Mapping Lens Metabolites:Effects of HBO on GSH levels
Since a major goal of the Molecular Vision Laboratory (MVL) is to develop novel therapies to delay the onset of ARN cataract, considerable time and effort has been invested into establishing and characterising an in vitro animal model that utilises HBO to induce acute oxidative stress in organ cultured bovine lenses. The initial characterisation of this bovine lens HBO model was published in Lim et al. (2016), and my contributions to this published study are presented in Chapter 6. A major finding of Lim et al. (2016) was that a regionally specific depletion of GSH level occurs in response to HBO exposure, leading in turn to an elevation of protein mixed disulphides (PSSGs), two changes known to precede cataract formation. In this study GSH and GSSG levels were quantified by HPLC, and PSSGs were detected by Western blotting. In this Chapter, I have utilised imaging mass spectrometry (IMS) to visualise changes to bovine lens metabolites in response to acute in vitro HBO exposure. This experimental approach has produced a large data set, but I have concentrated primarily on GSH metabolites in an effort to more fully understand this initial deletion of GSH in both HBO and by extension cataract. This work has been published in Nye-Wood et al. (2017). Before presenting these results I first review the major findings of Lim et al. (2016) to provide a context for understanding the need to more fully understand the effects of HBO on lens metabolites in general and GSH in particular.
Validating the HBO Model
HBO exposure affects the lens by increasing the partial pressure of dissolved O 2 and bringing molecular oxygen to deeper regions of the lens that are normally deprived of O2 by the activity of mitochondria in the outermost layers of the lens. This disruption of the normal O2 gradient (see Figure 1.9) generates ROS that disrupts redox homeostasis in deeper FCs (Lim et al., 2016). In a similar manner, HBN will cause inert N2 to diffuse across the lens, but will not form ROS. Thus by comparing the lenses exposed to HBN and HBO we can assess what effect an acute exposure to oxidative stress has on lens structure and function, independent of the effect of high pressure. As outlined in the introduction (section 1.5), in vivo HBO exposure causes nuclear GSH depletion and an increase in mixed disulfides in humans, and model systems (Giblin et al., 1988). These effects are also induced in the in vitro bovine lens
(Cappiello et al., 1995, Padgaonkar et al., 2000). Raman spectroscopy of lens thiols and disulfides reveals that in the bovine lens nucleus, but not the cortex, reduced thiol groups like GSH decrease in abundance, and disulfide bridges accumulate between proteins (Gosselin et al., 2007). In the bovine lens, HBO accelerates the physiological symptoms of lens aging (Raju et al., 2015).
After many 5- and 15-hour HBO treatments, in conjunction with HBN controls, the bovine lens was seen to resist HBO-induced opacification (Lim et al., 2016). This inability to induce cataract in the in vitro bovine lens resembles the in vivo guinea pig exposures, though many morphological and biochemical changes were seen in these lenses, including immature cataracts (Giblin et al., 1995). Lim et al. (2016) investigated the effect of 100atm pressure on cell morphology using confocal microscopy and found no evidence of membrane damage caused by HBN or HBO treatment for 5 or 15 hours, indicating that neither 100 atm exposure nor HBO-induced oxidative stress compromises the regular hexagonal cross section or cell integrity of FCs (Lim et al., 2016).
HBO treatment and glutathione
Giblin et al (1995) reported that HBO exposure has a dose-response relationship with in vivo GSH content. The bovine lens HBO model corroborates this effect in that 15 hour exposures cause significantly more GSH loss than 5 hour exposures (Figure 3.1, from Lim et al., (2016)). The bovine lens is large enough to manually dissect into regional fractions of outer cortex (OC), inner cortex (IC), and core, and measure concentrations of GSH and its oxidised dimer, glutathione disulfide (GSSG), using biochemical assays. After 5 hours HBO exposure, GSH decreases in all lens regions. Interestingly, the magnitude of this change is largest in the OC, and the effect intensifies after 15 hours. Curiously, GSSG also decreases in abundance after HBO treatment. In the OC it takes 15 hours exposure to reach significance. This suggests oxidative stress does not directly oxidise GSH to form GSSG (Figure 3.1), and instead has an alternative destination in the HBO model.
HBO and protein glutathionylation
The observation that both GSH and GSSG concentrations decrease in the HBO model suggests an alternative fate for GSH. While we cannot dismiss the loss of GSSG to the incubation solution, Lim et al. (2016) saw that the decline in GSSG concentration was in part explained by elevated glutathionylation of urea-soluble (US) proteins, which are assessed by probing a Western blot with an antibody specific for the protein-glutathione disulfide bond, PSSG (Figure 3.2). In control lenses (HBN) some glutathionylation was evident in the OC and IC after 5 hours, and became apparent in the core (C) only after 15 hours of exposure. HBO-induced oxidative stress led to higher levels of PSSGs after 5 hours (Figure 3.2A, arrowed). In the 15 hour treatment group, HBN induced changes are limited to faint labelling of core proteins in the range 37-45kDa, and a slight decrease in labelling intensity in the OC. On the other hand HBO-induced changes involved a dramatic increase in cortical PSSGs (Figure 3.2B).
