Transport pathways for clearance of human Alzheimer's amyloid beta-peptide and apolipoproteins E and J in the mouse central nervous system

Abstract

Amyloid beta-peptide (Abeta) clearance from the central nervous system (CNS) maintains its low levels in brain. In Alzheimer's disease, Abeta accumulates in brain possibly because of its faulty CNS clearance and a deficient efflux across the blood-brain barrier (BBB). By using human-specific enzyme-linked immunosorbent assays, we measured a rapid 30 mins efflux at the BBB and transport via the interstitial fluid (ISF) bulk flow of human-unlabeled Abeta and of Abeta transport proteins, apolipoprotein E (apoE) and apoJ in mice. We show (i) Abeta40 is cleared rapidly across the BBB via low-density lipoprotein receptor-related protein (LRP)1 at a rate of 0.21 pmol/min g ISF or 6-fold faster than via the ISF flow; (ii) Abeta42 is removed across the BBB at a rate 1.9-fold slower compared with Abeta40; (iii) apoE, lipid-poor isoform 3, is cleared slowly via the ISF flow and across the BBB (0.03-0.04 pmol/min g ISF), and after lipidation its transport at the BBB becomes barely detectable within 30 mins; (iv) apoJ is eliminated rapidly across the BBB (0.16 pmol/min g ISF) via LRP2. Clearance rates of unlabeled and corresponding 125I-labeled Abeta and apolipoproteins were almost identical, but could not be measured at low physiologic levels by mass spectrometry. Amyloid beta-peptide 40 binding to apoE3 reduced its efflux rate at the BBB by 5.7-fold, whereas Abeta42 binding to apoJ enhanced Abeta42 BBB clearance rate by 83%. Thus, Abeta, apoE, and apoJ are cleared from brain by different transport pathways, and apoE and apoJ may critically modify Abeta clearance at the BBB.

Fig. 1

a, SDS-PAGE analysis of crude extract of human plasma apoJ. b, HPLC purification of the crude apoJ extract. c, SDS-PAGE analysis of the apoJ peak in b (reduced conditions). ApoAI, apolipoprotein A one.

Fig. 2

a, Time-disappearance curves of unlabeled human Aβ40 (0.0866 ng) and ¹⁴C-inulin from brain ISF after their simultaneous administration into the caudate nucleus in mice. Each pair of time points represents data from individual mice for Aβ and inulin. b, Clearance of unlabeled human synthetic Aβ40 and Aβ42 from brain ISF within 30 min of administration into the caudate nucleus. Aβ40 and Aβ42 were infused simultaneously with ¹⁴C-inulin. c, Levels of Aβ40 in brain after 30 min of its simultaneous administration with inulin (not shown) in the presence and absence of anti-LRP1 (N-20, 60 μg/mL), non-immune IgG (60 μg/mL) and RAP (5 μM). d, Intact human unlabeled Aβ40 and Aβ42 in plasma 30 min after local CNS administration of peptides simultaneously with ¹⁴C-inulin in the presence and absence of centrally administered anti-LRP1 (N-20, 60 μg/mL). Inulin levels were barely detectable (not shown). Aβ levels were determined focally in brain and plasma by using human specific ELISAs, as described in Methods. In b-d, values are mean ± s.e.m. from 3 to 5 independent experiments.

Fig. 3

a, Clearance of unlabeled human apoE (isoform 3; 0.70 ng), non-lipidated and lipidated, from brain ISF within 30 min of administration into the caudate nucleus. ApoE was infused simultaneously with ¹⁴C-inulin. b, Clearance of native human apoJ (1.6 ng) from brain ISF within 30 min of its simultaneous administration into the caudate nucleus with ¹⁴C-inulin. c, Clearance of unlabeled Aβ40 and apoE (isoform 3) administered into brain ISF simultaneously with ¹⁴C-inulin either alone (closed bars) or in the form of Aβ- apoE complex (open bars). Inulin values not shown. ApoE, apoJ and Aβ levels were determined focally in brain and plasma by using human specific ELISAs, as described in Methods. Values are mean ± s.e.m. from 3 to 5 independent experiments.

Fig. 4

a, Brain clearance of unlabeled (solid bars) and ¹²⁵I-labeled (open bars) Aβ and apolipoproteins from brain ISF. The same amounts of unlabeled and the respective ¹²⁵I-labeled Aβ40, Aβ42, apoE non-lipidated and lipidated isoform 3 and apoJ were infused with ¹⁴C-inulin into brain ISF in the caudate nucleus. The levels of unlabeled Aβ and apoE were determined by human specific ELISAs (solid bars) and of ¹²⁵I-labeled Aβ and apoE by gamma counting (open bars). The percentage recovery was calculated as N_t/N₀ × 100 (eq. 1, Methods). Mean ± s.e.m., n = 3-8. b, Clearance of ¹²⁵I-apoJ (1.6 ng/0.5 μL; TCA-precipitable radioactivity) in the absence and presence of RAP (5 μM), anti-LRP-2 (Rb 6286, 60 μg/ml, from Dr. S. Argraves ) and anti-LRP-1 (N20, 60 μg/ml) simultaneously infused with ¹⁴C-inulin. c, Effects of apoJ on ¹²⁵I-Aβ42 clearance from brain ISF and effect of anti-LRP2 (Rb 6286, 60 μg/ml ). For b-c, values are mean ± s.e.m., n = 3-5.