First, we used our brain tissue clearance technique (16
) to compare the disappearance curves from brain interstitial fluid (ISF) of 125
I-radiolabeled lipid-poor recombinant human apoE isoforms, astrocyte-derived lipo-apoE isoforms (33
), unbound free monomeric synthetic human Aβ40 and Aβ42 peptides, and complexes of various apoEs with Aβ40 and Aβ42. Different apoE and Aβ test tracers and their complexes were microinfused into brain ISF at equimolar concentration of 40 nM simultaneously with 14
C-inulin (reference marker). Clearance was measured over a period of 30 to 300 minutes. It is of note that clearance rates of unlabeled and corresponding 125
I-labeled apolipoproteins and Aβ have been shown to be almost identical (19
). Total efflux from brain ISF of lipid-poor apoE isoforms corrected for degradation (see below) was significantly slower than that of Aβ40 or Aβ42 (Figure A).
apoE isoform–specific clearance across the mouse BBB in vivo.
The analysis of 2 transport components contributing to total efflux of undegraded ligands from brain indicated less efficient efflux across the BBB of apoE isoforms compared with Aβ isoforms, whereas transport by ISF bulk flow was very slow and similar for all test tracers studied (Figure B). apoE4 was cleared at a considerably slower rate across BBB compared with apoE3 or apoE2, as indicated by the respective slopes of the radioactivity disappearance curves at the BBB (Figure B). Lipidation favored apoE retention in the brain in an isoform-specific manner, i.e., lipo-apoE4>lipo-apoE3 or lipo-apoE2, and thereby further diminished apoE BBB clearance compared with their respective lipid-poor isoforms (Figure B). Since lipo-apoE was a mixture of different size particles, i.e., 7–12 nm and 12–17 nm (33
), in a separate study, we compared clearance of different size lipo-apoE particles. As illustrated for lipo-apoE3, there was not a significant difference in clearance from the brain between 7–12 nm and 12–17 nm particles compared with a mixture of 7–17 nm particles (Supplemental Figure 1; supplemental material available online with this article; doi:
). Therefore, in all studies with lipo-apoE, we used a mixture of apoE particles.
According to our model (see Methods), the elimination of inulin from brain ISF (Figure A) reflects a passive drainage of molecules via the ISF bulk flow, as reported (16
). The fractional transport rate constants (k
) for different apoE lipid–poor and lipidated isoforms were calculated from 72 individual experiments (as shown in Figure A) by using Equations 2 and 4 (see Methods). The rates of the total efflux, elimination via transport across the BBB, elimination by the ISF bulk flow, and retention in the brain corrected for degradation as well as the half-times for clearance and retention in the brain are given in Supplemental Table 1. Figure B and Supplemental Table 1 show that the transport rate via the BBB of lipo-apoE4 was 8.3-fold, 4.9-fold, and 2.9-fold lower than that for free Aβ40, lipid-poor apoE2 or apoE3, and apoE4, respectively, and 2.6-fold and 2.4-fold lower than for lipo-apoE2 and lipo-apoE3, respectively. Conversely the retention rate of free Aβ40 in the brain was the shortest, i.e., 4.1 × 10–3
, as reported (16
). This was 1.7-fold faster than for Aβ42, consistent with the previous report demonstrating a 1.9-fold faster BBB efflux rate for Aβ40 compared with Aβ42 (19
). Aβ40 retention rate was 3.8-fold and 9.5-fold less than for lipid-poor apoE2 and apoE4, respectively, or 11.7 and 15.9 times less than for lipo-apoE2 and lipo-apoE4, respectively (Supplemental Table 1). These data indicate that lipo-apoE4 has by far the greatest retention rate in the brain and very slow efflux across the BBB compared with other apoE isoforms or Aβ peptides.
During these relatively short-term transport kinetic experiments, apoE was minimally degraded in the brain ISF at 30 or 300 minutes (less than 10%), as shown by TCA-precipitation and SDS-PAGE analyses of brain tissue supernatants after 125I-apoE2 and 125I-apoE4 microinfusion (Supplemental Figure 2, A and B). However, there was a significant time-dependent progressive degradation of both apoE2 and apoE4 in plasma, as shown by a significant increase in their respective TCA nonprecipitable fractions (Supplemental Figure 2C), indicating metabolism either during transport across the BBB and/or during systemic clearance in the circulation. There was also very low degradation of lipo-apoE isoforms ranging from 10%–15%, as indicated by the TCA-precipitation analysis of brain supernatants after 125I–lipo-apoE2 and 125I–lipo-apoE4 microinfusion (Supplemental Figure 2D). The relative contributions to clearance of apoE isoforms by transport across the BBB, ISF flow and degradation, and retention in the brain of undegraded and uncleared apoE ligands indicated a reciprocal relationship between transport across the BBB and retention of apoE ligands in the brain, namely, the higher the BBB transport, the lower the retention in the brain and vice versa (Figure C). The slow clearance via the ISF flow and low rates of degradation were similar between different lipid-poor and lipo-apoE isoforms and did not influence significantly BBB transport or retention. This analysis importantly suggests that a failure in effective removal across the BBB is a key to high retention of lipo-apoE4 in the brain compared with apoE3 or apoE2, which exhibit moderate transport across the BBB.
