Degradation of Amyloid β-Protein by a Metalloprotease Secreted by Microglia and Other Neural and Non-neural Cells

Wei Qiao Qiu,Zhen Ye,Dora Kholodenko,Peter Seubert,Dennis J Selkoe

doi:10.1074/jbc.272.10.6641

Abstract

Amyloid beta-protein (Abeta) is the major component of neuritic (amyloid) plaques in Alzheimer's disease, and its deposition is an early and constant event in the complex pathogenetic cascade of the disease. Although many studies have focused on the biosynthetic processing of the beta-amyloid precursor protein and on the production and polymerization of Abeta, understanding the degradation and clearance of Abeta has received very little attention. By incubating the conditioned medium of metabolically labeled Abeta-secreting cells with media of various cultured cell lines, we observed a time-dependent decrease in the amount of Abeta in the mixed media. The factor principally responsible for this decrease was a secreted metalloprotease released by both neural and non-neural cells. Among the cells examined, the microglial cell line, BV-2, produced the most Abeta-degrading activity. The protease was completely blocked by the metalloprotease inhibitor, 1,10-phenanthroline, and partially inhibited by EDTA, whereas inhibitors of other protease classes produced little or no inhibition. Substrate analysis suggests that the enzyme was a non-matrix metalloprotease. The protease cleaved both Abeta1-40 and Abeta1-42 peptides secreted by beta-amyloid precursor protein-transfected cells but failed to degrade low molecular weight oligomers of Abeta that form in the culture medium. Lipopolysaccharide, a stimulator of macrophages/microglia, activated BV-2 cells to increase their Abeta-degrading metalloprotease activity. We conclude that secreted Abeta1-40 and Abeta1-42 peptides are constitutively degraded by a metalloprotease released by microglia and other neural cells, providing a potential mechanism for the clearance of Abeta in brain tissue.

Highlights

The defining pathological features of Alzheimer’s disease (AD)1 are extracellular deposits of amyloid ␤-proteins (A␤) that form senile plaques and amyloid angiopathy and intraneuronal deposits of modified tau proteins that form neurofibrillary tangles
Decrease of A␤ Is Mediated by the Conditioned Media of Non-neural and Neural Cells—We examined several non-neural and neural cell lines for the secretion of an A␤-degrading protease activity: COS monkey kidney cells; Chinese hamster ovary (CHO) cells; the human neuroblastoma cell lines, M17 and SY5Y; and the murine microglial line, BV-2
All cultures were passaged by brief incubation with EDTA rather than trypsin, in order to avoid the formation of a serine protease/␣2macroglobulin complex in the medium, which we recently showed can occur during tryptic passage of cells and degrade secreted A␤ [15]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Chinese hamster ovary (CHO) cells stably transfected with ␤APP770 cDNA containing the Val 3 Phe mutation at residue 717 (7PA2 cells) [13] were routinely cultured in Dulbecco’s modified Eagle’s. Untransfected CHO cells, monkey kidney COS cells, and the human neuroblastoma cell lines, M17 and SY5Y, were grown in Dulbecco’s modified Eagle’s medium, 10% FBS. All these cells were passaged by adding 3 ml of 50 mM EDTA, immediately aspirating it off, and incubating the cells at 37 °C for 2 min followed by washing with Hanks’ balanced salt solution (Life Technologies, Inc.). Assays of A␤-degrading Activity—To obtain conditioned media (CM) containing A␤-degrading activity, different cultured cells were washed 3 times with serum-free N2 medium (N2 supplement (Life Technologies, Inc.), 1% ovalbumin, 1 mM pyruvate in MEM) (N2). Conditioned media were collected and centrifuged before assaying A␤-degrading activity as described above

RESULTS

Cysteine Aspartic Metallo Metallo Serine Serine Serine Serine Serine

DISCUSSION