to-blood transfer ().
Fig. 3 Levels of human Aβ40 in the brain (a) and plasma (b) 30 min after microinjection of human Aβ40 (40 nM) and 14C-inulin (0.023 μCi) into the mouse caudate nucleus in the presence and absence of RAP (5 μM) with and without (more ...)
Consistent with a previous report (Ito et al. 2010
), Aβ recovery in brain and its appearance in plasma after intracerebral administration were not affected by aprotinin alone (), suggesting that aportinin alone does not influence LRP1-mediated Aβ clearance from brain and that at least in vivo
Aβ and aprotinin likely bind to different exosites on LRP1. Overall, this data shows that only in the presence of RAP aprotinin substantially affects the measurement of Aβ clearance from the mouse brain.
As aprotinin prevented RAP-mediated inhibition of Aβ40 clearance from the brain, we next explored whether aprotinin interacts directly with RAP. Here, we have demonstrated by ELISA that aprotinin binds to immobilized RAP (). By incubating aprotinin and RAP for 1 h at 37°C we have additionally confirmed by immunoblotting analysis after cross-linking with bis[sulfosuccinimidyl]suberate (BS3) that aprotinin interacts directly with RAP (). Thus, binding of RAP to aprotinin would prevent RAP from blocking 125I-Aβ40 binding to LRP1 at the abluminal side of the BBB in vivo which in turn may confound measurements of Aβ clearance and data interpretation.
Level 2 – Soluble LRP1 in plasma
We have demonstrated that circulating plasma sLRP1 provides a key endogenous peripheral ‘sink’ activity for Aβ by promoting a continuous removal of Aβ from brain (Sagare et al. 2007a
). In neurologically healthy humans and mice, sLRP1 normally binds > 70% of circulating Aβ preventing free Aβ access to the brain (Sagare et al. 2007a
) (). In AD patients and AD transgenic mice, however, Aβ binding to sLRP1 is compromised because of increased levels of oxidized sLRP1 which does not bind Aβ (Sagare et al. 2007a
) resulting in elevated levels of free Aβ40 and Aβ42 that can re-enter the brain via RAGE-mediated transport (Deane et al. 2003
; Ujiie et al. 2003
; Donahue et al. 2006
; Sagare et al. 2007a
In the human hippocampus, it was shown that RAGE expression in brain endothelium is increased with advanced AD compared to early stage AD and/or individuals with mild cognitive impairment (MCI) (Miller et al. 2008
) which may further contribute to Aβ accumulation into the brain via enhanced Aβ influx from blood to brain. Moreover it has been recently shown that a diminished sLRP1-Aβ ‘sink’ activity precedes an increase in the tau/Aβ42 CSF ratio and a drop in global cognitive decline in individuals with MCI converting into AD, and is therefore a useful early biomarker for AD-type dementia (Sagare et al. 2009
). It has also been shown that recombinant LRP1 fragments can effectively replace oxidized sLRP1 and sequester free Aβ in plasma in AD patients and AD transgenic mice ultimately reducing Aβ-related pathology in the brain (Sagare et al. 2007a
). It is of note, a recent Phase II clinical trial in patients with mild AD with Baxter's intravenous immunoglobulin preparation Gammagard Liquid containing both sLRP1 and anti-RAGE immunoglobulins (Weber et al. 2009
) has shown encouraging results (Relkin et al. 2009
). It has been suggested that both sLRP1 and anti-RAGE may contribute to the observed beneficial effects of GGL by improving peripheral sink for Aβ and preventing Aβ influx into the brain, respectively (Dodel et al. 2010
Level 3 – LRP1 in liver
In aged Squirrel monkeys, Aβ systemic clearance is reduced and is associated with increased Aβ levels in the brain (Mackic et al. 1998b
). Also, an increased entry of circulating Aβ42 into the brain and its deposition onto senile plaques in aged Rhesus monkeys (Mackic et al. 2002
) or accumulation of circulating Aβ40 in the cerebral vessels in aged Squirrel monkeys with CAA (Ghilardi et al. 1996
), have been demonstrated. An age-dependent reduction in the systemic Aβ clearance may diminish the ‘sink action’ for Aβ clearance from the brain which in turn could increase the RAGE-dependent free Aβ transport across the BBB into brain regions expressing RAGE.
It has also been reported that a rapid peripheral clearance of Aβ is mediated mainly by LRP1 in the liver (Tamaki et al. 2006
). Using the perfused rat liver preparation, Tamaki et al. (2007)
were able to convincingly show that RAP blocks 125
I-Aβ uptake by the liver likely because in these experiments RAP was pre-infused into the liver before administration of 125
I-labeled Aβ and aprotinin, and was not mixed with Aβ/aprotinin. In addition, the dilution of aprotinin in the liver uptake measurements (Tamaki et al. 2007
) was at least by ~ 40-fold greater than in the brain clearance measurements (Ito et al. 2010
). These differences in the experimental design were likely to minimize the interaction between RAP and aprotinin in the Aβ liver uptake study.
It is of note that reduced hepatic LRP1 levels have been shown to be associated with decreased peripheral Aβ clearance in the aged rats (Tamaki et al. 2006
). In addition, both sLRP1-Aβ complexes and free Aβ are eliminated via the kidneys (Sagare et al. 2007a
), but whether LRP1 is involved in Aβ clearance by the kidneys as in the liver is presently unknown.