Fibrillar amyloid beta-protein mediates the pathologic accumulation of its secreted precursor in human cerebrovascular smooth muscle cells.
Cerebrovascular deposition of the amyloid beta-protein (Abeta) 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 Abeta has pronounced deleterious effects on smooth muscle cells within the cerebral vessel wall. We have previously shown that Abeta(1-40) possessing the E22Q HCHWA-D mutation extensively assembles into fibrils on the surface of cultured human cerebrovascular smooth muscle (HCSM) cells. This cell-surface Abeta fibril formation induces a series of pathologic responses in cultured HCSM cells, including a marked increase in the levels of cell-associated amyloid beta-protein precursor (AbetaPP) and cell death. In the present study, we investigated the relationship between HCSM cell-surface Abeta fibril formation and the striking increase in cell-associated AbetaPP. Time course studies showed that cell-surface HCHWA-D Abeta(1-40) fibril formation occurred rapidly, whereas both the increase in cell-associated AbetaPP 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 AbetaPP was secreted AbetaPP (sAbetaPP). Localization studies showed that the sAbetaPP was present on the HCSM cell surface. This result raised the possibility that sAbetaPP may bind back to HCSM cell-surface fibrils formed by HCHWA-D Abeta(1-40). Indeed, binding of biotinylated sAbetaPP to fibrillar HCHWA-D Abeta(1-40) was demonstrated by transmission electron microscopy. Furthermore, solid-phase binding assays showed that biotinylated sAbetaPP exhibited dose-dependent, saturable binding to fibrillar (but not soluble) HCHWA-D Abeta(1-40) with k(d) approximately 28 nM. Exon deletion experiments further defined a fragment of sAbetaPP (AbetaPP(18-119)), encoded by AbetaPP exons 2 and 3, to contain the fibrillar Abeta-binding domain. In addition, AbetaPP(18-119) effectively blocked the cell-surface accumulation of sAbetaPP and subsequent cell death in HCSM cells treated with pathogenic Abeta. Together, these findings could explain the accumulation of AbetaPP in cerebrovascular Abeta deposits observed both in vitro and in vivo and may contribute to the pathologic responses evoked by pathogenic forms of Abeta in HCSM cells.
Melchor
J P
JP
Departments of Medicine and Pathology, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8153, USA.
Van Nostrand
W E
WE
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000502:physiology*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D001921:Brain / Q000098:blood supply*; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D011993:Recombinant Fusion Proteins; D001710:Biotin; D005982:Glutathione Transferase