Fibrillar amyloid β-protein mediates the pathologic accumulation of its secreted precursor in human cerebrovascular smooth muscle cells

Abstract

Cerebrovascular deposition of the amyloid β-protein (Aβ) is a key pathologic lesion seen in patients with Alzheimer's disease and certain related disorders, including hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). The deposition of Aβ has pronounced deleterious effects on smooth muscle cells within the cerebral vessel wall. We have previously shown that Aβ1-40 possessing the E22Q HCHWA-D mutation extensively assembles into fibrils on the surface of cultured human cerebrovascular smooth muscle (HCSM) cells. This cell-surface Aβ fibril formation induces a series of pathologic responses in cultured HCSM cells, including a marked increase in the levels of cell-associated amyloid β- protein precursor (AβPP) and cell death. In the present study, we investigated the relationship between HCSM cell-surface Aβ fibril formation and the striking increase in cell-associated AβPP. Time course studies showed that cell-surface HCHWA-D Aβ1-40 fibril formation occurred rapidly, whereas both the increase in cell-associated AβPP and loss of cell viability were delayed responses. Domain analysis using site-specific antibodies indicated that the vast majority of the increase in cell- associated AβPP was secreted AβPP (sAβPP). Localization studies showed that the sAβPP was present on the HCSM cell surface. This result raised the possibility that sAβPP may bind back to HCSM cell-surface fibrils formed by HCHWA-D Aβ1-40. Indeed, binding of biotinylated sAβPP to fibrillar HCHWA-D Aβ1-40 was demonstrated by transmission electron microscopy. Furthermore, solid-phase binding assays showed that biotinylated sAβPP exhibited dose-dependent, saturable binding to fibrillar (but not soluble) HCHWA-D Aβ1-40 with k(d) ≃ 28 nM. Exon deletion experiments further defined a fragment of sAβPP (AβPP18-119), encoded by AβPP exons 2 and 3, to contain the fibrillar Aβ-binding domain. In addition, AβPP18-119 effectively blocked the cell-surface accumulation of sAβPP and subsequent cell death in HCSM cells treated with pathogenic Aβ. Together, these findings could explain the accumulation of AβPP in cerebrovascular Aβ deposits observed both in vitro and in vivo and may contribute to the pathologic responses evoked by pathogenic forms of Aβ in HCSM cells.