Intravitreally delivered neprilysin reduces amyloid-beta in the mouse eye

Abstract

Purpose: Amyloid-beta peptide (Aβ), generated in the eye and other tissues, has been hypothesized to exert toxic effects that contribute to progression and pathology of multiple retinal degenerative diseases. Neprilysin (NEP), a native endopeptidase that cleaves Aβ into inactive products, is a membrane-anchored protein. However, the extracellular domain of NEP (sNEP) is soluble and retains catalytic activity. We tested the ability of intravitreally injected recombinant sNEP to reduce, in vivo, ocular levels of Aβ₄₀ and Aβ₄₂ (respectively, 40 and 42 amino acids in length), two principal Aβ forms.

Methods: Anesthetized 10-month wildtype (C57BL/6J) mice, and 2-3-month 5XFAD transgenic mice expressing human Aβ₄₂, received intravitreal injections (2 µL) of phosphate-buffered saline (PBS) containing sNEP (0.004 to 10 µg). The fellow, untreated eye served as control. Treated mice were maintained for 30 min up to 12 weeks. Shortly before euthanasia, the treated eye intravitreally received sNEP inhibitor (phosphoramidon; PA) to block further sNEP activity. Harvested eye tissues (combined lens/vitreous, retina and RPE/choroid) were homogenized and extracted with PBS. Extracts were analyzed for protein (Bradford) and for Aβ₄₀ and Aβ₄₂ (ELISA). sNEP activity remaining at defined post-treatment times (with no PA delivery) was analyzed by a fluorometric assay. Retinal function in sNEP-treated eyes was analyzed by electroretinography (ERG).

Results: Untreated C57BL/6J eyes (n=78) exhibited Aβ₄₀ at 42.8 ± 26.3 (mean ± SEM) pmol per g protein (pmol/g); those of 5XFAD mice (n=10) exhibited substantial Aβ₄₂ (14.6 ± 8.4 pmol/g) as well as Aβ₄₀ (73.4 ± 35.5 pmol/g). In C57BL/6J mice, increasing sNEP yielded progressively greater Aβ₄₀ reductions at 2 hr post-treatment [reductions of 12% ± 3% (n=3) with 4 ng sNEP, and 85% ± 3% (n=5) with 10 µg sNEP]. sNEP activity declined by 50% within ~8 hr after delivery and was undetectable after 2 days, but Aβ₄₀ remained low (~80% reduction) for up to ~8 weeks. In 5XFAD mice at 24 hr after 10 µg sNEP treatment, the reduction of Aβ₄₀ (99% ± 1%) exceeded that for Aβ₄₂ (42% ± 36%) (n=4; p=0.002). sNEP treatment of C57BL/6J and 5XFAD eyes preserved robust ERG responsiveness.

Conclusions: sNEP delivery to the mouse eye yields substantial in vivo reductions in Aβ₄₀ and Aβ₄₂ levels. The results encourage further study of intravitreal sNEP treatment for investigational and, potentially, therapeutic applications.