Metallothionein-3 modulates the amyloid β endocytosis of astrocytes through its effects on actin polymerization

BACKGROUND: Astrocytes may play important roles in the pathogenesis of Alzheimer’s disease (AD) by clearing extracellular amyloid beta (Aβ) through endocytosis and degradation. We recently showed that metallothionein 3 (Mt3), a zinc-binding metallothionein that is enriched in the central nervous system, contributes to actin polymerization in astrocytes. Because actin is likely involved in the endocytosis of Aβ, we investigated the possible role of Mt3 in Aβ endocytosis by cortical astrocytes in this study. RESULTS: To assess the route of Aβ uptake, we exposed cultured astrocytes to fluorescently labeled Aβ(1–40) or Aβ(1–42) together with chloropromazine (CP) or methyl-beta-cyclodextrin (MβCD), inhibitors of clathrin- and caveolin-dependent endocytosis, respectively. CP treatment almost completely blocked Aβ(1–40) and Aβ(1–42) endocytosis, whereas exposure to MβCD had no significant effect. Actin disruption with cytochalasin D (CytD) or latrunculin B also completely blocked Aβ(1–40) and Aβ(1–42) endocytosis. Because the absence of Mt3 also results in actin disruption, we examined Aβ(1–40) and Aβ(1–42) uptake and expression in Mt3(−/−) astrocytes. Compared with wild-type (WT) cells, Mt3(−/−) cells exhibited markedly reduced Aβ(1–40) and Aβ(1–42) endocytosis and expression of Aβ(1-42) monomers and oligomers. A similar reduction was observed in CytD-treated WT cells. Finally, actin disruption and Mt3 knockout each increased the overall levels of clathrin and the associated protein phosphatidylinositol-binding clathrin assembly protein (PICALM) in astrocytes. CONCLUSIONS: Our results suggest that the absence of Mt3 reduces Aβ uptake in astrocytes through an abnormality in actin polymerization. In light of evidence that Mt3 is downregulated in AD, our findings indicate that this mechanism may contribute to the extracellular accumulation of Aβ in this disease.