Lipopolysaccharide alters the blood-brain barrier transport of amyloid beta protein: a mechanism for inflammation in the progression of Alzheimer's disease

Abstract

Alzheimer's disease (AD) brains are characterized by accumulation of amyloid beta protein (Abeta) and neuroinflammation. Increased blood-to-brain influx and decreased brain-to-blood efflux across the blood-brain barrier (BBB) have been proposed as mechanisms for Abeta accumulation. Epidemiological studies suggest that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the progression of AD. We hypothesized that inflammation alters BBB handling of Abeta. Mice treated with lipopolysaccharide (LPS) had increased brain influx and decreased brain efflux of Abeta, recapitulating the findings in AD. Neither influx nor efflux was mediated by LPS acting directly on BBB cells. Increased influx was mediated by a blood-borne factor, indomethacin-independent, blocked by the triglyceride triolein, and not related to expression of the blood-to-brain transporter of Abeta, RAGE. Serum levels of IL-6, IL-10, IL-13, and MCP-1 mirrored changes in Abeta influx. Decreased efflux was blocked by indomethacin and accompanied by decreased protein expression of the brain-to-blood transporter of Abeta, LRP-1. LPS paradoxically increased expression of neuronal LRP-1, a major source of Abeta. Thus, inflammation potentially increases brain levels of Abeta by three mechanisms: increased influx, decreased efflux, and increased neuronal production.

Figure 1. LPS Increases BBB Transport of I-Aβ in the Blood-to-brain Direction (Influx)

(a) Dose response curve of the effect of LPS on the brain uptake of intravenously injected I-Aβ. ANOVA showed means were significantly different (p < 0.001). Newman-Keuls post analysis showed that only the 3 mg/kg dose was associated with significantly increased brain uptake of I-Aβ (*** = p < 0.001 vs. all other treatment groups). (b) Serum clearance of intravenously injected I-Aβ. LPS pretreatment (solid circles) increased serum concentrations of I-Aβ in comparison to saline injected mice (open circles), possibly because of volume contraction (n = 24 control and n = 19 LPS). (c) Multiple-time regression analysis for brain uptake of intravenously injected I-Aβ. LPS treatment (closed circles) was associated with an increase in brain uptake of I-Aβ. (n = 24 control and n = 10 LPS). Data is from the same mice as in panel b. (d) LPS treatment significantly increased brain uptake of intravenous I-Aβ 30 min after iv injection (n = 6-7/group, * = p < 0.05). The brain/serum ratio for simultaneously injected radioactive albumin was subtracted, thus correcting for any disruption of the BBB induced by LPS.

Figure 2. LPS Decreases the BBB Transport of I-Aβ in the Brain-to-blood Direction (Efflux)

(a) Dose response curve of the effects of LPS on brain clearance of I-Aβ given by brain intraventricular (ICV) administration. One-way ANOVA showed means are significantly different (p < 0.01) and Newman-Keuls post analysis showed that only the 3 mg/kg dose produced a significant reduction in brain clearance of I-Aβ (*** = p < 0.01 vs. all other treatment groups). For all experiments, n = 4-8 mice/group. (b) Time curve of brain clearance of I-Aβ after its ICV administration(n = 2-4/time point). Statistical comparison of the lines showed them to be significantly different: [F(1,16) = 9.39,p < 0.01]. LPS increased the half-time clearance of I-Aβ from brain from 19.1 min to 52 min.

Figure 3. Effect of LPS on I-Aβ Transport is Indirect

(a) LPS treatment did not alter brain uptake of I-Aβ perfused in buffer for 5 min through the left ventricle of the heart (n = 5-6/group). This method perfuses the I-Aβ directly through the vasculature of the brain in buffer, eliminating the influence of serum factors on BBB transport. Lack of effect in LPS-treated mice demonstrates that a serum factor is an immediate mediator of the LPS effect on efflux. Panels B-D: LPS effects on monolayer cultures of brain microvascular endothelial cell(BMEC) cultures. The TEER measures were taken at the end of the influx and efflux experiments shown in panels c-d. (b) LPS had a dose-dependent effect on transendothelial electrical resistance (TEER), demonstrating that LPS disrupted the BBB through a paracellular pathway. (c) LPS did not have an effect on I-Aβ influx in the BMEC model (n = 13-14 wells/group). (d) LPS did not have an effect on I-Aß efflux in the BMEC model (n = 14-15 wells/group).

Figure 4. Role of the Triglyceride Triolein on Aß Blood-to-brain Transport

Injection of triolein with I-Aß had no effect on BBB transport in control mice, but significantly blocked the effect of LPS (n = 5-9/group, * = p < 0.05 for LPS treated vs. all other groups).

Figure 5. Effects of Indomethacin on LPS-mediated changes in Aβ transport

Mice were pretreated with indomethacin (20 mg/kg) 30 min before administration of LPS (a) Pretreatment with indomethacin did not significantly affect brain influx of I-Aβ (n = 6-8/group, # = p < 0.05 vs. Indomethcain and ** = p < 0.01 vs. Saline). (b) Indomethacin pretreatment inhibited the LPS-mediated effects on I-Aβ efflux (n = 6-10 mice/group, # = p < 0.05 vs. Saline and Indomethacin and ** = p < 0.01 vs. Indomethacin + LPS).

Figure 6. Effects of LPS on Expression of BBB RAGE, BBB LRP, and Neuronal LRP

Brain microvessels (a-b) or brain homogenate (c-d) were obtained from mice trerated with subacute LPS. (a) Western blot analysis for RAGE showed no effect of LPS on the protein levels in brain microvessels of this blood-to-brain transporter of Aß. (b) Western blot analysis for 515 kDa subunit of the BBB Aβ clearance transporter, LRP-1, in isolated mouse brain microvessels from mice treated with saline, LPS and indomethacin as indicated (n = 20-22 cortices/microvessel isolation with 1 isolation/band, analyzed 5 times). LPS decreased LRP protein expression (*p<0.05) but indomethacin was without effect. (c) LRP mRNA and LRP protein (d) levels from whole brain in mice treated with saline (n = 6) or LPS (n = 6-7). LPS increased both mRNA (*p<0.05) and protein (**p<0.01) expression in whole brain, indicating upregulation of neuronal LRP.