Gelsolin restores A beta-induced alterations in choroid plexus epithelium

Abstract

Histologically, Alzheimer's disease (AD) is characterized by senile plaques and cerebrovascular amyloid deposits. In previous studies we demonstrated that in AD patients, amyloid-beta (A beta) peptide also accumulates in choroid plexus, and that this process is associated with mitochondrial dysfunction and epithelial cell death. However, the molecular mechanisms underlying A beta accumulation at the choroid plexus epithelium remain unclear. A beta clearance, from the brain to the blood, involves A beta carrier proteins that bind to megalin, including gelsolin, a protein produced specifically by the choroid plexus epithelial cells. In this study, we show that treatment with gelsolin reduces A beta-induced cytoskeletal disruption of blood-cerebrospinal fluid (CSF) barrier at the choroid plexus. Additionally, our results demonstrate that gelsolin plays an important role in decreasing A beta-induced cytotoxicity by inhibiting nitric oxide production and apoptotic mitochondrial changes. Taken together, these findings make gelsolin an appealing tool for the prophylactic treatment of AD.

Figure 1

Gelsolin expression in choroid plexus epithelial cells. (a) Antimegalin immunoprecipitation of rat choroid plexus cell extracts, followed by blotting with respective antibodies, revealed an association between megalin, endogenous gelsolin, the exogenously added secreted gelsolin form, and the exogenously added Aβ. Immunoprecipitation with nonspecific serum showed no unspecific Aβ association. Binding of gelsolin with exogenously added Aβ was also observed. Representative blots are shown (n = 4). (b) Megalin colocalized with gelsolin and exogenously added Aβ in choroid plexus cultures. Confocal images also show gelsolin colocalization with Aβ. Scale  bars = 10 μm. IP: Immunoprecipitation; NRS: normal rabbit serum.

Figure 2

Secreted gelsolin inhibits Aβ-induced disruption on choroid plexus epithelial cell cytoskeleton. (a) Representative confocal images of choroid plexus confluent monolayer labeled with anti-ZO-1 antibody. Under control conditions, ZO-1 immunostaining is distributed along the plasma membrane. In contrast, after exposure to Aβ_1–42 for 48 hours, a disruption of the plasma membrane pattern of ZO-1 was observed, resulting in increased cytoplasmic localization. Note the ability of gelsolin treatment to prevent this Aβ-induced alteration in ZO-1 pattern. (n = 3). (b) Aβ_1–42 treatment resulted in increased serine phosphorylation of ZO-1 and decreased ZO-1 expression in choroid plexus epithelial cells. Gelsolin coadministration markedly attenuated Aβ_1–42 alteration in ZO-1 (n = 3). (c) BIODIPY FL phallacidin staining of choroid plexus epithelial cells showed a disruption of the actin cytoskeleton after treatment with Aβ_1–42 for 48 hours, and reversion when gelsolin was simultaneously added. Magnification: ×40. Scale bars = 10 μm.

Figure 3

Secreted gelsolin expression modulates NO production and cell death in choroid plexus epithelial cells. (a) Choroid plexus epithelial cells treated with Aβ_1–42 for 48 hours exhibited a significantly enhanced NO production compared with untreated cells. Secreted gelsolin coadministration completely blocked this effect (n = 3); **P < .01. (b) Increased cell death was observed in choroid plexus cell cultures 48 hours after Aβ_1–42 treatment, and gelsolin addition totally reversed this toxic effect (n = 3); *P < .05. (c and d) Aβ_1–42 treatment reduced mitochondrial complex IV in-gel activity in choroid plexus epithelial cells, whereas secreted gelsolin administration increased complex IV activity and reversed this decrease in Aβ_1–42-induced activity. Blue native analysis of these culture samples showed altered protein expression in the mitochondrial complex IV. Representative blue native blots and quantitative histograms are shown (n = 4 per group); *P < .05.