Brain plasmin enhances APP alpha-cleavage and Abeta degradation and is reduced in Alzheimer's disease brains

Abstract

The proteolytic processing of amyloid precursor protein (APP) has been linked to sphingolipid-cholesterol microdomains (rafts). However, the raft proteases that may be involved in APP cleavage have not yet been identified. In this work we present evidence that the protease plasmin is restricted to rafts of cultured hippocampal neurons. We also show that plasmin increases the processing of human APP preferentially at the alpha-cleavage site, and efficiently degrades secreted amyloidogenic and non-amyloidogenic APP fragments. These results suggest that brain plasmin plays a preventive role in APP amyloidogenesis. Consistently, we show that brain tissue from Alzheimer's disease patients contains reduced levels of plasmin, implying that plasmin downregulation may cause amyloid plaque deposition accompanying sporadic Alzheimer's disease.

Fig. 1. Detection of plasminogen on the surface of and in rafts from mature hippocampal neurons, and specific activation in rafts. (A) Surface staining of mature hippocampal neurons was performed using a polyclonal antibody against plasminogen. White arrowheads indicate sites with plasminogen on the membrane. (B) Western blot analysis of all fractions of the Optiprep gradient used to obtain rafts. Plasminogen is present in all lanes (80 kDa). The 50 and 30 kDa cleavage products appear only in the raft fraction (shown by asterisks). Molecular weight markers on the left.

Fig. 2. Plasmin and tPA induce a preferential increase in APP α-cleavage in hippocampal neurons expressing human APP. (A) Autoradiography of a tricine gel loaded with [³⁵S]methionine-labeled hippocampal neurons previously infected with SFV encoding the human APP695 isoform. The samples correspond to non-treated infected neurons (control) and infected neurons incubated with 1 unit/ml of plasmin (plasmin). The 12 kDa fragment (β) and the 10 kDa fragment (α) were immunoprecipitated using a polyclonal antibody against the C-terminal region of APP. (B) Percentage of the fragments corresponding to the β-cleavage (β) or the α-cleavage (α) with respect to the control (considered as 0). Error bars represent the standard error. While the α C-terminal fragment of APP showed a consistent 25% increase in plasmin-treated neurons, the β C-terminal fragment increased to a lower extent (only 8%) and with a higher variation. (C) Analysis of the α N-terminal secreted fragment of APP. Stage 5 neurons were infected with SFV-APP and subsequently treated with (tPA) or without (control) tPA. Equivalent aliquots from the medium were taken after 5 min, 1 h or 2.5 h incubation. The samples were analyzed by western blotting using the monoclonal antibody recognizing the APP secreted form produced by the α-cleavage. (D) Western blot of the extracted cells using the same antibody. The total amount of viral-induced APP expression is similar.

Fig. 3. Activation of plasmin reduces Aβ levels in cells overexpressing human APP. (A) The levels of Aβ and p3 secreted from HEK cells stably transfected with human APP695 in the absence (C) or presence of tPA (tPA). The mean value and standard error from three independent experiments are shown. Activation of plasmin resulted in a consistent 24% decrease in the levels of secreted Aβ with respect to the control (indicated as 100%). The results with p3 were more variable, although a 15% reduction was observed. (B) Amounts of Aβ and p3 immunoprecipitated from media of labeled HEK cells stably transfected with human APP695 and treated with different concentrations of plasmin (indicated in units/ml).

Fig. 4. Plasmin is reduced in AD brains. (A) Western blot analysis of human neocortex extracts using an antibody against plasminogen. The 30 kDa plasmin is shown for: two control brains (40 and 63 years old) without amyloid plaques (none); one brain (83 years old) corresponding to a non-demented individual with a moderate number of plaques (++); two brains (77 and 84 years old) corresponding to diagnosed AD patients containing a high number of plaques [++++ (AD)]. (B) Western blot of human hippocampus extracts from AD brains (AD) and control brains (C) using an antibody against plasminogen that recognizes the 30 kDa plasmin.