All 3 lipid-poor apoE isoforms (Figure D) as well as lipo-apoE isoforms (not shown) appeared in the cerebrospinal fluid (CSF) with a pattern comparable to that of inulin, a reference molecule that is cleared from brain ISF into CSF by passive diffusion via ISF bulk flow (16
). Therefore, apoE clearance from brain ISF to CSF did not exhibit an isoform-specific effect. In contrast, apoE isoforms microinjected into brain ISF appeared in plasma with a significantly different pattern, i.e., apoE2 and apoE3 greater than apoE4 (TCA precipitable), compared with almost negligible levels of inulin at the corresponding time points between 100 and 300 minutes (Figure E). These data confirmed that (a) the reference molecule inulin is not transported across the BBB, as shown previously (16
), (b) there is an in vivo transcytosis of apoE2 and apoE3 across the BBB into the blood, and (c) apoE4 transport across the BBB from brain to blood is negligible. It is of note that the time-appearance curves of apoE isoforms in plasma cannot be used to estimate total recovery of apoE ligands in plasma because apoE entering the plasma compartment is continuously removed from the plasma by systemic clearance via liver, kidney, and other organs (19
). Therefore, the areas under curves in Figure E underestimate apoE recovery in plasma. Similarly, the time-appearance curves of apoE in the CSF are influenced by the CSF’s rapid turnover rate, which continuously clears molecules into blood by nonspecific absorption across the arachnoid granulations (2
Since apoE binds to different lipoprotein receptors, e.g., VLDLR, LDL receptor (LDLR), and LRP1 (35
) that are expressed at the BBB and may have roles in signaling, endocytosis, and/or transcytosis of their respective ligands (36
), we next used lipoprotein receptor–specific antibodies (Fab2
) against VLDLR, LDLR, and LRP1 to determine whether blocking these receptors influences the efflux of apoE isoforms across the BBB. Specific receptor–blocking antibodies were infused in the ISF 15 minutes prior to tracer infusion and then simultaneously with the tracer mixture containing test apolipoproteins at their physiologic CSF concentration of 40 nM. Figure A shows that anti-VLDLR– and anti-LRP1–blocking antibodies inhibited the BBB efflux of lipo-apoE2 and lipo-apoE3 by 50% and 30%, and 58% and 40%, respectively, while anti-LDLR did not have an effect. A combination of anti-VLDLR and anti-LRP1 almost completely (~85%) inhibited apoE2 efflux at the BBB, whereas adding anti-LDLR to anti-VLDLR did not have an effect on apoE2 efflux inhibition greater than that of adding anti-VLDLR alone. The BBB clearance of both lipo-apoE2 and lipo-apoE3 was almost completely inhibited (>90%) by excess unlabeled ligand. These data suggest that VLDLR and LRP1 are likely to have a role in mediating apoE2 and apoE3 efflux at the BBB, whereas a nonspecific clearance accounts for less than 10% of the specific receptor–mediated clearance. In contrast, blocking LRP1 or LDLR did not have an effect on lipo-apoE4 efflux at the BBB (Figure A), whereas blocking VLDLR resulted in more than 85% inhibition. Adding anti-LRP1 or anti-LDLR to anti-VLDLR did not result in greater inhibition of lipo-apoE4 efflux compared with inhibition seen with adding anti-VLDLR alone. As with apoE3 and apoE2, excess unlabeled ligand inhibited 125
I–lipo-apoE4 clearance by more than 85%. These data suggest that VLDLR is a major receptor mediating lipo-apoE4 efflux at the BBB, whereas LRP1 is not involved. A minor portion (~10%) of BBB apoE4 clearance was by a nonspecific unsaturable transport, as with apoE2 and apoE3. A similar pattern for the receptors’ involvement was obtained with lipid-poor apoE2 and apoE4 (Supplemental Figure 3), suggesting VLDLR and LRP1 are required for efflux of apoE2 across the BBB, whereas VLDLR, but not LRP1, mediates very slow efflux of apoE4.
apoE isoform–specific clearance across the mouse BBB in vivo depends on differential contributions of VLDLR-mediated and LRP1-mediated transport.
