Clearance of extracellular and cell-associated amyloid β peptide through viral expression of neprilysin in primary neurons

Amyloid beta peptide (A beta), the pathogenic agent of Alzheimer's disease (AD), is a physiological metabolite constantly anabolized and catabolized in the brain. We previously demonstrated that neprilysin is the major A beta -degrading enzyme in vivo. To investigate whether or not manipulation of neprilysin activity in the brain would be an effective strategy for regulating A beta levels, we expressed neprilysin in primary cortical neurons using a Sindbis viral vector and examined the effect on A beta metabolism. The corresponding recombinant protein, expressed in the cell bodies and processes, exhibited thiorphan-sensitive endopeptidase activity, whereas a mutant neprilysin with an amino acid substitution in the active site did not show any such activity. Expression of the wildtype neprilysin, but not the mutant, led to significant decreases in both the A beta 40 and 42 levels in the culture media in a dose-dependent manner. Moreover, neprilysin expression also resulted in reducing cell-associated A beta, which could be more neurotoxic than extracellular A beta. These results indicate that the manipulation of neprilysin activity in neurons, the major source of A beta in the brain, would be a relevant strategy for controlling the A beta levels and thus the A beta -associated pathology in brain tissues.