Protein Disulfide Levels and Lens Elasticity Modulation: Applications for Presbyopia

Abstract

Purpose: The purpose of the experiments described here was to determine the effects of lipoic acid (LA)-dependent disulfide reduction on mouse lens elasticity, to synthesize the choline ester of LA (LACE), and to characterize the effects of topical ocular doses of LACE on mouse lens elasticity.

Methods: Eight-month-old mouse lenses (C57BL/6J) were incubated for 12 hours in medium supplemented with selected levels (0-500 μM) of LA. Lens elasticity was measured using the coverslip method. After the elasticity measurements, P-SH and PSSP levels were determined in homogenates by differential alkylation before and after alkylation. Choline ester of LA was synthesized and characterized by mass spectrometry and HPLC. Eight-month-old C57BL/6J mice were treated with 2.5 μL of a formulation of 5% LACE three times per day at 8-hour intervals in the right eye (OD) for 5 weeks. After the final treatment, lenses were removed and placed in a cuvette containing buffer. Elasticity was determined with a computer-controlled instrument that provided Z-stage upward movements in 1-μm increments with concomitant force measurements with a Harvard Apparatus F10 isometric force transducer. The elasticity of lenses from 8-week-old C57BL/6J mice was determined for comparison.

Results: Lipoic acid treatment led to a concentration-dependent decrease in lens protein disulfides concurrent with an increase in lens elasticity. The structure and purity of newly synthesized LACE was confirmed. Aqueous humor concentrations of LA were higher in eyes of mice following topical ocular treatment with LACE than in mice following topical ocular treatment with LA. The lenses of the treated eyes of the old mice were more elastic than the lenses of untreated eyes (i.e., the relative force required for similar Z displacements was higher in the lenses of untreated eyes). In most instances, the lenses of the treated eyes were even more elastic than the lenses of the 8-week-old mice.

Conclusions: As the elasticity of the human lens decreases with age, humans lose the ability to accommodate. The results, briefly described in this abstract, suggest a topical ocular treatment to increase lens elasticity through reduction of disulfides to restore accommodative amplitude.

Figure 1

Flow charts for methods used to determine lens elasticity (left) and lens protein disulfide levels (right) for lens culture experiments.

Figure 2

Differential alkylation results for BSA. Trace a, all of the cysteine side chains were reacted with ST322. Trace b, all of the cysteine side chains were reacted with FL492. Trace c, cysteines, not involved in disulfide bonds, were reacted with ST322; then disulfide bonds were reduced with TCEP and alkylated with FL492. Trace d, treated blank without BSA.

Figure 3

Computer-controlled apparatus for lens elasticity measurements and representative photographic results. The Z-stage, lens holder, probe, force transducer imaging mirror, and microscope, depicted in the schematic on the right, are labeled in the photograph of the apparatus on the left. An example of a lens being studied is presented as three photos. In the top photograph, the lens is positioned on Z-stage in modified cuvette with HBSS; the force probe is not in contact with the lens. In the center photograph, the force probe is in contact with the top surface of the lens. In the bottom photograph, the lens is shown after 405-μm upward stage movement in 15-μm increments. Note that the equatorial diameter has increased and the Z-axis of the lens has decreased.

Figure 4

The effect of LA on lens elasticity and lens disulfide levels in vitro. (a) Scatter plot of the change in diameter for each individual lens studied after 12-hour exposure, in vitro, to media or media + LA (x-axis, μM). (b) Dose-response curve of the change in D-D0 (mean ± SD, from data in [a]) versus the Log10 of the LA concentration. (c) Scatter plot of the percent SS of each individual lens in the study with the LA concentrations (μM) indicated on the x-axis. (d) Dose-response curve of the change in the percent SS (mean ± SD from data in [c]) versus Log10 of the LA concentration (μM). All lenses were from old mice (8 months).

Figure 5

Proposed fragmentation of LA determined by MS. The first step in the fragmentation is the opening of the dithiolane ring by loss of H₂S and C₂H₅ in the negative ion and positive ion modes, respectively.

Figure 6

Proposed fragmentation of LACE in the positive ion mode based on the atomic masses of the most prevalent ions. Where F2 forms by break-up of the dithiolane ring, the ring is intact in F3 and totally lost, presumably as a fragment, when F4 is formed.

Figure 7

Comparison force-displacement plots of lenses from OD (filled symbols) and OS (unfilled symbols) eyes. Group 1, old mice (OD treated, OS untreated).

Figure 8

Intergroup and treated versus untreated force-displacement curve comparisons. Force-displacement plots (mean ± SD) for intergroup comparisons (b, c) and for comparisons of lens elasticity from treated (OD, filled symbols) and untreated (OS, unfilled symbols) eyes of the group 1 old mice (a).