Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens.

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

Regulation of water flow in the ocular lens: new roles for aquaporins.

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 ( P H 2 O ${P_{{{\mathrm{H}}_{\mathrm{2}}}{\mathrm{O}}}}$ ) 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.

Piezo1 channel causes lens sclerosis via transglutaminase 2 activation.

Presbyopia is caused by age-related lenticular hardening, resulting in near vision loss, and it occurs in almost every individual aged ≥50 years. The lens experiences mechanical pressure during for focal adjustment to change its thickness. As lenticular stiffening results in incomplete thickness changes, near vision is reduced, which is known as presbyopia. Piezo1 is a mechanosensitive channel that constantly senses pressure changes during the regulation of visual acuity, and changes in Piezo1 channel activity may contribute to presbyopia. However, no studies have reported on Piezo1 activation or the onset of presbyopia. To elucidate the relevance of Piezo1 activation and cross-linking in the development of presbyopia, we analysed the function of Piezo1 in the lens. The addition of Yoda1, a Piezo1 activator, induced an increase in transglutaminase 2 (TGM2) mRNA expression and activity through the extra-cellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH2-terminal kinase1/2 pathways. In ex vivo lenses, Yoda1 treatment induced γ-crystallin cross-linking via TMG2 activation. Furthermore, Yoda1 eye-drops in mice led to lenticular hardening via TGM2 induction and activation in vivo, suggesting that Yoda1-treated animals could serve as a model for presbyopia. Our findings indicate that this presbyopia-animal model could be useful for screening drugs for lens-stiffening inhibition.

Modulation of Membrane Trafficking of AQP5 in the Lens in Response to Changes in Zonular Tension Is Mediated by the Mechanosensitive Channel TRPV1.

In mice, the contraction of the ciliary muscle via the administration of pilocarpine reduces the zonular tension applied to the lens and activates the TRPV1-mediated arm of a dual feedback system that regulates the lens' hydrostatic pressure gradient. In the rat lens, this pilocarpine-induced reduction in zonular tension also causes the water channel AQP5 to be removed from the membranes of fiber cells located in the anterior influx and equatorial efflux zones. Here, we determined whether this pilocarpine-induced membrane trafficking of AQP5 is also regulated by the activation of TRPV1. Using microelectrode-based methods to measure surface pressure, we found that pilocarpine also increased pressure in the rat lenses via the activation of TRPV1, while pilocarpine-induced removal of AQP5 from the membrane observed using immunolabelling was abolished by pre-incubation of the lenses with a TRPV1 inhibitor. In contrast, mimicking the actions of pilocarpine by blocking TRPV4 and then activating TRPV1 resulted in sustained increase in pressure and the removal of AQP5 from the anterior influx and equatorial efflux zones. These results show that the removal of AQP5 in response to a decrease in zonular tension is mediated by TRPV1 and suggest that regional changes to PH2O contribute to lens hydrostatic pressure gradient regulation.

Intracellular hydrostatic pressure regulation in the bovine lens: a role in the regulation of lens optics?

The optical properties of the bovine lens have been shown to be actively maintained by an internal microcirculation system. In the mouse lens, this water transport through gap junction channels generates an intracellular hydrostatic pressure gradient, which is subjected to a dual feedback regulation that is mediated by the reciprocal modulation of the transient receptor potential vanilloid channels TRPV1 and TRPV4. Here we test whether a similar feedback regulation of pressure exists in the bovine lens and whether it regulates overall lens optics. Lens pressure was measured using a microelectrode/pico-injector-based pressure measurement system, and lens optics were monitored in organ cultured lenses using a laser ray tracing system. Like the mouse, the bovine lenses exhibited a similar pressure gradient (0 to 340 mmHg). Activation of TRPV1 with capsaicin caused a biphasic increase in surface pressure, while activation of TRPV4 with GSK1016790A caused a biphasic decrease in pressure. These biphasic responses were abolished if lenses were preincubated with either the TRPV1 inhibitor A-889425 or the TRPV4 inhibitor HC-067047. While modulation of lens pressure by TRPV1 and TRPV4 had minimal effects on lens power and overall vision quality, the changes in lens pressure did induce opposing changes to lens geometry and its gradient of refractive index that effectively kept lens power constant. Hence, our results suggest that the TRPV1/4-mediated feedback control of lens hydrostatic pressure operates to ensure that any fluctuations in lens water transport, and consequently water content, do not result in changes in lens power and therefore overall vision quality.

Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens.

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.

Verification and spatial mapping of TRPV1 and TRPV4 expression in the embryonic and adult mouse lens.

The transient receptor protein vanilloid channels, TRPV1 and TRPV4, have recently been shown to be mechanosensors in the ocular lens that act to transduce physical changes in lens volume and internal hydrostatic pressure into the activation of signalling pathways in lens epithelial cells. These pathways in turn regulate ion and water transport to ensure that the optical properties of the lens remain constant. Despite the functional evidence that implicate the roles of TRPV1 and TRPV4 in the lens, their respective cellular expression patterns in the different regions of the lens has to date not been fully characterised. Using Western blotting we have confirmed that TRPV1 and TRPV4 are expressed throughout all regions (epithelium, outer cortex, inner cortex/core) of the adult mouse lens. Subsequent immunolabeling of lens cryosections confirmed that TRPV1 and TRPV4 are expressed throughout all regions of the lens, but revealed differentiation-dependent differences in the subcellular expression of the two channels in the different regions. In the epithelium and outer cortex, intense TRPV1 and TRPV4 labeling was predominately associated with the cytoplasm. In a discrete zone in the inner cortex, labeling for both proteins was greatly diminished, but could be enhanced by incubating sections with the detergent Triton X-100 to reveal TRPV1 and TRPV4 labelling that was associated with the membrane. This suggests that in this region of the lens there is a potential interacting protein that masks the binding of the TRPV1 and TRPV4 antibodies to their respective epitopes in the lens inner cortex. In the core of the lens, which contains the embryonic nucleus, TRPV1 and TRPV4 labelling was associated exclusively with fibre cell membranes. This labelling in the lens core of the adult mouse lens appeared to originate in early development as a similar membrane labelling was observed at embryonic day 10 (E10) of the cells in the lens vesicle that subsequently forms the embryonic nucleus in the adult lens. During subsequent stages of embryonic development TRPV1 and TRPV4 remained membranous in the inner cortex and core, while showing labelling that was associated with the cytoplasm in the superficial outer cortical region. The extent of cytoplasmic labelling for TRPV4, but not TRPV1, in this cortical region could however be dynamically regulated by cutting the zonules that normally attach the lens to the ciliary body. We have shown an early onset and continuous expression of TRPV1 and TRPV4 across all lens regions, and that TRPV4 can be dynamically trafficked into the membranes of differentiating fibre cells, results that suggests that these mechanosensitive channels may also be functionally active in lens fibre cells.

Dynamic functional contribution of the water channel AQP5 to the water permeability of peripheral lens fiber cells.

Although the functionality of the lens water channels aquaporin 1 (AQP1; epithelium) and AQP0 (fiber cells) is well established, less is known about the role of AQP5 in the lens. Since in other tissues AQP5 functions as a regulated water channel with a water permeability (PH2O) some 20 times higher than AQP0, AQP5 could function to modulate PH2O in lens fiber cells. To test this possibility, a fluorescence dye dilution assay was used to calculate the relative PH2O of epithelial cells and fiber membrane vesicles isolated from either the mouse or rat lens, in the absence and presence of HgCl2, an inhibitor of AQP1 and AQP5. Immunolabeling of lens sections and fiber membrane vesicles from mouse and rat lenses revealed differences in the subcellular distributions of AQP5 in the outer cortex between species, with AQP5 being predominantly membranous in the mouse but predominantly cytoplasmic in the rat. In contrast, AQP0 labeling was always membranous in both species. This species-specific heterogeneity in AQP5 membrane localization was mirrored in measurements of PH2O, with only fiber membrane vesicles isolated from the mouse lens, exhibiting a significant Hg2+-sensitive contribution to PH2O. When rat lenses were first organ cultured, immunolabeling revealed an insertion of AQP5 into cortical fiber cells, and a significant increase in Hg2+-sensitive PH2O was detected in membrane vesicles. Our results show that AQP5 forms functional water channels in the rodent lens, and they suggest that dynamic membrane insertion of AQP5 may regulate water fluxes in the lens by modulating PH2O in the outer cortex.

The Role of Aquaporins in Ocular Lens Homeostasis.

