Functional characterization of the brain-to-blood efflux clearance of human amyloid-β peptide (1–40) across the rat blood–brain barrier
Introduction
An abnormally elevated level of amyloid-β peptide (Aβ) in the brain is one of the prominent features of Alzheimer's disease (AD) (Hardy and Selkoe, 2002). Aβ is a 38–43 amino acid peptide derived from the proteolytic processing of amyloid precursor protein (APP), and Aβ(1–40) is the major component of amyloid plaques in brain. When [125I] human Aβ(1–40) (hAβ(1–40)) was microinjected into the brain, it was eliminated from the brain to the blood via the blood–brain barrier (BBB) (Shibata et al., 2000, Shiiki et al., 2004), which consists of brain capillary endothelial cells (Terasaki and Ohtsuki, 2005). This elimination process could play an important role in preventing the accumulation of hAβ(1–40) in the brain. On the other hand, exogenous [125I]hAβ(1–40) administered to the circulating blood or via the carotid artery was transferred into the brain (Martel et al., 1997, Wengenack et al., 2000, Deane et al., 2003), suggesting that peripheral hAβ(1–40) is also transported into the brain across the BBB. Therefore, both blood-to-brain and brain-to-blood transport determine the net flux of Aβ transport across the BBB and would play an important role in the accumulation of cerebral Aβ.
In vivo cerebral Aβ clearance has been reported to involve a specific receptor (low-density lipoprotein receptor-related protein 1 (LRP-1)) (Shibata et al., 2000) and proteases (neprilysin (NEP) (Iwata et al., 2001), insulin-degrading enzyme (IDE) (Farris et al., 2003) and endothelin-converting enzyme (ECE) (Eckman et al., 2003)). Since the involvement of each of these in cerebral hAβ(1–40) clearance has been examined in different in vivo systems, the contribution that each of these molecules makes to the clearance system is still unclear. Indeed, it has been reported that LRP-1 is involved in hAβ(1–40) efflux transport in the mouse brain using human receptor-associated protein (RAP) (Shibata et al., 2000), while our recent report failed to demonstrate a significant contribution of LRP-1 to hAβ(1–40) efflux transport in the rat brain using rat RAP (Shiiki et al., 2004).
P-glycoprotein (P-gp) is expressed on the luminal membrane of brain capillary endothelial cells (BCEC) and excretes substrates into the circulation. Recent studies have reported that hAβ(1–40) and hAβ(1–42) could be substrates of P-gp (Lam et al., 2001) and the expression levels of P-gp inversely correlate with Aβ deposition in the brain in elderly humans and P-gp knockout (multidrug resistance 1a/b−/−) mice (Vogelgesang et al., 2002, Cirrito et al., 2005). However, P-gp gene deficiency reduced the expression level of LRP-1 in brain capillaries (Cirrito et al., 2005). Furthermore, since P-gp is expressed in the liver which is the major organ responsible for systemic hAβ(1–40) clearance (Ghiso et al., 2004), P-gp gene deficiency is likely to affect systemic hAβ(1–40) clearance. Therefore, it is still unknown whether P-gp directly contributes to brain-to-blood transport of hAβ(1–40) at the BBB in vivo. It is of crucial importance to characterize in vivo BBB Aβ efflux transport and, particularly, the contribution of LRP-1 and/or P-gp as part of the efflux system preventing the accumulation of Aβ in the brain.
The purpose of this study was to clarify the brain-to-blood efflux clearance of hAβ(1–40) by combining brain efflux index (BEI) and brain slice uptake studies. We also investigated the contribution of LRP-1 and P-gp to cerebral Aβ clearance in rats by means of a BEI study.
Section snippets
Animals
Male and female Sprague–Dawley rats (7–8 weeks old) were purchased from Charles River Laboratories (Yokohama, Japan). All experiments were approved by the Animal Care Committee of the Graduate School of Pharmaceutical Sciences, Tohoku University.
Reagents
Monoiodinated and non-oxidized [125I]hAβ(1–40) (2200 Ci/mmol) was purchased from Perkin-Elmer Life Sciences (Boston, MA, USA). [3H]Dextran was obtained from American Radiolabeled Chemicals (St. Louis, MO, USA). [carboxy-14C]inulin ([14C]inulin, 1.92
The apparent brain-to-blood efflux clearance of [125I]hAβ(1–40) in rats
The in vivo brain-to-blood efflux transport of [125I]hAβ(1–40) was determined using the BEI method with [3H]dextran as a reference compound. [125I]hAβ(1–40) was injected into rat brain at a concentration of 18.2 nM in the injectate (0.6 nM as the cerebral concentration), which was lower than the half saturation concentration of [125I]hAβ(1–40) elimination we have reported (247 nM in injectate and 8.15 nM as cerebral concentration; Shiiki et al., 2004). The microinjected [125I]hAβ(1–40) was
Discussion
The present study shows, firstly, that the apparent brain-to-blood efflux clearance of hAβ(1–40) from the male rat cortex is 11.0 μL/(min g brain) using a combination of BEI and brain slice uptake studies. The elimination of hAβ(1–40) from rat brain consists of partial LRP-1-mediated transport and P-gp does not significantly contribute to the elimination.
The microinjected [125I]hAβ(1–40) was eliminated from the rat brain in a time-dependent manner. In the BEI study, [3H]mannitol (MW 182), [14
Acknowledgements
The authors would like to thank Drs. T. Iwatsubo and T. Hashimoto for valuable discussions, and Ms. N. Funayama for secretarial assistance. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas 17025005 from The Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and a 21st Century Center of Excellence (COE) Program grant from the Japan Society for the Promotion of Science.
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