The involvement of receptors was next tested using mice with specific deletions of the VLDLR and LDLR genes. First, we showed that deletion of the VLDLR gene does not alter the expression of LDLR and LRP1 proteins in brain capillaries and, similarly, that LDLR deletion does not alter the expression of VLDLR and LRP1 in brain capillaries (Figure B). Deletion of the VLDLR gene, however, reduced clearance of lipo-apoE2 and lipo-apoE3 by about 60% and clearance of lipo-apoE4 at the BBB by more than 80% (Figure C). Addition of an LRP1-specific blocking antibody led to an approximately 90% inhibition of apoE2 and apoE3 BBB efflux in VLDLR–/– mice compared with values in the wild-type mice (Figure C) but did not have an effect on lipo-apoE4 efflux (Figure C). These data confirmed that VLDLR is a major receptor for apoE4 clearance from brain, whereas both LRP1 and VLDLR clear apoE2 and apoE3 at the BBB. We performed a similar experiment in LDLR–/– mice and found that deletion of LDLR did not affect either lipo-apoE2 or lipo-apoE4 efflux at the BBB (Figure D). The addition of VLDLR and LRP1 antibodies decreased efflux of lipo-apoE2 in LDLR–/– mice by 58% and 32%, thus confirming the role of these 2 receptors in apoE2 clearance. Conversely, blocking LRP1 did not have any effect on lipo-apoE4 efflux in LDLR–/– mice, whereas VLDLR-specific antibodies diminished efflux of lipo-apoE4 by 85%, confirming that VLDLR is a major receptor required for slow apoE4 clearance at the BBB.
Since apoE binds Aβ with high affinity and is known to be an Aβ-binding protein (21
), we next determined whether binding of Aβ to apoE alters Aβ clearance across the BBB from preformed apoE-Aβ complexes. The formation of apoE2-Aβ40 and apoE4-Aβ40 complexes was demonstrated by 4%–20% Tris-glycine nondenaturing gradient gel electrophoresis for lipidated complexes and 10%–20% Tris-tricine native PAGE analysis for lipid-poor complexes (not shown), as we reported previously (33
). Size exclusion chromatography was used to remove excess free Aβ from all apoE-Aβ preparations. For example, in the case of a lipid-poor apoE2-Aβ40 complex, a peak eluting at 29 minutes that was positive for both apoE (3D12 antibody) and Aβ (6E10) represented an Aβ40-apoE complex (Figure A), whereas excess free Aβ eluted later with a peak at 32 minutes that was positive only for 6E10 (Aβ) and negative for 3D12 (apoE), indicating free Aβ. We then compared clearance of free Aβ40 versus Aβ40-apoE complexes with either apoE2 or apoE4 at equimolar physiologic CSF concentrations (40 nM). In contrast to free Aβ40, Aβ-apoE2 or Aβ-apoE4 complex was not cleared significantly at the BBB within 30 minutes (not shown). At 90 minutes, more than 85% of free Aβ40 was eliminated at the BBB exclusively through an LRP1-mediated transport (i.e., blockade or lack of VLDLR and LDLR did not influence Aβ efflux), as reported (16
). This clearance was much greater than the approximately 38% and 24% clearance of Aβ40 seen when it was complexed with lipid-poor apoE2 and apoE4, respectively (Figure B). The same results were obtained regardless of whether the label (125
I) was on apoE or Aβ. apoE lipidation further diminished the BBB efflux of Aβ40 to 15% and 9% via apoE2 and apoE4, respectively. Even more pronounced differences were obtained between Aβ42-apoE2 and Aβ42-apoE4 complexes (Figure C). For example, only 25% and 12% of Aβ42 was cleared via lipid-poor apoE2 and apoE4, respectively, whereas 9% and 3% of Aβ42 was cleared by lipo-apoE2 and lipo-apoE4, respectively, compared with 38% as seen for free unbound Aβ42.
apoE isoforms disrupt Aβ clearance across the mouse BBB in vivo (apoE4>apoE3 or apoE2) by redirecting differentially redirecting transport of Aβ-apoE complexes from LRP1 to VLDLR.
