ReviewLens glutathione homeostasis: Discrepancies and gaps in knowledge standing in the way of novel therapeutic approaches
Introduction
The lens has evolved as an anaerobic biological system with millimolar concentrations of glutathione (GSH). The critical role of GSH in maintaining lens redox status and transparency is well recognized and has been, over the years, the subject of several excellent reviews (Giblin, 2000, Lou, 2000, Reddy, 1990, Truscott, 2005). While one could argue that nothing new was to be expected concerning the protective role of GSH in the lens, our interest in GSH homeostasis in the lens was rekindled with the unexpected finding that lenticular GSH levels were not completely suppressed in the LEGSKO mouse in spite of complete absence of γ-glutamylcysteine ligase (Fan et al., 2012). This issue, which is the subject of intense investigation in our laboratory, is closely linked to lenticular ascorbate metabolism and cataractogenesis and the work of David Beebe who has pioneered the importance of the vitreous as a source of oxidative stress to the lens (Beebe et al., 2014, Holekamp et al., 2005, Li et al., 2013b, Shui et al., 2009). These paradigm shifting studies inspired us to study lens biology in connection not only to the aqueous humor but also to the vitreous humor. Additionally, Dr. Beebe’s pioneer studies provide the mechanistic framework for a potential therapeutic treatment of high risk (>90%) and rapid (within two years) nuclear cataract formation after vitrectomy surgery (Petrash, 2013).
In order to provide a complete coverage of lens GSH homeostasis, we have to discuss the lenticular GSH dynamics from the perspective of both protein conjugated GSH and free GSH/oxidized GSH (GSSG). Since several excellent reviews have covered the protein glutathionylation (Lou, 2000, Lou, 2003, Lou and Dickerson, 1992, Lou et al., 1990, Lou et al., 1995), we will mainly focus on the roles of latter. Below we review the established mechanisms and pathways that are involved in lens GSH homeostasis. We also provide a brief summary of recent progress regarding lens nucleus GSH homeostasis, as well as the impact of aging on lens GSH homeostasis, since age-related nuclear cataract (ARNC) is often believed to be, in part, associated with declining nuclear GSH levels in the aging human lens (Giblin, 2000).
Intracellular GSH is synthesized by two ATP-dependent enzymes: γ-glutamylcysteine ligase (GCL) and glutathione synthase (GS) to produce γ-glutamylcysteine and GSH, respectively. The mammalian GCL is a heterodimer enzyme consisting of a 73-kDa catalytic subunit, Gclc, and a 28-kDa modulatory subunit, Gclm. The catalytic subunit, Gclc has the enzymatic activity and is regulated via a GSH feedback inhibition mechanism (Richman and Meister, 1975). Gclm has no enzymatic activity, but heterodimer formation of Gclm and Gclc significantly decreases the Km value for glutamate and increases the Ki value for the feedback inhibition by GSH (Chen et al., 2005).
