Paul J. Donaldson

Highlights: • SCOPE is a workflow for anatomically aligned, cross-sample matrix-assisted laser desorption/ionization mass spectrometry imaging comparison. • The “HexBin” framework preserves tissue microdomain heterogeneity across biological replicates. • Region-specific lens redox responses to oxidative stress and therapy are spatially mapped.

To establish an imaging approach capable of mapping Ca²⁺ dynamics across different regions of the intact ocular lens, and to determine the functional contributions of TRPV1 and TRPV4 channels to extracellular Ca²⁺ influx in epithelial and differentiating fiber cells. Rat lenses were mounted in a recording chamber with zonular attachments intact and imaged using confocal laser-scanning microscopy. Epithelial cells were loaded with Cal520 AM, while fiber cells were microinjected with Fluo-8 pentapotassium salt. Region-specific changes in intracellular Ca²⁺ were monitored in response to pharmacological manipulations designed to deplete endoplasmic reticulum (ER) Ca²⁺ stores, activate G-protein coupled receptors, or stimulate extracellular Ca²⁺ influx via mechanosensitive TRPV1 and TRPV4 channels. Central and equatorial epithelial cells exhibited biphasic Ca²⁺ responses to thapsigargin, consistent with ER-dependent release and store-operated Ca²⁺ entry, whereas differentiating fiber cells lacked detectable ER-dependent Ca²⁺ mobilization. In epithelial cells, TRPV4 activation with GSK1016790A (GSK) induced transient Ca²⁺ influx, whereas exposure to the TRPV1 agonist capsaicin (Cap) had no effect. In contrast, fiber cells displayed robust extracellular Ca²⁺ entry in response to TRPV1 and TRPV4 activation by Cap and GSK, respectively. These responses were abolished in Ca²⁺-free conditions or by pre-incubation with TRPV1- or TRPV4-specific antagonists. This study establishes a confocal imaging approach for simultaneous analysis of Ca²⁺ dynamics in epithelial and fiber cells of the intact lens. Our findings reveal region-specific differences in Ca²⁺ regulation and provide insight into how TRPV1 and TRPV4 activate signaling pathways that regulate lens ion homeostasis and water transport to maintain optical properties.

Purpose: To determine whether modulation of lens water content can alter the stiffness of the ex vivo bovine lens which have a similar stiffness profile to the presbyopic human lens.Methods: Bovine lenses cultured in isotonic artificial aqueous humor (AAH) were initially subjected to either MRI imaging using a clinical 3T scanner or a spin test to obtain baseline measurements of water content and shear modulus, respectively. Lenses were then exposed to either hypotonic or hypertonic stress to swell or shrink lenses, respectively, or isotonic AAH + ouabain or high extracellular potassium (AAH-High-K⁺) to inhibit lens water transport, for up to 4 hours before repeating the MRI scans and spin test.Results: In isotonic AAH both free and total water was higher in the outer cortex of the lens relative the central lens nuclear region, but the shear modulus profile had the opposite profile being highest in the lens nucleus. Exposure to hypertonic AAH that shrinks the lens caused a loss of lens water and an increase in the shear modulus in the lens nucleus that served to steepen the shear modulus profile. In contrast, exposure to hypotonic-AAH to sweel the lens increased both free and total water content through all regions of the lens and caused a reversal of the shear modulus so that the nucleus of the lens became less stiff than the outer cortex. These effects of osmotic stress on the shear modulus profile were partially reversed upon the return of lenses to isotonic AAH. Inhibiting lens water transport under isotonic conditions caused more subtle increases in lens water content than seen with hypotonic challenge but still cause a similar softening of the nucleus but had no major effect on the shear modulus in the outer cortex of the bovine lens.Conclusions: Our results demonstrate a link between lens water content and the stiffness of the nucleus of the bovine lens. This suggests that the modulation of lens water transport represents a novel strategy for the development of pharmacological interventions designed to restore accommodation in presbyopes by softening of the nucleus of the human lens.

Since the discovery of the first family member by Cosens and Mannings over 50 years ago

Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. We find that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. Investigation of MP20 localization in human lenses indicate that in young fiber cells MP20 is initially localized to the cytoplasm in differentiating fiber cells but upon fiber cell maturation is inserted into the plasma membrane, correlating with the restriction of the diffusion of extracellular tracers into the lens. Together these results suggest that MP20 forms lens thin junctions in vivo, confirming its role as a structural protein in the human eye lens essential for its optical transparency.