Extending HBO time from 5 to 15 hours led to PSSG bands appearing in the core sample, but reduced the number and intensity of bands overall. There were also several new bands detected in the nucleus of HBN treated lenses, suggesting those bands may not be HBO specific. Extending time of exposure causes a downwards trend in glutathionylation in the OC and an upwards trend in the core, though ultimately these changes did not reach statistical significance (Figure 3.2E). Because only the water insoluble fraction was analysed, the reduction in labelling intensity may indicate proteins shifting into the water soluble fraction by cleavage or conformational change, or to the urea-insoluble fraction by promoting insolubilisation. Importantly, these trends are consistent with those of Giblin et al. (1995) who exposed guinea pigs to 2 hours of 2.5atm HBO three times a week, for a total of 100 instances. They saw protein glutathionylation increase most rapidly between the fifteenth and thirtieth iteration of HBO exposure, before reaching a maximum concentration after 65 exposures, and declining after 100 exposures. Their measured changes were also more apparent in the cortex than the core.
HBO and total protein
The effect of HBO on the formation of protein aggregates (higher molecular weight) and protein truncation (lower molecular weight peptides) was assessed by SDS-PAGE followed by Coomassie Blue staining. Because Coomassie Blue dye binds to protein non-specifically, it can reveal large scale differences in abundance, or evidence of differential protein processing between treatment groups. The differences that were seen in each proteome were limited to large molecular weight bands that did not match the PSSG bands shown in Figure 3.2. Altogether, the similarity of the HBN and HBO proteome indicates large-scale changes in protein mass, as would be caused by covalent binding and aggregation, are not induced in the HBO model.
In summary, while exposure of the bovine lens to HBO replicates certain hallmarks of cataractogenesis, namely GSH and GSSG depletion and elevated PSSG labelling, it does not cause major protein aggregation or truncation that would lead to RI fluctuations, light scatter, and ultimately the loss of lens transparency observed in ARN cataract. Thus, bovine lens HBO exposure appears to be a good model to study the processes of ageing that occur as a natural precursor to the later development of ARN cataract.
Quantitative experimental data on HBO-induced oxidation of proteins and antioxidants therefore exist, although prior to my research spatial information remained limited to comparing nuclear and cortical homogenates. The emerging importance of transport between lens cortex and nucleus for lens homeostasis – and its dysregulation in cataract – has indicated a need to spatially resolve these pathological changes. The work of Lim et al. (2016) in the bovine lens suggests that the initial step in the response of the lens to oxidative stress is a depletion of the antioxidant GSH. Therefore, spatially resolving the effects of HBO on lens GSH metabolism is a logical first step in investigating the mechanism of action of oxidative stress on the lens. My efforts to spatially map the effects of HBO on GSH and its metabolites using imaging mass spectrometry (IMS) are present in the remainder of this Chapter.
Chapter 1: Introduction
1.1 The Major Tissues of the Eye
1.2 Lens Cellular Structure and Organisation
1.3 Lens Function in the Normal and Ageing Lens
1.4 The Microcirculation and Age: an Initiating factor in Lens Pathology?
1.5 Testing my Hypothesis: the Hyperbaric Oxygen Model of ARN Cataract
Chapter 2: Methods
2.1 General laboratory procedures
2.2 Bovine Lens Tissue and Hyperbaric Treatment
2.3 MALDI Imaging Mass Spectrometry
2.4 Liquid chromatography tandem mass spectrometry
2.5 Magnetic Resonance Imaging
2.6 Western Blotting
Chapter 3: Mapping Lens Metabolites: Effects of HBO on GSH levels
3.1 Validating the HBO Model.
3.2 Metabolome Mapping in the Normal Bovine Lens
3.3 Lens Metabolic Compartments
3.4 Effects of HBO on GSH Metabolism
3.5 Effects of HBO on GSH-Derived Metabolites
3.6 Effects of HBO on metabolite thiol/disulfide exchange
Chapter 4: Lens Crystallins: Mapping HBO induced PTMs
4.1 The Lens Crystallins
4.2 Crystallin Mapping in the Normal Bovine Lens
4.3 Effects of HBO on Lens Crystallins
Chapter 5: Effects of HBO on Water Content and Solute Transport
5.1 Use of MRI to Visualise Lens Water Content and Functionality
5.2 T1 measurements of Water Content in the Bovine Lens
5.3 MRI Visualisation of Solute Delivery by the Microcirculation System
5.4 Oxidative Damage to the Microcirculation System: Are Gap Junctions Involved?
Chapter 6: Effects of HBO on Lens Optics and Vision Quality
6.1 Use of MRI to Extract the Optical Properties of the Bovine Lens
6.2 Effects of HBO on the Optical Power of the Bovine Lens
6.3 Effects of HBO on Overall Vision Quality
Chapter 7: Conclusions & Significance
7.1 Summary of Main Findings
7.2 The Ageing Lens and in vivo Oxidative Stress
7.3 Changing Water in the Nucleus: an Initiating Event in the Pathological Loss of Lens Function?
7.4 Implications of my research for Lens Pathology
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Oxidative Stress and the Lens: Characterising a Hyperbaric Oxygen model of Lens Ageing