Our data suggest that one can measure accurately the clearance rates of unlabeled human Aβ and apolipoproteins across the BBB and via the ISF bulk flow in mice by using human specific ELISAs. MALDI-TOF mass spectrometry and LC ESI MS/MS were less sensitive than the ELISA measurements in determining the CNS clearance rates in mice of unlabeled Aβ and apolipoproteins at low physiological levels as found normally in the CSF and brain in mice and humans (DeMattos et al., 2003
). The present study shows for the first time that iodination does not alter the CNS clearance measurements of Aβ 1-40 and 1-42, as long as transport of 125
I-labeled Aβ is measured in its monoiodinated reduced form, as reported (Deane et al., 2003
; LaRue et al., 2004
; Deane et al., 2004
; Deane et al., 2005
). Moreover, we show for the first time that apoE and apoJ are cleared from brain by different transport routes and at substantially different rates, that the clearance of lipidated apoE is much slower than non-lipidated apoE, and that binding of Aβ to these apolipoproteins may critically alter its clearance from the CNS.
The present study shows unlabeled Aβ40, at the levels corresponding to those of mouse endogenous brain Aβ(Deane et al., 2005
), is removed rapidly from brain ISF via transport across the BBB at a rate of 0.21 pmol/min/g ISF. Transport of unlabeled Aβ40 at the BBB was mediated via LRP1, as found with 125
I-labeled Aβ40 (Shibata et al., 2000
; Deane et al., 2004
). Since the levels of soluble, rapidly exchangeable Aβ pool in brain ISF in AD mice are around 300 pmol/L ISF, as shown by microdialysis technique (DeMattos et al., 2003
), given the Aβ BBB efflux rate as determined in the present study, it would take about 40 seconds for LRP1 to clear this Aβ pool from brain ISF, assuming Aβ central production and re-entry of circulating Aβ into the brain (Deane et al., 2003
) stop. In AD models and AD the levels of total Aβ in brain including insoluble Aβ are in low micromolar range, as for example around 6 μmol/kg brain (Cirrito et al., 2003
; DeMattos et al., 2003
). Making assumptions as above, it would take under pathological conditions about 30 to 45 minutes for LRP1 at the BBB to eliminate all soluble free Aβ from brain ISF, and about 14 days to remove total Aβ from brain in AD, providing that all Aβ could be resolublized into its free form. Although these numbers suggest high BBB efflux capacity for free Aβ, the present study indicates that the major pathogenic species of Aβ, Aβ42 (Selkoe, 2001
), is cleared at the BBB at a rate 1.9-fold slower than Aβ40. Earlier work suggested LRP1 expression at the BBB may be substantially reduced in AD and in AD models (Deane et al., 2004
). Thus, both of these factors may further modify elimination times of free Aβ under pathological conditions.
We show lipid poor apoE (isoform 3) is cleared slowly from brain compared to Aβ40 or Aβ42 mainly because of its low transport at the BBB, i.e., 0.04 pmol/min/g ISF. Since a large part of the effect of apoE isoforms on AD and cerebral amyloid angiopathy risk is mediated by the interaction of apoE with Aβ (Holtzman and Zlokovic, 2006
), and neither mouse apoE nor human apoE have an impact on synthesis of brain Aβ in AD models (Bales et al., 1997
; Holtzman and Zlokovic, 2006
), we hypothesized apoE must affect clearance of Aβ within or from brain. Our data show that binding of Aβ40 to apoE (isoform 3) reduces by 5.7-fold its efflux rate at the BBB. The present study has been focused on apoE3 and used relatively short clearance times within the periods of 30 min. There may be isoform-specific differences in apoE clearance from brain and in apoE-mediated retention of Aβ in brain that could possibly be revealed over the longer times allowed for the clearance measurements (> 30 min) than in the present study. It is well recognized that the ε4 allele has a gene-dose effect on the risk and age of onset of AD and the amount of deposited Aß40 and vascular Aβ load, as reviewed by Holtzman and Zlokovic (2006)
. The isoform-specific effect of apoE on clearance of lipid-poor and lipidated apoE, the effects of longer efflux times on apoE clearance and the receptors involved in mediating slow removal of apoE and apoE-Aβ complexes across the BBB, remain to be determined by future studies. It is interesting to note, it has been recently suggested that low density lipoprotein receptor (LDLR) may be involved in regulating the CNS levels of human and mouse endogenous apoE (Fryer et al., 2005
) and amyloid pathology in AD mice (Cao et al., 2006
Finally, we demonstrate that native apoJ is eliminated rapidly from brain ISF across the BBB at a rate lower than that of Aβ40, but significantly higher than the rate of Aβ42. ApoJ is the major carrier protein for Aß in biological fluids (Calero et al., 2000
), and its receptor LRP2 is expressed at the BBB (Zlokovic et al., 1996
; Chun et al., 1999
). We hypothesized LRP2 may be involved in efflux of apoJ out of the CNS, and Aβ binding to apoJ may enhance clearance of highly pathogenic Aβ42. Our data show that both RAP and LRP2-specific antibody block apoJ clearance, indicating LRP2 is required for apoJ efflux at the BBB. We next show that binding of Aβ42 to apoJ accelerates Aβ42 clearance rate at the BBB by 83%, which again requires LRP2. It has been reported that LRP2 at the BBB is saturated from the blood side by physiological levels of apoJ in plasma which precludes brain influx of circulating Aβ bound to apoJ across the BBB (Zlokovic et al., 1996
). In contrast, efflux of Aβ42-apoJ complex from brain ISF to blood is substantial at physiological apoJ CSF levels as shown in the present study suggesting the net-transport of Aβ via apoJ at the BBB favors its efflux from brain. Consistent with the present study are findings demonstrating that lack of apoJ in AD mice may increase levels of soluble Aβ in brain (DeMattos et al., 2004
). Since apoJ increases Aβ neurotoxicity in AD mice (De Mattos et al., 2002
), clearance of Aβ-apoJ complexes from brain could be neuroprotective.
In conclusion, the present study highlights the importance of Aβ clearance mechanisms in the CNS suggesting that efflux of Aβ form brain is controlled by different transport pathways at the BBB. The lipoprotein receptors seem to play a major role in determining the rate of Aβ efflux at the BBB, either in its free form via LRP1 and/or in its bound form as a complex with apoJ via LRP2. Whether other lipoprotein receptors such as LDLR participate in slow clearance of Aβ-apoE complexes at the BBB remains to be explored. Future studies should characterize in greater detail possible role of apoE/LDLR transport interactions at the BBB and of apoJ/LRP2 interactions in regulating the levels of soluble, as well as of deposited Aβ in brain.