Lead-induced accumulation of beta-amyloid in the choroid plexus: role of low density lipoprotein receptor protein-1 and protein kinase C

Abstract

The choroid plexus (CP), constituting the blood-cerebrospinal fluid barrier, has the capacity to remove beta-amyloid (Abeta) from the cerebrospinal fluid. Our previous work indicates that exposure to lead (Pb) results in Abeta accumulation in the CP by decreasing the expression of low density lipoprotein receptor protein-1 (LRP1), a protein involved in the transport and clearance of Abeta. The current study was designed to explore the relationship between Abeta accumulation, protein kinase C (PKC) activity, and LRP1 status in the CP following Pb exposure. Confocal microscopy revealed that LRP1 was primarily localized in the cytosol of the CP in control rats and migrated distinctly towards the apical surface and the microvilli following acute Pb exposure (27 mg Pb/kg, i.p., 24h). Co-immunostaining revealed a co-localization of both PKC-delta and LRP1 in the cytosol of control rats, with a distinct relocalization of both towards the apical membrane following Pb exposure. Preincubation of the tissues with PKC-delta inhibitor rottlerin (2 microM) prior to Pb exposure in vitro, resulted in abolishing the Pb-induced relocalization of LRP1 to the apical surface. Importantly, a significant elevation in intracellular Abeta levels (p<0.01) was observed in the cytosol of the CP following Pb exposure, which was abolished following preincubation with rottlerin. In addition, rottlerin caused a relocalization of Abeta from the cytosol to the nucleus in both Pb-treated and control CP tissues. Finally, co-immunoprecipitation studies revealed a strong protein-protein interaction between LRP1 and PKC-delta in the CP. These studies suggest that Pb exposure disrupts Abeta homeostasis at the CP, owing partly to a Pb-induced relocalization of LRP1 via PKC-delta.

Fig. 1

Intracellular distribution of LRP1 in rat CP tissues following in vivo acute Pb exposure. Rats received a single ip injection of Pb at a concentration of 27 mg Pb/kg as Pb acetate. The control rats received an equivalent concentration of Na acetate. Twenty-four hours later, the CP tissues were collected. (A–B) Representative CP tissues from a control rat. The arrow indicates a relatively even distribution of LRP1 in the cytosol of choroidal epithelia. (D–E) Representative CP tissues from Pb-exposed rats. The arrow indicates relocalization of LRP1 from the cytoplasm towards the apical pole and in the brush border of the choroidal epithelia. (C, F) Overlays of A & B and B & E, respectively. (G) represents a negative control. The tissue was treated in the same way as in (D) except that it was not exposed to any of the primary antibodies. (B, E, H) Representive corresponding transmission images. The data indicate a normal morphology of the tissues. All images are representative of experiments performed in triplicates.

Fig. 2

Co-localization of LRP1 and PKC-δ in the rat CP tissues following acute Pb exposure in vivo. (A–C) Confocal image of LRP1 and PKC-δ in the cytosol of a representative CP tissue from a control rat. (D–F) Confocal image of LRP1 and PKC-δ in a representative CP tissue from a Pb exposed rat. LRP1 is stained in green (A, D) and PKC-δ in red (B, E). (G–I) represents a negative control which was treated similarly except that it was not exposed to any of the primary antibodies. (C, F) represents an overlay of LRP1 and PKC-δ in controls and Pb-treated rats, respectively. Please see the legend in Fig. 1 for Pb-dose regimen. The data are representative of experiments performed in triplicates.

Fig. 3

Inhibition of Pb-induced migration of LRP1 by rottlerin. Rat CP tissues were isolated and pre-treated with rottlerin (2 µM, 20 min) alone or followed by Pb (10 µM, 1 h) exposure in vitro. (A–C) Confocal image of LRP1 and PKC-δ in the cytosol of a representative CP tissue from a control rat. (D–F) Confocal image of LRP1 and PKC-δ in a representative CP tissue from a Pb-exposed tissue in absence of rottlerin pre-treatment. (G–I) Confocal image of a representative CP tissue pre-treated with rottlerin in the absence of Pb. (J–L) Representative image of a CP tissue pre-treated with rottlerin followed by Pb exposure. LRP1 is stained in green (A, D, G, J), and PKC-δ in red (B, E, H, K). The data are representative of experiments performed in triplicate.

Fig. 4

Co-immunoprecipitation of LRP1 and PKC-δ for protein-protein interaction. Lysates from control Z310 cells were captured with anti-LRP1 in the presence of protein-A agarose beads and the complex was run on SDS-PAGE. (A) immunoblot with anti-LRP1. (B) immunoblot with anti- PKC-δ. The data are representative of experiments performed in triplicate.

Fig. 5

Rottlerin attenuated the Pb-induced increase in Aβ accumulation. (A,C) Confocal image from a representative CP tissue of a control rat demonstrating the accumulation of Aβ primarily in the cytosol with minimal staining in the nuclei. (D, F). Confocal image from a representative CP tissue in a Pb-exposed rat demonstrating an increase in Aβ signal. (G, I) Confocal image from a representative CP tissue pre-treated with rottlerin in absence of Pb. (J, L) Confocal image from a representative CP tissue pre-treated with rottlerin in presence of Pb. (B, E, H, K) represent transmission images revealing normal morphology of the tissues. Please see the legend to Fig.3 for Pb-dose regimen. The data are representative of experiments from four replicates.

Fig. 6

Quantification of the fluorescent signals using Laser Scanning Cytometry. The fluorescent signals in Fig. 5 were quantified using software Image J. The bar designated Pb(−)/rottlerin (−) represents untreated controls, Pb(+)/rottlerin(−) represents treatment with Pb in absence of rottlerin, Pb(−)/rottlerin (+) represents incubation with rottlerin in absence of Pb, and Pb (+)/rottlerin(+) represents tissues pre-treated with rottlerin followed by Pb. Data represent mean ± SD, n=16 (a total of 16 cells per group taken from 4 tissue samples with fluorescence averaged from 4 cells per sample). Bars with different superscripts labeled as a,b,c indicate significant difference from one another.