Like other tissue systems, the lens has a functional GSH de novo synthesis machinery, which mostly lies in the epithelial and cortical layers, while mature fibers cells sit in inner layers of the lens that have lost cell organelles such as nucleus and mitochondria (Bassnett and Beebe, 1992). However, due to the avascularity of the lens, for GSH biosynthesis to take place, the constituent amino acids, glutamic acid, glycine and cysteine have to be transported to the epithelial and outer cortical fibers cells. Pioneering work from Reddy et al. has demonstrated that these amino acids are delivered to the lens from the plasma via the aqueous humor and the lens epithelium, from which they are delivered to the rest of the lenticular system (Reddy, 1973, Reddy, 1979) (Fig. 1). For GSH synthesis, the Km value of GCL for cysteine is ∼0.15 mM, while that for glutamate is ∼1.7 mM, and that of GS for glycine is ∼0.8 mM (McBean, 2012). In order for GSH biosynthesis to take place, the required intracellular concentration of these amino acids is thought to be close to their Km value. However, various studies suggest species-specific results regarding these amino acids levels in the lens epithelium and outer cortex. For example, Lim et al. (Lim et al., 2007) find that the concentration of the three amino acids in the rat lens cortex is lower than that required for the GCL and GS Km value of its proper constituent amino acids. In contrast, other studies in human, rabbit and bovine lens demonstrate much higher values than these required for the Km (Barber, 1968, Kern and Ho, 1973, Reddy, 1973). It has to be pointed out that these studies measured the total GSH from the homogenate of the lens tissue, and that this does not exclude the possibility that intracellular amino acid level might be much higher to fulfill the needs of GSH de novo synthesis. Nevertheless, similar to other body systems, such as the central nervous system (CNS) (Aoyama et al., 2012), cysteine level is relatively lower than glutamic acid and glycine. It is, therefore, the rate-limiting substance in lenticular de novo GSH synthesis and an adequate cysteine supply is essential wherever GSH de novo synthesis is taking place.
From the above considerations, it is clear that cellular cysteine and GSH synthesis are tightly linked. Multiple mechanisms have been postulated in terms of intracellular cysteine homeostasis in studies of the central nervous system (CNS) or hepatocytes (Lu, 1999, McBean and Flynn, 2001). The sodium independent cystine/glutamate exchanger (Xc−) has been shown to take up cystine into cells, which is subsequently reduced into cysteine for GSH synthesis (Lewerenz et al., 2013, Lim and Donaldson, 2011). In one set of studies, the Xc− exchanger was found present in the entire rat lens, predominantly in the cytoplasm in the outer cortex cells, while it was more membranous in the inner cortex region (Lim et al., 2005). In the human lens, Xc− is present in the entire lens region at a young age, but no immunoreactivity is found in the central lens region of aged human lenses (Lim et al., 2013). In contrast, in other studies, Xc− was reported to be predominately present in the membranes of outer cortex cells, and no detection was observed in the nuclear region of the dog lens(Lall et al., 2008). These studies provide evidence for the presence of Xc−, but whether this exchanger is important for lens cysteine homeostasis is still not very clear. Several findings point to quite different research directions. In a vascular eye perfusion study in guinea pigs, radiolabeled cysteine, cystine and methionine were injected through the common carotid artery (Mackic et al., 1997). In this study, cysteine, but not cystine, was readily taken up by the lens epithelial and cortical fiber layers, while infused cystine failed to incorporate into GSH synthesis. Other evidence in support of cysteine rather than cystine uptake is that high levels of free cysteine, but not cystine, are found in human (Barber, 1968), monkey (Gaasterland et al., 1979) and calf (Kern and Ho, 1973) aqueous humor, though different results was reported in the aqueous humor of guinea pigs (Mackic et al., 1997). Furthermore, recent reports (Martis et al., 2015) indicate no impact on lens GSH level based on tests with Xc− knockout mice.
On the other hand, earlier studies indicate that over 90% of cystine transport is actually processed by sodium-dependent high affinity glutamate transporters (XAG−), based on rat brain tissue uptake experiments (Flynn and McBean, 2000) and a cultured astrocytes study (Bender and Norenberg, 2000). Also, cysteine was found to be able to inhibit XAG-facilitated transport (McBean and Flynn, 2001). Altogether, five subtypes of high-affinity glutamate transporters (excitatory amino acid transporters 1–5 (EAAT1–5)) have been identified in mammalian tissues(Aoyama and Nakaki, 2013, Bridges and Esslinger, 2005). More recent studies demonstrate that EAAT3, also named “excitatory amino acid carrier 1” (EAAC1), is more functional in cysteine transport than the control of extracellular glutamate levels (Aoyama and Nakaki, 2013, Holmseth et al., 2012). The brain cysteine and GSH levels are significantly reduced in EAAC1-deficent mice, and this can be attenuated by the treatment with N-acetylcysteine (NAC), whose uptake proceeds via different uptake mechanisms than cysteine/cystine (Aoyama et al., 2006). All five types of EAATs are found present in rat lens based on RT-PCR and western-blot analysis (Lim et al., 2005). We therefore speculate that lenticular cysteine transport from aqueous humor occurs, most likely via a sodium-dependent transporter system, such as EAAC1, and that high cysteine transport activity occurs at the lens equator region, as reported by Truscott’s group (Sweeney et al., 2003).
