Exogenous Neprilysin for Amyloid-beta Degradation

Abundant evidence indicates a relationship between the progression of Alzheimer’s disease and the tissue build-up of amyloid-beta (Aâ), a ~4 kDa peptide with a strong propensity to form soluble, highly neurotoxic oligomers. The existence of Aâ in the retina and retinal pigment epithelium (RPE), and the similarity of certain features of age-related macular degeneration (AMD) as well as glaucoma to Alzheimer’s, suggest that selectively decreasing the level of Aâ in the posterior eye tissues likely represents a therapeutic strategy for AMD/glaucoma. We propose to investigate a novel technology for reducing Aâ build-up in the retina/RPE: namely, to promote Aâ degradation by locally introducing neprilysin (NEP), an Aâ-degrading enzyme. The experiments will be conducted on 5XFAD mice, an established model that overexpresses Aâ in the retina/RPE. In Aim 1 we will deliver a recombinant soluble form of NEP (sNEP) to one eye of the anesthetized mouse by intra-vitreal or supra-choroidal injection of ~1 ìL of sNEP at defined concentration (nM-ìM range). sNEP is prepared by expression in Pichia pastoris, recovery of sNEP secreted into the medium following induction by methanol, concentration of the protein, and purification to homogeneity. In Aim 2, we will intra-ocularly deliver adeno-associated virus (AAV) carrying the gene for sNEP. In both Aims, the fellow, control eye will remain untreated or receive vehicle only. The primary screen for analyzing the effect of a given NEP treatment will be Aâ determination by sandwich ELISA of posterior eye tissues recovered upon euthanasia of the mouse at hours to weeks after treatment. Using homogenized retina/RPE/choroid preparations extracted with defined medium, we will determine the amounts of Aâ40 and Aâ42 (the two major forms of Aâ). Preliminary experiments involving intra-ocular or homogenate “spiking” with Aâ standard demonstrate the efficiency of native Aâ extraction. We will also analyze extracts of tissue homogenates for NEP activity using an assay based on NEP-catalyzed cleavage of the fluorescent substrate glutaryl-Ala-Ala-Phe-methoxy coumarin (GAAF-MNA). Success in the project will establish conditions of intra-ocular NEP treatment that can be further refined in subsequent studies of AMD and glaucoma animal models to investigate NEP-induced Aâ degradation in relation to known morphological/biochemical/immunological markers of disease status. We hypothesize that results obtained in this project will lay the foundation for longer-term studies that could lead to NEP-based, Aâ-targeting therapeutic strategies for multiple degenerative diseases of the retina.