As we reported, there was minimal degradation of free monomeric Aβ40 or Aβ42 microinjected into the brain ISF (16
). In these relatively short-term kinetic studies and at apoE levels corresponding to physiological concentrations of apoE in the CSF, degradation of Ab was not significantly influenced by its binding to either apoE2, apoE3, or apoE4 (either lipid poor or lipidated) at 30 and 300 minutes. Degradation of both Aβ40 and Aβ42 was approximately 10% (Supplemental Figure 2, E and F; Figure D). Figure D shows the relative contributions of transport across the BBB, ISF flow, and degradation to the clearance of Aβ40 and Aβ42 when in complex with apoE2, apoE3, or apoE4 isoforms compared with free Aβ40 and Aβ2. The data indicate that binding of Aβ to apoE inhibits rapid efflux of Aβ40 and Aβ42 across the BBB in an isoform-specific fashion, i.e., Aβ clearance was inhibited to the greatest degree when in complex with apoE4 compared with clearance of Aβ-apoE3 and Aβ-apoE2, and this inhibition was significantly enhanced by apoE lipidation. There was a reciprocal relationship between reductions in BBB transport and accumulations of undegraded Aβ-apoE complexes in the brain, whereas the ISF flow and degradation were similar for all studied complexes. Aβ40 and Aβ42 efflux across the BBB was inhibited to the greatest degree when either was complexed with lipo-apoE4; efflux was 3-fold lower for such complexes compared with Aβ complexed with lipo-apoE3 or lipo-apoE.
We next used a panel of lipoprotein receptor–specific antibodies to determine whether the same receptors mediating apoE2, apoE3, and apoE4 efflux at the BBB are required for efflux of Aβ complexes with apoE2, apoE3, and apoE4. Clearance of 125
I-Aβ40–lipo-apoE2 and 125
I-Aβ40–lipo-apoE3 complexes at the BBB was inhibited by both VLDLR and LRP1 antibodies (Figure E); the involvement of VLDLR was confirmed in VLDLR–/–
mice, which exhibited a 60% reduction in 125
I-Aβ40–lipo-apoE2 clearance compared with littermate controls (Figure F). As seen with apoE2, anti-LRP1 inhibited the efflux of 125
I-Aβ40–lipo-apoE2 from brains in VLDLR–/–
mice by an additional 30%. In contrast, 125
I-Aβ42–lipo-apoE4 BBB clearance was inhibited by more than 80% in VLDLR–/–
mice compared with controls and was not affected by an LRP1-specific antibody (Figure G). Efflux of 125
I-Aβ40–lipo-apoE2 was significantly reduced (by approximately 40%) in RAP–/–
mice (Figure F), a functional LRP1 knockout with severely depleted (~80%) LRP1 levels at the BBB (17
). In contrast, 125
I-Aβ40–lipo-apoE4 efflux at the BBB was not affected in RAP–/–
mice (Figure G). These experiments confirm the results obtained with LRP1-specific blocking antibodies.
We then asked whether isoform-specific differences in apoE clearance across the BBB in vivo may reflect differences among the internalization rates of different apoE isoforms by their respective lipoprotein receptors at the abluminal side of the BBB. To address this question, we used isolated mouse brain microvessels as a model, as reported (17
). Lipid-poor apoE bound to the abluminal surfaces of isolated mouse brain microvessels in an isoform-specific manner, e.g., apoE2>apoE3>apoE4, and was almost displaced by excess unlabeled ligand (Figure A). Receptor-bound apoE2 and apoE3 were internalized by endocytosis with a t1/2
of about 3.9 ± 0.3 and 3.6 ± 0.4 minutes, respectively (Figure , B and C). Specific lipoprotein receptor–blocking antibodies were then used to identify the respective contributions of VLDLR and LRP1 in apoE2 and apoE3 endocytosis. First, we showed that apoE2 internalization was inhibited completely when both VLDLR and LRP1 were blocked as well as when there was excess unlabeled apoE2 (Figure B). When VLDLR only was blocked, apoE2 internalization reflected endocytosis via LRP1 that was extremely rapid, with a t1/2
of less than 30 seconds, consistent with the previously shown rapid endocytic rate of LRP1 (17
). In contrast, when LRP1 was blocked, the apoE2 internalization was much slower, with a t1/2
of 8.5 ± 1.5 minutes. This is consistent with a previous study demonstrating that VLDLR has the slowest internalization rate of all lipoprotein receptors (32
). Similar results suggesting a rapid efflux component via LRP1 and a slow efflux component via VLDLR were obtained for apoE3 (Figure C). We next repeated the same experiment with apoE4 and found that its internalization rate was much slower than that of apoE2 and apoE3, with a t1/2
of 8.7 ± 1.5 minutes (Figure D). Blockade of VLDLR resulted in almost complete inhibition of apoE4 internalization, whereas blockade of LRP1 did not affect apoE4 endocytosis, consistent with our in vivo findings. LRP1- and VLDLR-specific antibodies together did not have a greater effect on inhibition of apoE4 internalization than VLDLR antibody alone. Therefore, in the presence of an LRP1 antibody, apoE4 endocytosis was mediated via VLDLR, with a t1/2
of 8.9 ± 1.3 minutes, which was comparable to a t1/2
of VLDLR-mediated internalization for the Aβ-apoE2 and Aβ-apoE3 complexes. These results suggest that LRP1 contributed to a substantially faster internalization rate at the BBB of apoE2 and apoE3 compared with apoE4, which was internalized slowly by VLDLR only. During these short-term kinetic internalization studies, there was low degradation (<5%) of apoE2 and apoE4, as determined by their respective TCA nonprecipitable fractions in brain vessel lysates and in the incubation medium over the studied short periods of time (not shown).