Abstract: Aquaporins (AQPs), by playing essential roles in the maintenance of ocular lens homeostasis, contribute to the establishment and maintenance of the overall optical properties of the lens over many decades of life. Three aquaporins, AQP0, AQP1 and AQP5, each with distinctly different functional properties, are abundantly and differentially expressed in the different regions of the ocular lens. Furthermore, the diversity of AQP functionality is increased in the absence of protein turnover by age-related modifications to lens AQPs that are proposed to alter AQP function in the different regions of the lens. These regional differences in AQP functionality are proposed to contribute to the generation and directionality of the lens internal microcirculation; a system of circulating ionic and fluid fluxes that delivers nutrients to and removes wastes from the lens faster than could be achieved by passive diffusion alone. In this review, we present how regional differences in lens AQP isoforms potentially contribute to this microcirculation system by highlighting current areas of investigation and emphasizing areas where future work is required.

Spatial distributions of AQP5 and AQP0 in embryonic and postnatal mouse lens development.

The expression of the water channel protein aquaporin (AQP)-5 in adult rodent and human lenses was recently reported using immunohistochemistry, molecular biology, and mass spectrometry techniques, confirming a second transmembrane water channel that is present in lens fibre cells in addition to the abundant AQP0 protein. Interestingly, the sub-cellular distribution and level of post-translational modification of both proteins changes with fibre cell differentiation and location in the adult rodent lens. This study compares the sub-cellular distribution of AQP0 and AQP5 during embryonic and postnatal fibre cell development in the mouse lens to understand how the immunolabelling patterns for both AQPs observed in adult lens are first established. Immunohistochemistry was used to map the cellular and sub-cellular distribution of AQP5 and AQP0 throughout the lens in cryosections from adult (6 weeks-8 months) and postnatal (0-2 weeks) mouse lenses and in sections from paraffin embedded mouse embryos (E10-E19). All sections were imaged by fluorescence confocal microscopy. Using antibodies directed against the C-terminus of each AQP, AQP5 was abundantly expressed early in development, being found in the cytoplasm of cells of the lens vesicle and surrounding tissues (E10), while AQP0 was detected later (E11), and only in the membranes of elongating primary fibre cells. During the course of subsequent embryonic and postnatal development the pattern of cytoplasmic AQP5 and membranous AQP0 labelling was maintained until postnatal day 6 (P6). From P6 AQP5 labelling became progressively more membranous initially in the lens nucleus and then later in all regions of the lens, while AQP0 labelling was abruptly lost in the lens nucleus due to C-terminal truncation. Our results show that the spatial distribution patterns of AQP0 and AQP5 observed in the adult lens are established during a narrow window of postnatal development (P6-P15) that precedes eye opening and coincides with regression of the hyaloid vascular system. Our results support the hypothesis that, in the older fibre cells, insertion of AQP5 into the fibre cell membrane may compensate for any change in the functionality of AQP0 induced by truncation of its C-terminal tail.

Verification and spatial localization of aquaporin-5 in the ocular lens.

Until recently, the lens was thought to express only two aquaporin (AQP) water channels, AQP1 and AQP0. In this study we confirm lenticular AQP5 protein expression by Western blotting and mass spectrometry in lenses from a variety of species. In addition, confocal microscopy was used to map cellular distributions of AQP5 in mouse, rat and human lenses. Tandem mass spectrometry of a human lens membrane preparation revealed extensive sequence coverage (56.2%) of AQP5. Western blotting performed on total fiber cell membranes from mouse, rat, bovine and human lenses confirmed AQP5 protein expression is conserved amongst species. Western blotting of dissected lens fractions suggests that AQP5 is processed in the lens core by C-terminal truncation. Immunohistochemistry showed that AQP5 signal was most abundant in the lens outer cortex and decreased in intensity in the lens core. Furthermore, AQP5 undergoes differentiation-dependent changes in subcellular location from an intracellular localization in differentiating fiber cells to the plasma membrane of mature fiber cells upon the loss of fiber cell nuclei. Our results show that AQP5 is a significant component of lens fiber cell membranes, representing the second most abundant water channel in these cells. Together, the changes to AQP5 distribution and structure are likely to modulate the functional role of AQP5 in different regions of the lens.

Regulation of lens water content: Effects on the physiological optics of the lens.

