Reduction of amyloid-beta levels in mouse eye tissues by intra-vitreally delivered neprilysin

Abstract Amyloid-beta (Aβ) is a group of aggregation-prone, 38- to 43-amino acid peptides generated in the eye and other organs. Numerous studies suggest that the excessive build-up of low-molecular-weight soluble oligomers of Aβ plays a role in the progression of Alzheimer's disease and other brain degenerative diseases. Recent studies raise the hypothesis that excessive Aβ levels may contribute also to certain retinal degenerative diseases. These findings, together with evidence that a major portion of Aβ is released as monomer into the extracellular space, raise the possibility that a technology enabling the enzymatic break-down of monomeric Aβ in the living eye under physiological conditions could prove useful for research on ocular Aβ physiology and, perhaps ultimately, for therapeutic applications. Neprilysin (NEP), an endopeptidase known to cleave Aβ monomer into inactive products, is a membrane-associated protein. However, sNEP, a recombinant form of the NEP catalytic domain, is soluble in aqueous medium. With the aim of determining the Aβ-cleaving activity of exogenous sNEP in the microenvironment of the intact eye, we analyzed the effect of intra-vitreally delivered sNEP on ocular Aβ levels in mice that exhibit readily measurable, aqueous buffer-extractable Aβ40 and Aβ42, two principal forms of Aβ. Anesthetized 10-month wild-type (C57BL/6J) and 2–3-month 5XFAD transgenic mice received intra-vitreal injections of sNEP (0.004–10 μg) in one eye and were sacrificed at defined post-treatment times (30 min - 12 weeks). Eye tissues (combined lens, vitreous, retina, RPE and choroid) were homogenized in phosphate-buffered saline, and analyzed for Aβ40 and Aβ42 (ELISA) and for total protein (Bradford assay). The fellow, untreated eye of each mouse served as control, and concentrations of Aβ (pmol/g protein) in the treated eye were normalized to that of the untreated control eye. In C57BL/6J mice, as measured at 2 h after sNEP treatment, increasing amounts of injected sNEP yielded progressively greater reductions of Aβ40, ranging from 12% ± 3% (mean ± SEM; n = 3) with 4 ng sNEP to 85% ± 13% (n = 5) with 10 μg sNEP. At 4 ng sNEP the average Aβ40 reduction reached >70% by 24 h following treatment and remained near this level for about 8 weeks. In 5XFAD mice, 10 μg sNEP produced an Aβ40 decrease of 99% ± 1% (n = 4) and a substantial although smaller decrease in Aβ42 (42% ± 36%; n = 4) within 24 h. Electroretinograms (ERGs) were recorded from eyes of C57BL/6J and 5XFAD mice at 9 days following treatment with 4 ng or 10 μg sNEP, conditions that on average led, respectively, to an 82% and 91% Aβ40 reduction in C57BL/6J eyes, an 87% and 92% Aβ40 reduction in 5XFAD eyes, and a 23% and 52% Aβ42 reduction in 5XFAD eyes. In all cases, sNEP-treated eyes exhibited robust ERG responses, consistent with a general tolerance of the posterior eye tissues to the investigated conditions of sNEP treatment. The sNEP-mediated decrease of ocular Aβ levels reported here represents a possible approach for determining effects of Aβ reduction in normally functioning eyes and in models of retinal degenerative disease.