The metabolic pathway transsulfuration can also supply cysteine from methionine via the transmethylation pathway (McBean, 2012). The transsulfuration pathway is also present in the lenticular system (Persa et al., 2004). Cystathionine-beta-synthase (CBS), one of the enzymes utilizing methionine to produce cysteine via transmethylation is elevated in human lens nucleus but decreased in the epithelial layer with aging. CBS expression can also be stimulated with oxidative stress, such as H2O2 in lens epithelial cell culture (Persa et al., 2004). In addition, the betaine-homocysteine S-methyltransferase 1(BHMT1), a remethylation enzyme that converts homocysteine to methionine, is found to be down-regulated in aged human lens nuclei (Zhou et al., 2015). This may explain the findings that the free cysteine levels are elevated in the lens nucleus under oxidative stress (Giblin et al., 1995, Lou, 2000). However, in a vascular eye perfusion study in guinea pigs (Mackic et al., 1997), methionine failed to produce cysteine and incorporate into GSH, suggesting minimal contribution of methionine in circulation to lenticular cysteine homeostasis (Mackic et al., 1997). Apparently, more study is needed to clarify whether the transsulfuration pathway plays a significant role in lens cysteine and GSH homeostasis.
As mentioned above, high affinity sodium-dependent glutamate transporters have been located in rat lenses (Lim et al., 2005), though no report exists so far about their expression level in the human lens. We anticipate that these transporters are also present in human lenses. Extensive studies and reviews have addressed their roles in the CNS (Divito and Underhill, 2014, Vandenberg and Ryan, 2013). On the other hand, it is well established that cellular glutamate originates from glutamine, which is readily taken up by the lens via a transport mechanism that is hundreds of times more efficient than that of glutamate. Glutamine was shown to convert into glutamate and be incorporated into GSH synthesis in ex vivo calf and rat lens culture systems (Kern and Ho, 1973). However, no in vivo confirmation of this in vitro finding was reported. Since the anterior, but not posterior, part of the lens is equipped with a monolayer of epithelial cells (Beebe, 2008), it is anticipated that the anterior will be more selective and specific for amino acid transport than the posterior of the lens. In that regard, it is our point of view that ex vivo lens culture systems using total immersion are convenient, but not suitable for lens transporter studies because lens anterior and posterior surfaces have completely different structures. More work is needed to test whether glutamine uptake and conversion to glutamate plays a significant role in lens GSH homeostasis. At least one neutral amino acid transporter, ASCT2, has been found present in rat lenses (Lim et al., 2006), and free glutamine measured in calf aqueous humor is more than double that of glutamate (Kern and Ho, 1973).
Intracellular glycine homeostasis, like other neurotransmitters, is mainly regulated by the high affinity sodium-dependent transporters GLYT1 and GLYT2, which belong to the solute transporter family (SLC6) (Chen et al., 2004). Both glycine transporters have been identified in rat lenses (Lim et al., 2006, Lim et al., 2007) (Fig. 1).
