Neprilysin-dependent Reduction of Amyloid-beta in Eye Tissues

Amyloid-beta (Aβ), a 38-43 amino acid peptide generated in the eye and other tissues, has been hypothesized to exert toxic effects that contribute to the progression and pathology of age-related macular degeneration and other retinal degenerative diseases. My thesis study addresses the engineering of a treatment approach that enables the control of Aβ levels in the living eye. Neprilysin (NEP), a native endopeptidase that cleaves Aβ into inactive products, is a membrane-anchored protein. However, the recombinant-catalytic domain of NEP (termed sNEP) is soluble and retains catalytic activity. We have investigated the ability of intravitreally injected sNEP to reduce, in vivo, ocular levels of Aβ40 and Aβ42, (40 and 42 amino acids respectively) two principal Aβ forms. Anesthetized 10-month wildtype (C57BL/6J) mice, and 2-3-month 5XFAD transgenic mice overexpressing human Aβ42, received intravitreal injections of phosphate-buffered saline (PBS) containing sNEP. Treated mice were maintained for varying periods. Harvested eye tissues (combined lens, vitreous, retina, retinal pigment epithelium and choroid) were homogenized, extracted with PBS and analyzed for Aβ (ELISA) and protein (Bradford assay) to enable determinations of Aβ concentrations (pmol per g protein). Analytical procedures developed in the study included those for terminating sNEP activity prior to animal euthanasia, using the sNEP inhibitor phosphoramidon. sNEP activity remaining at defined post-sNEP-treatment times was analyzed by fluorometric assay. Retinal function in sNEP-treated eyes was analyzed by electroretinography (ERG). Key results obtained in the study include the following. (1) Untreated C57BL/6J eyes and 5XFAD eyes exhibit substantial Aβ levels. (2) Intravitreally delivered sNEP exhibits a concentration-dependent and time-dependent activity in reducing ocular Aβ levels. (3) Although sNEP is cleared rapidly from the eye after delivery, Aβ levels remain low for up to several weeks. (4) As determined at a fixed time following sNEP treatment that substantially reduces Aβ, C57BL/6J and 5XFAD mouse eyes exhibit robust ERG responsiveness, indicating good tolerance of the eye tissues to the sNEP treatment. Overall, the results of this study establish that sNEP delivery to the mouse eye enables substantial in vivo reductions in Aβ levels. The data encourage further study of intravitreal sNEP treatment for investigational and, potentially, therapeutic applications.