Mary F. Knauer

Preferential adsorption, internalization and resistance to degradation of the major isoform of the Alzheimer's amyloid peptide, Aβ1–42, in differentiated PC12 cells

A central question in Alzheimers disease (AD) is the role of amyloid in pathogenesis. Recent discoveries implicating the longer A beta 1-42 form of amyloid in pathogenesis led us to characterize the interaction of A beta with cells to elucidate differences that might account for these observations. We characterized the adsorption, internalization and degradation of radiolabeled A beta in NGF-differentiated PC12 cells under conditions that are not acutely toxic. All A beta peptides examined absorb to the surface of PC12 cells and are internalized; however the adsorption and internalization of A beta 1-42 is significantly greater than that of A beta 1-40 and A beta 1-28. The adsorption of A beta 1-42 is decreased by treatment of the cells with neuraminidase, but not heparitinase. The fate of the internalized A beta 1-42 is also very different than shorter A beta peptides; a fraction of the internalized A beta 1-42 accumulates intracellularly and is resistant to degradation for at least 3 days while A beta 1-40 and shorter peptides are eliminated with a half life of about 1 h. A beta 1-42 does not appear to inhibit lysosomal hydrolases, since A beta 1-28 is degraded at the same rate in the presence or absence of A beta 1-42. The intracellular A beta 1-42 is located in a dense organellar compartment and colocalizes with the lysosomal markers Lucifer Yellow and horseradish peroxidase. These data indicate that there are significant differences in the cell surface adsorption, internalization and catabolism of A beta 1-42 compared to A beta 1-40 and A beta 1-28. These differences may be important for the preferential accumulation of the longer A beta 1-42 isoform and its association with AD pathogenesis.

Cell surface APP751 forms complexes with protease nexin 2 ligands and is internalized via the low density lipoprotein receptor-related protein (LRP)

The secreted isoforms of the amyloid precursor protein (APP) that contain the Kunitz domain are also known as protease nexin 2 (PN2). Normal proteolytic processing of transmembrane APP, which results in the majority of soluble PN2, cleaves within the Alzheimers A beta peptide, precluding A beta formation. Recent data indicate that soluble PN2 is internalized by cells via the low density lipoprotein receptor-related protein (LRP), which binds multiple ligands including apolipoprotein E (apoE) [23]. However, soluble PN2 cannot contribute to amyloid accumulation, so we examined whether the unprocessed, transmembrane form of APP751 containing the intact A beta sequence would form complexes with a PN2 ligand, EGF binding protein (EGFBP), and be internalized by LRP. We found that the addition of EGFBP to cells overexpressing APP751 induced the internalization of this amyloidogenic form of APP. The 39 kDa LRP receptor associated protein (RAP), an antagonist for LRP, blocked the internalization of APP751/PN2, suggesting a common LRP-mediated internalization pathway for both soluble and transmembrane APP751/PN2 after protease complex formation. Previous work has shown that internalization of transmembrane APP can lead to the formation of amyloidogenic carboxyl-terminal fragments and increased secretion of the Alzheimers A beta peptide. Our data suggest the protease ligands for PN2 may play an important role in altering APP processing pathways to favor amyloid formation, and that LRP may be a point at which the apoE and amyloid processing pathways intersect.

The efficient catabolism of thrombin-protease nexin 1 complexes is a synergistic mechanism that requires both the LDL receptor-related protein and cell surface heparins.

Protease nexin 1 (PN1) is a serine protease inhibitor (SERPIN) that acts as a suicide substrate for thrombin (Th) and urokinase-type plasminogen activator (uPA). PN1 forms 1:1 stoichiometric complexes with these proteases, which are then rapidly bound, internalized, and degraded. The low density lipoprotein receptor-related protein (LRP) is the receptor responsible for the internalization of protease-PN1 complexes. However, we found that the LRP is not significantly involved in the initial cell surface binding of thrombin-PN1, leading us to investigate what cellular component was responsible for this initial interaction. Since Th-PN1 complexes retain a high-affinity for heparin after complex formation, unlike several of the other SERPINs, we tested the possibility that cell surface heparins were involved in initial complex binding. Soluble heparin was found to be a potent inhibitor of the binding of Th-PN1 to the cell surface and greatly facilitated the dissociation of Th-PN1 complexes pre-bound in the absence of soluble heparin. To ascertain the role of cell surface heparins, further studies were done using complexes of thrombin and PN1(K7E), a variant of PN1 in which the heparin binding site was rendered non-functional. When added at equal initial concentrations of complexes, Th-PN1(K7E) was catabolized 5- to 10-fold less efficiently than Th-PN1, a direct result of the greatly diminished initial binding of the Th-PN1(K7E) complexes. These data demonstrate the sizable contribution of cell surface heparins to Thrombin-PN1 complex binding and support a model in which these heparins act to concentrate the complexes at the cell surface facilitating their subsequent LRP-dependent endocytosis.

Analysis of a Structural Determinant in Thrombin-Protease Nexin 1 Complexes That Mediates Clearance by the Low Density Lipoprotein Receptor-related Protein

We recently identified a synthetic peptide, Pro47–Ile58, derived from the mature protease nexin 1 (PN1) sequence, that inhibited the low density lipoprotein receptor-related protein (LRP)-mediated internalization of thrombin-PN1 (Th-PN1) complexes. Presently, we have analyzed this sequence in Th-PN1 complex catabolism using two independent approaches: 1) An antibody was generated against Pro47–Ile58, which inhibited complex degradation by 70% but had no effect on the binding of the complexes to cell surface heparins. This places the structural determinant in PN1 mediating complex internalization by the LRP outside of the heparin-binding site. 2) Site-directed genetic variants of PN1 with a single Ala substitution at His48, or two Ala substitutions, one at His48 and another at Asp49, were expressed in Sf9 insect cells. The catabolic rate of complexes formed between Th and the singly substituted and doubly substituted variants was lowered to 50 and 15%, respectively, when compared with the catabolic rate of native Th-PN1 complexes. This is the first analysis of a structural determinant in a serineprotease inhibitor (SERPIN) required for LRP-mediated internalization and in part may explain the cryptic nature of this site in the unreacted serine protease inhibitor.

Mechanism of Thrombin Clearance by Human Astrocytoma Cells

Abstract : Astroglial cells secrete a variety of factors that contribute to the regulation of neurite initiation and continued outgrowth, among them proteases and protease inhibitors. An alteration in the balance between these proteins has been implicated in Alzheimers disease, resulting in an accumulation of thrombin : protease nexin 1 (PN1) complexes in the brains of these patients. This report aims at providing a biochemical explanation for this phenomenon. We show that human astrocytoma cells bind and internalize thrombin and thrombin : PN1 complexes efficiently by a PN1‐dependent mechanism. Binding was potently inhibited by soluble heparin and did not occur with the mutant PN1 (K7E) deficient in heparin binding. Receptor‐associated protein, an antagonist of the low‐density lipoprotein receptor‐related protein (LRP), inhibited internalization of thrombin by the astrocytoma cells, but did not affect cell‐surface binding. The results are consistent with a mechanism by which astrocytoma cells clear thrombin in a sequential manner : thrombin is first complexed with PN1, then bound to cell‐surface heparins, and finally internalized by LRP. This mechanism provides a link between the neuronal growth regulators thrombin and PN1 and proteins genetically associated with Alzheimers disease such as LRP.