Isoform-specific lipid-poor apoE clearance at the abluminal surface of mouse brain capillaries in vitro is regulated by differential internalization rates of VLDLR and LRP1.
Next, we used astrocyte-derived lipo-apoE particles to determine whether the same internalization receptor requirements held as for the lipid-poor apoE isoforms. There was again an isoform-specific difference in lipo-apoE2 versus lipo-apoE4 binding (Figure A). The internalization rate of lipo-apoE2 was significantly faster than that of lipo-apoE4 (Figure , B and C), with the respective t1/2 values of 3.9 ± 0.4 minutes and 8.4 ± 1.4 minutes, which were comparable to the t1/2 values of their lipid-poor counterparts (see above). A combination of VLDLR- and LRP1-specific blocking antibodies resulted in complete inhibition of lipo-apoE2 internalization, whereas inhibition of VLDLR revealed a fast LRP1 component of lipo-apoE2 internalization, with a t1/2 of less than 30 seconds (Figure B). Internalization of lipo-apoE4 was almost completely blocked with a VLDLR-specific antibody, revealing no fast LRP1 component, as seen for lipid-poor apoE4 (Figure C). By using isolated capillaries from VLDLR–/– and LDLR–/– mice, we confirmed that LDLR was not involved in uptake of lipo-apoE2 or lipo-apoE4 (not shown), whereas deletion of VLDLR resulted in a greater than 60% reduction in apoE2 binding (Figure D) and internalization (Figure E) as well as in an approximately 60% inhibition in lipo-apoE3 internalization (Figure F). In VLDLR–/– mice, the internalization of lipo-apoE2 or lipo-apoE3 was inhibited up to 90% by addition of an LRP1-specific antibody (Figure , E and F). Internalization of lipo-apoE4 was inhibited by approximately 80% in VLDLR–/– mice (Figure G).
Isoform-specific lipo-apoE clearance at the abluminal surface of mouse brain capillaries in vitro is regulated by differential internalization rates of VLDLR and LRP1.
Binding and internalization of apoE-Aβ complexes at the abluminal surface of brain microvessels was next studied using the fast protein liquid chromatography–purified (FPLC-purified) apoE2-Aβ40 and apoE4-Aβ40 complexes as above. Aβ40-apoE2 and Aβ42-apoE2 complexes bound to both VLDLR and LRP1, whereas Aβ40-apoE4 and Aβ42-apoE4 complexes bound only to VLDLR, not to LRP1, as shown with the lipoprotein receptor–specific blocking antibodies (Figure A). Binding of radiolabeled complexes was inhibited by more than 90% by excess unlabeled ligand. The internalization rate of free Aβ40 was rapid, i.e., t1/2
was less than 30 seconds and was completely inhibited by an LRP1-specific antibody, as reported (17
). The internalization rates of Aβ40 complexes with lipo-apoE2 and lipo-apoE3 were comparable but substantially lower than for Aβ40 alone, as indicated by their respective internalization curves (Figure B). There was a clear isoform-specific effect, i.e., lipo-apoE2 and lipo-apoE3 internalized Aβ40 at rates significantly higher than lipo-apoE4 (Figure B). As shown in Figure C, both VLDLR and LRP1 were involved in endocytosis of Aβ40 via lipo-apoE2 and lipo-apoE3, whereas VLDLR was the key receptor for internalization of Aβ40–lipo-apoE4 complex. LRP1-dependent internalization of Aβ40 was shown by comparison.
apoE isoform–specific inhibition (apoE4>apoE3 and apoE2) of Aβ internalization at the abluminal surface of mouse brain capillaries in vitro is mediated by VLDLR.