Gamma-glutamyl transpeptidase (GGT), also known as gamma-glutamyl transferase, a glycoprotein, is localized at the cell surface and anchored to the cell membrane via a single N-terminus transmembrane domain. It cleaves only the extracellular GSH, oxidized GSH (GSSG), as well as glutathione S-conjugates (Ikeda et al., 1995, Wickham et al., 2012), therefore providing the cells with the amino acids necessary for intracellular GSH synthesis (Hanigan, 2014). GGT was reported present in the lens, ciliary body and cornea over 40 years ago in two independent studies (Reddy and Unakar, 1973, Ross et al., 1973) (Fig. 1). GGT activity enables the cells to maintain their intracellular GSH levels, thus coping with reactive oxygen species (ROS) attack. Both GGT knockout and mutant mice develop cataract in a very short time period after birth (Chevez-Barrios et al., 2000, Yamada et al., 2013). GGT deficient mice were found to have severe cysteine deficiency (∼20% of WT plasma cysteine level) (Lieberman et al., 1996). Interestingly, despite high plasma GSH level in GGT deficient mice, eye and lens GSH levels are markedly reduced (∼5% of WT) (Chevez-Barrios et al., 2000). The drastic reduction of lens GSH content in GGT KO mice cannot simply be explained by impaired GSH de novo synthesis due to cysteine deficiency. The lens Gclc conditional knockout mouse (LEGSKO mouse) recently created by our group (Fan et al., 2012) is able to maintain ∼50% GSH levels (1–2 mM) relative to wild type (WT) mice on the C57BL/6 background (unpublished), while levels were lower in the FVB/B6 hybrid strain (Fan et al., 2012). We believe that both low cysteine and GSH supply to the lens are necessary for drastic GSH reduction. In other words, lenses lacking efficient de novo GSH synthesis may take up either GSH or GSSG from surrounding ocular structures, i.e. the aqueous humor or the vitreous humor. We will discuss this aspect in the following section. Needless to say, the gamma-glutamyl cycle is an important amino acid recycling mechanism for maintenance of lens GSH homeostasis, particularly in the metabolically active regions of the lens.
Section snippets
Lens GSH/GSSG uptake
An in situ vascular eye perfusion study in guinea pig has come to a very surprising conclusion that the de novo GSH synthesis from circulating and aqueous sulfur amino acids, such as cysteine, cystine or methionine can be only a minor source of the millimolar concentration of GSH in the epithelium (Mackic et al., 1997). A t1/2 of 5480 h was estimated for lens epithelium GSH to be replaced entirely if solely based on circulating sulfur amino acids, the limiting amino acids in intracellular GSH
Lens nucleus GSH homeostasis
From the inner core to the periphery, the lens nucleus is constituted by an embryonic nucleus, a fetal nucleus, and an adult nucleus formed by differentiated fiber cells. Each of these can be delicately peeled off the human lens using simple tweezers (Beebe, 2008). These mature fibers cells have no capacity for protein or small molecule synthesis, including that of GSH. However, the lens nucleus still contains substantial level of GSH for maintenance of lens nucleus redox status (Giblin, 2000,
Aging impact on lens GSH homeostasis
The GSH level declines with age in the lens, particularly in the lens nucleus, and this is widely believed to be a major mechanism for age-related nuclear cataract formation (Giblin, 2000, Lou, 2003, Truscott, 2005). Multiple causes have been proposed for this decrease:
- 1)
The age-related loss of synthetic enzymes for de novo GSH synthesis is considered one of the major mechanisms. GCL, the GSH de novo synthesis rate limiting enzyme, has a 16-fold decrease in activity over a 83-year time frame (
Concluding remarks and perspectives
The lens is an avascular ocular tissue equipped with an unusually high level of GSH. The high concentration of GSH works with its antioxidant partners to keep the lens redox system in check and maintain lens transparency for several decades in humans. In addition to GSH de novo synthesis that only occurs in the epithelium and outer cortex, the lens is believed to also take up large portions of GSH from surrounding ocular tissues, i.e. the aqueous humor and the vitreous humor. However, more
Acknowledgment
We thank the National Eye Institute (Grant EY024553 to XF, and EY 07099 to VMM), and the Case Western Reserve University Visual Science Research Center (NEI P30EY-11373) for supporting our research.
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