Oxidative stress plays a major role in the formation of the cataract that is the result of advancing age, diabetes or which follows vitrectomy surgery. Glutathione (GSH) is the principal antioxidant in the lens, and so supplementation with GSH would seem like an intuitive strategy to counteract oxidative stress there. However, the delivery of glutathione to the lens is fraught with difficulties, including the limited bioavailability of GSH caused by its rapid degradation, anatomical barriers of the anterior eye that result in insufficient delivery of GSH to the lens, and intracellular barriers within the lens that limit delivery of GSH to its different regions. Hence, more attention should be focused on alternative methods by which to enhance GSH levels in the lens. In this review, we focus on the following three strategies, which utilize the natural molecular machinery of the lens to enhance GSH and/or antioxidant potential in its different regions: the NRF2 pathway, which regulates the transcription of genes involved in GSH homeostasis; the use of lipid permeable cysteine-based analogues to increase the availability of cysteine for GSH synthesis; and the upregulation of the lens’s internal microcirculation system, which is a circulating current of Na⁺ ions that drives water transport in the lens and with it the potential delivery of cysteine or GSH. The first two strategies have the potential to restore GSH levels in the epithelium and cortex, while the ability to harness the lens’s internal microcirculation system offers the exciting potential to deliver and elevate antioxidant levels in its nucleus. This is an important distinction, as the damage phenotypes for age-related (nuclear) and diabetic (cortical) cataract indicate that antioxidant delivery must be targeted to different regions of the lens in order to alleviate oxidative stress. Given our increasing aging and diabetic populations it has become increasingly important to consider how the natural machinery of the lens can be utilized to restore GSH levels in its different regions and to afford protection from cataract.

Cataracts are the world’s leading cause of blindness, and diabetes is the second leading risk factor for cataracts after old age. Despite this, no preventative treatment exists for cataracts. The altered metabolism of excess glucose during hyperglycaemia is known to be the underlying cause of diabetic cataractogenesis, resulting in localised disruptions to fibre cell morphology and cell swelling in the outer cortex of the lens. In rat models of diabetic cataracts, this damage has been shown to result from osmotic stress and oxidative stress due to the accumulation of intracellular sorbitol, the depletion of NADPH which is used to regenerate glutathione, and the generation of fructose metabolites via the polyol pathway. However, differences in lens physiology and the metabolism of glucose in the lenses of different species have prevented the translation of successful treatments in animal models into effective treatments in humans. Here, we review the stresses that arise from hyperglycaemic glucose metabolism and link these to the regionally distinct metabolic and physiological adaptations in the lens that are vulnerable to these stressors, highlighting the evidence that chronic oxidative stress together with osmotic stress underlies the aetiology of human diabetic cortical cataracts. With this information, we also highlight fundamental gaps in the knowledge that could help to inform new avenues of research if effective anti-diabetic cataract therapies are to be developed in the future.

The ocular lens is an important determinant of overall vision quality whose refractive and transparent properties change throughout life. The lens operates an internal microcirculation system that generates circulating fluxes of ions, water and nutrients that maintain the transparency and refractive properties of the lens. This flow of water generates a substantial hydrostatic pressure gradient which is regulated by a dual feedback system that uses the mechanosensitive channels TRPV1 and TRPV4 to sense decreases and increases, respectively, in the pressure gradient. This regulation of water flow (pressure) and hence overall lens water content, sets the two key parameters, lens geometry and the gradient of refractive index, which determine the refractive properties of the lens. Here we focus on the roles played by the aquaporin family of water channels in mediating lens water fluxes, with a specific focus on AQP5 as a regulated water channel in the lens. We show that in addition to regulating the activity of ion transporters, which generate local osmotic gradients that drive lens water flow, the TRPV1/4‐mediated dual feedback system also modulates the membrane trafficking of AQP5 in the anterior influx pathway and equatorial efflux zone of the lens. Since both lens pressure and AQP5‐mediated water permeability () can be altered by changes in the tension applied to the lens surface via modulating ciliary muscle contraction we propose extrinsic modulation of lens water flow as a potential mechanism to alter the refractive properties of the lens to ensure light remains focused on the retina throughout life. image