Abstract:
The progressive accumulation of á-amyloid (Aá) in senile plaques and in the cerebral vasculature is the hallmark of Alzheimer's disease (AD) and related disorders. Several major pathways for Aá clearance include receptor-mediated cellular uptake, blood-brain barrier transport and direct proteolytic degradation. My thesis focused on two novel Aá degrading enzymes- Membrane type 1 matrix metalloproteinase (MT1-MMP) and myelin basic protein (MBP). Matrix metalloproteinase 2 (MMP2) was shown to be expressed in reactive astrocytes surrounding amyloid plaques and may contribute to Aá degradation. MT1-MMP is the physiological activator for the zymogen pro-MMP2. In addition to MMP2, its activator MT1-MMP was also expressed in reactive astrocytes surrounding amyloid deposits in transgenic mice. Using a cell-based system MT1-MMP overexpression can degrade exogenous Aá peptides. Purified MT1-MMP degraded both soluble and fibrillar Aá peptides and this activity was blocked by specific MMP inhibitors. Mass spectrometry analysis identified multiple cleavage sites on Aá. Furthermore, in situ experiments showed that purified MT1-MMP degraded parenchymal fibrillar amyloid plaques that form in the brains of transgenic mice. Together, these findings indicate that MT1-MMP possesses Aá degrading activity in vitro.MBP is the major structural protein component of myelin sheath. MBP possesses endogenous serine proteinase activity and can undergo autolysis. Recently, our lab showed that MBP binds Aá and inhibits Aá fibril formation. Aá peptides were degraded by purified human brain MBP and recombinant human MBP, but not an MBP fragment without autolytic activity. Similarly, cells expressing MBP degraded exogenous Aá peptides. In addition, purified MBP also degraded assembled fibrillar Aá. Mass spectrometry analysis identified multiple cleavage sites on Aá. Further, in situ experiments showed that purified MBP can degrade parenchymal amyloid plaques and cerebral vascular amyloid that form in the brains of transgenic mice. Lastly, series of C-terminal deletion MBP proteins were tested for autolysis and Aá degradation activities to identify the responsible region. Together, these findings indicate that purified MBP possesses Aá degrading activity in-vitro.To conclude, I characterized two novel Aá degrading enzymes in-vitro. Further in vivo experiments are needed to investigate the role of these two Aá degrading enzymes in the pathology of AD.
