Modulation of plasmin activity for the control of prion propagation

Abstract

The self-propagation of the scrapie prion protein, PrPSc, the misfolded form of the cellular prion protein, PrPC, leads to an array of rapidly progressive, neurodegenerative disorders, known as prion diseases. Under physiological conditions, PrPC is mainly subjected to α-cleavage to generate C1 and N1 fragments. The α-cleavage of PrPC could disrupt the region necessary for the conformational conversion of PrPC to PrPSc. Recently, plasmin, a serine protease, was reported to effectively cleave PrPC at α-site and sustainably inhibit prion conversion. In this study, the effect of modulation of plasmin activity on prion propagation was investigated. First, the role of α2AP, a physiological inhibitor of plasmin, in prion propagation was addressed. In the test tube and cell-based assays, α2AP facilitated prion propagation through hindering plasmin activity. Furthermore, the level of α2AP was remarkably increased in the brain of mice infected with prions. Especially, α2AP was highly expressed in the reactive astrocytes in the hippocampal region of these mice. α2AP was found to localize in the C3+- reactive astrocytes, which are known to be related to neurotoxic pathway, suggesting a potential role of α2AP during prion pathogenesis. Although it remains elusive to fully understand the role of α2AP during prion conversion, these data suggest α2AP may be one of the target molecules for future investigation to control prion diseases. Second, the effect of aspirin, which was previously shown to prevent prion propagation through enhancing plasmin activity in the test tube assay and cell-based model, was assessed in an in vivo model of prion diseases. The level of C1 fragment, an α-cleavage of PrPC product, was increased in prion-infected mice that received aspirin via the oral route. Aspirin significantly increased plasmin activity in the serum and remarkably reduced the PrPSc level, prolonged incubation time, and prevented disease-associated neuropathology in the brain of these mice. Besides, the expression of functional synaptic and dendritic structural proteins significantly increased in these mouse brains even at the asymptomatic stage of prion diseases. These results suggest that prion suppression is feasible by control of plasmin activity associated with cellular processing of PrPC and propose a potential target for prion therapy. Third, in addition to its enzyme activity, another mechanism of plasmin during prion propagation was investigated. At the concentration insufficiently preventing prion aggregation in vitro, plasmin was found to associate with prion aggregates. Plasmin interacted with prion aggregates at two different sites with a much higher affinity than that to PrP monomers. The super high-resolution microscopy revealed that plasmin bound to prion aggregates both within and at the surface of aggregates. Additionally, plasmin activity was significantly reduced due to the binding to prion aggregates in test tube assay and cultured cells with permanent prion infection. However, plasmin remarkably increased at the protein level in prion-infected cells and in the brain of mice infected with prions. This suggests that the increase in plasmin protein level might be a cellular response to the decrease in its activity by prion aggregates. Taken together, these data postulate that prion propagation can be controlled by physical interaction of prion aggregates with plasmin, besides its enzymatical effect on PrPC processing