The lens is an important determinant of overall vision quality whose refractive and transparent properties change throughout life. Alterations to the refractive properties of the lens contribute to the process of emmetropisation in early childhood, and then the gradual loss in lens power that occurs throughout adulthood. In parallel to these changes to lens refractive power, age-dependent increases in lens stiffness and light scattering result in presbyopia and cataract, respectively. In recent years it has been confirmed that the lens operates an internal microcirculation system that generates circulating fluxes of ions, water and nutrients that maintain the refractive properties and transparency of the lens. By actively regulating lens water content, the microcirculation system controls two key parameters, lens geometry and the gradient of refractive index, which together determine the refractive properties of the lens. Furthermore, by delivering nutrients and antioxidants to the lens nucleus, the microcirculation system maintains lens transparency by preventing crystallin aggregation. Interestingly, the solubility, intramolecular packing and refractive index increment of crystallin proteins can be modulated by the ability of crystallin proteins to dynamically bind water, a processed called syneresis. In a series of previous studies it has been shown that the application of external pressure to the lens can induce syneresis. Since it is now known that lens water transport generates a substantial internal hydrostatic pressure gradient, we speculate that the microcirculation is capable of regulating crystallin function by altering the amount of water bound to lens proteins in the nucleus, where the pressure gradient and protein concentrations are the highest. Here we present evidence for the links between lens transport, pressure, syneresis and protein function. Furthermore, because the lens pressure gradient can be regulated by intrinsic and extrinsic stimuli, we suggest mechanisms via which this integrative system can be used to effect the changes to the refractive and transparent properties of the lens that are observed across our lifetime.

Lens Aquaporins in Health and Disease: Location is Everything!

Cataract and presbyopia are the leading cause of vision loss and impaired vision, respectively, worldwide. Changes in lens biochemistry and physiology with age are responsible for vision impairment, yet the specific molecular changes that underpin such changes are not entirely understood. In order to preserve transparency over decades of life, the lens establishes and maintains a microcirculation system (MCS) that, through spatially localized ion pumps, induces circulation of water and nutrients into (influx) and metabolites out of (outflow and efflux) the lens. Aquaporins (AQPs) are predicted to play important roles in the establishment and maintenance of local and global water flow throughout the lens. This review discusses the structure and function of lens AQPs and, importantly, their spatial localization that is likely key to proper water flow through the MCS. Moreover, age-related changes are detailed and their predicted effects on the MCS are discussed leading to an updated MCS model. Lastly, the potential therapeutic targeting of AQPs for prevention or treatment of cataract and presbyopia is discussed.

Changes to Zonular Tension Alters the Subcellular Distribution of AQP5 in Regions of Influx and Efflux of Water in the Rat Lens.

Purpose: The lens uses circulating fluxes of ions and water that enter the lens at both poles and exit at the equator to maintain its optical properties. We have mapped the subcellular distribution of the lens aquaporins (AQP0, AQP1, and AQP5) in these water influx and efflux zones and investigated how their membrane location is affected by changes in tension applied to the lens by the zonules. Methods: Immunohistochemistry using AQP antibodies was performed on axial sections obtained from rat lenses that had been removed from the eye and then fixed or were fixed in situ to maintain zonular tension. Zonular tension was pharmacologically modulated by applying either tropicamide (increased) or pilocarpine (decreased). AQP labeling was visualized using confocal microscopy. Results: Modulation of zonular tension had no effect on AQP1 or AQP0 labeling in either the water efflux or influx zones. In contrast, AQP5 labeling changed from membranous to cytoplasmic in response to both mechanical and pharmacologically induced reductions in zonular tension in both the efflux zone and anterior (but not posterior) influx zone associated with the lens sutures. Conclusions: Altering zonular tension dynamically regulates the membrane trafficking of AQP5 in the efflux and anterior influx zones to potentially change the magnitude of circulating water fluxes in the lens.

The acute-phase protein serum amyloid A3 is expressed in the bovine mammary gland and plays a role in host defence.

The serum amyloid A protein is one of the major reactants in the acute-phase response. Using representational difference analysis comparing RNA from normal and involuting quarters of a dairy cow mammary gland, we found an mRNA encoding the SAA3 protein (M-SAA3). The M-SAA3 mRNA was localized to restricted populations of bovine mammary epithelial cells (MECs). It was expressed at a moderate level in late pregnancy, at a low level through lactation, was induced early in milk stasis, and expressed at high levels in most MECs during mid to late involution and inflammation/mastitis. The mature M-SAA3 peptide was expressed in Escherichia coli, antibodies made, and shown to have antibacterial activity against E. coli, Streptococcus uberis and Pseudomonas aeruginosa. These results suggest that the mammary SAA3 may have a role in protection of the mammary gland during remodelling and infection and possibly in the neonate gastrointestinal tract.