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. 2012 Jun 29:9:150.
doi: 10.1186/1742-2094-9-150.

Lipopolysaccharide impairs amyloid β efflux from brain: altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood-brain barrier

Michelle A Erickson  1 , Pehr E Hartvigson, Yoichi Morofuji, Joshua B Owen, D Allan Butterfield, William A Banks
Affiliations

Lipopolysaccharide impairs amyloid β efflux from brain: altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood-brain barrier

Michelle A Erickson et al. J Neuroinflammation. .

Abstract

Background: Defects in the low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein (Pgp) clearance of amyloid beta (Aβ) from brain are thought to contribute to Alzheimer's disease (AD). We have recently shown that induction of systemic inflammation by lipopolysaccharide (LPS) results in impaired efflux of Aβ from the brain. The same treatment also impairs Pgp function. Here, our aim is to determine which physiological routes of Aβ clearance are affected following systemic inflammation, including those relying on LRP-1 and Pgp function at the blood-brain barrier.

Methods: CD-1 mice aged between 6 and 8 weeks were treated with 3 intraperitoneal injections of 3 mg/kg LPS at 0, 6, and 24 hours and studied at 28 hours. 125I-Aβ1-42 or 125I-alpha-2-macroglobulin injected into the lateral ventricle of the brain (intracerebroventricular (ICV)) or into the jugular vein (intravenous (IV)) was used to quantify LRP-1-dependent partitioning between the brain vasculature and parenchyma and peripheral clearance, respectively. Disappearance of ICV-injected 14 C-inulin from brain was measured to quantify bulk flow of cerebrospinal fluid (CSF). Brain microvascular protein expression of LRP-1 and Pgp was measured by immunoblotting. Endothelial cell localization of LRP-1 was measured by immunofluorescence microscopy. Oxidative modifications to LRP-1 at the brain microvasculature were measured by immunoprecipitation of LRP-1 followed by immunoblotting for 4-hydroxynonenal and 3-nitrotyrosine.

Results: We found that LPS: caused an LRP-1-dependent redistribution of ICV-injected Aβ from brain parenchyma to brain vasculature and decreased entry into blood; impaired peripheral clearance of IV-injected Aβ; inhibited reabsorption of CSF; did not significantly alter brain microvascular protein levels of LRP-1 or Pgp, or oxidative modifications to LRP-1; and downregulated LRP-1 protein levels and caused LRP-1 mislocalization in cultured brain endothelial cells.

Conclusions: These results suggest that LRP-1 undergoes complex functional regulation following systemic inflammation which may depend on cell type, subcellular location, and post-translational modifications. Our findings that systemic inflammation causes deficits in both Aβ transport and bulk flow like those observed in AD indicate that inflammation could induce and promote the disease.

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Figures

Figure 1
Figure 1
Distribution of ICV-injected murine125I-Aβ1-42(a–c) or activated 125I-a2M (d,–f) in the vasculature (a,d), parenchyma (b,e), and serum (c,f) after treatment with LPS. Data analyzed by two-tailed t-test, n = 10–11 per group, *P < 0.05; ***P < 0.001.
Figure 2
Figure 2
Peripheral clearance of murine125I-Aβ1-42 from blood (a), and uptake by liver (b) or kidney (c) following LPS. Clearance rate (%Inj/ml-min) of Aβ from serum and unidirectional influx rates for liver and kidney (μl/g-min) are shown in (d). Data analyzed by linear regression analysis, n = 7–10 per group, *P < 0.05, **P < 0.01.
Figure 3
Figure 3
Brain efflux of ICV-injected14 C inulin (a) and corresponding appearance in serum (b) after treatment with LPS. Lower values indicate slower efflux. Data analyzed by two-tailed t-test, n = 10 per group, *P < 0.05, **P < 0.01.
Figure 4
Figure 4
Protein expression of LRP-1 and Pgp in pooled brain microvessels isolated from mice treated with saline or LPS. Representative immunoblot images shown for LRP-1 (a) and Pgp (d). Results from densitometric analysis of the small (b) and large (c) subunits of LRP-1, and Pgp (e) are shown as bar graphs. Data analyzed by two-tailed t-test, n = 4–5 microvessel pools (3 brains/pool) per group.
Figure 5
Figure 5
Oxidative modifications to brain microvascular LRP-1 following LPS treatment. Representative immunoblots for 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (HNE)-modified LRP-1 and immunoprecipitated LRP-1 are shown in (a). Results from densitometric analysis of 3-NT (b) and HNE (c)-modified LRP-1 normalized to total LRP-1 levels are shown as bar graphs. Data analyzed by two-tailed t-test, n = 4–5 microvessel pools (10 brains/pool) per group.
Figure 6
Figure 6
Decreased protein expression of LRP-1 in cultured HBECs and RBE4 cells following LPS treatment. Immunoblots of LRP-1 and γ-tubulin from HBECs are shown in (a), and densitometric analysis of LRP-1 small subunit expression shown in (b). Data from dot blot analysis of LRP-1 in RBE4 cells is shown in (c). Data analyzed by two-tailed t-test, n = 3 per group (HBEC) or 6 per group (RBE4), *P < 0.05.
Figure 7
Figure 7
Mislocalization of LRP-1 and ZO-1 in cultured MBECs following LPS treatment. MBECs grown on Transwell inserts were treated with LPS and stained for LRP-1 (red), ZO-1 (green), and DAPI (blue). Images captured at 400× magnification.
Figure 8
Figure 8
Schematic of mechanisms at the neurovascular unit which contribute to LPS-induced decreases in Aβ efflux by the BBB. Decreased efflux at the brain endothelial cell (EC) is due to functional impairment of LRP-1 (a) that may involve (b) or not involve (c) Pgp (b). Impairment of Pgp could also result in increased vascular uptake of Aβ from the periphery (d). Increased internalization of Aβ in the pericyte (PC) through upregulation of LRP-1 may also contribute to Aβ partitioning in the brain vasculature (e). Dashed line across endothelial cell indicates unknown subcellular routes in the translocation of Aβ.
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