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

Amyloid-beta (Aß) 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 Aß plays a role in the progression of Alzheimer's disease and other brain degenerative diseases. Recent studies raise the hypothesis that excessive Aß levels may contribute also to certain retinal degenerative diseases. These findings, together with evidence that a major portion of Aß is released as monomer into the extracellular space, raise the possibility that a technology enabling the enzymatic break-down of monomeric Aß in the living eye under physiological conditions could prove useful for research on ocular Aß physiology and, perhaps ultimately, for therapeutic applications. Neprilysin (NEP), an endopeptidase known to cleave Aß 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 Aß-cleaving activity of exogenous sNEP in the microenvironment of the intact eye, we analyzed the effect of intra-vitreally delivered sNEP on ocular Aß levels in mice that exhibit readily measurable, aqueous buffer-extractable Aß40 and Aß42, two principal forms of Aß. 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 Aß40 and Aß42 (ELISA) and for total protein (Bradford assay). The fellow, untreated eye of each mouse served as control, and concentrations of Aß (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 Aß40, 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 Aß40 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 Aß40 decrease of 99% ± 1% (n = 4) and a substantial although smaller decrease in Aß42 (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% Aß40 reduction in C57BL/6J eyes, an 87% and 92% Aß40 reduction in 5XFAD eyes, and a 23% and 52% Aß42 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 Aß levels reported here represents a possible approach for determining effects of Aß reduction in normally functioning eyes and in models of retinal degenerative disease.

Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and ß2 GABAA subunits. We isolated three anti-ß2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.