SEARCH

SEARCH BY CITATION

Keywords:

  • aging;
  • α-crystallin;
  • lens stiffness;
  • presbyopia;
  • protein denaturation;
  • thermal aggregation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Presbyopia, the inability to focus up close, affects everyone by age 50 and is the most common eye condition. It is thought to result from changes to the lens over time making it less flexible. We present evidence that presbyopia may be the result of age-related changes to the proteins of the lens fibre cells. Specifically, we show that there is a progressive decrease in the concentration of the chaperone, α-crystallin, in human lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and insoluble protein. This is accompanied by a large increase in lens stiffness. Stiffness increases even more dramatically after middle age following the disappearance of free soluble α-crystallin from the centre of the lens. These alterations in α-crystallin and aggregated protein in human lenses can be reproduced simply by exposing intact pig lenses to elevated temperatures, for example, 50 °C. In this model system, the same protein changes are also associated with a progressive increase in lens stiffness. These data suggest a functional role for α-crystallin in the human lens acting as a small heat shock protein and helping to maintain lens flexibility. Presbyopia may be the result of a loss of α-crystallin coupled with progressive heat-induced denaturation of structural proteins in the lens during the first five decades of life.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Presbyopia is the most common eye condition, affecting almost everyone by age 50. It is the inability to focus on near objects. The underlying aetiology of presbyopia, however, remains controversial. More than 150 years ago, Helmholtz postulated that presbyopia resulted from a hardening of the lens (1855); however, in a recent review of the topic, it was proposed that growth of the lens was the primary cause (Strenk et al., 2005). Others have suggested roles for the ciliary muscle (Eskeridge, 1984), capsule (Krag et al., 1997) and the vitreous (Coleman, 1970).

Experimental evidence in support of a lenticular basis for presbyopia has accumulated recently (Glasser & Campbell, 1999; Heys et al., 2004; Weeber et al., 2005) following on from earlier studies (Fisher, 1971; Pau & Kranz, 1991). It has become apparent that even if all the other ocular factors play some role in the gradual loss of focussing ability, the age-related loss in elasticity of the lens would, of itself, be sufficient to explain presbyopia (Glasser & Campbell, 1999). One of the main arguments against lens stiffening was that, until recently, the very limited data on isolated lenses seemed to show little evidence of sclerosis prior to middle age (Pau & Kranz, 1991), whereas it is well known that our focal length changes continuously from childhood (Duane, 1912; Bruckner et al., 1986).

A dynamic mechanical analysis (DMA) method for measuring the stiffness of lenses revealed that there is a dramatic increase in stiffness as we age (Heys et al., 2004) and that changes were detected well before the age of onset of presbyopia. The nucleus, that part of the lens present at birth, becomes approximately 500- to 1000-fold stiffer over our lifetime and there was a marked increase in stiffness up to age 50. These findings were supported by the results of a separate study (Weeber et al., 2005). These DMA measurements were performed on human lenses that had been frozen and there was some concern that freezing and thawing may affect the values (Schachar & Pierscionek, 2007), particularly because lens water alters with age (Lahm et al., 1985).

Here we report the measurement of stiffness in individual human lenses across the age range. These lenses were not frozen and were analysed as quickly as possible after the death of the donor. Our aim was to correlate the stiffness data with biochemical studies to try to discover the underlying cause for the changes in physical properties of the aging lens. The lens is composed of three structural proteins: α-, β- and γ-crystallin. α-Crystallin, which comprises approximately 40% of the total protein in the human lens, is a member of the small heat shock protein family (Horwitz, 1992).

Our results suggest that a progressive loss of soluble α-crystallin, which is associated with the formation of large protein aggregates, appears to contribute to lens stiffness. Heat-induced denaturation of crystallins in the lens fibre cells may be an important factor in the aetiology of presbyopia as model experiments using intact pig lenses incubated at 50 °C showed changes in stiffness, and soluble protein profiles that mimic those of the aging human lens. Such laboratory findings may provide a basis for epidemiological data that has revealed a correlation between the age of onset of presbyopia and the mean ambient temperature (Miranda, 1979).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The summary of an examination of human lens stiffness as a function of age is shown in Fig. 1. These data were obtained using a dynamic mechanical analyser with a custom-made probe that allowed several measurements of stiffness in the nuclear region of each lens. Only the figures for the core are illustrated because this is the region of the lens where shape change is detected during accommodation (Brown, 1974; Koretz et al., 1997; Dubbelman et al., 2003). The cortex also increased in stiffness with age but to a lesser degree (data not shown), which is in agreement with a previous study (Heys et al., 2004).

image

Figure 1. Influence of the age of the human lens on stiffness. Stiffness measurements in the centre of individual fresh lenses were taken using dynamic mechanical analysis (DMA). Each point is the mean value of nine separate measurements ± standard deviation. Figure 1 inset. The same data points plotted in a log format to illustrate the increase in stiffness prior to age 50.

Download figure to PowerPoint

In the current study, lenses were not frozen and were analysed as soon as possible following removal of the eye; typically less than 48 h after death. The appearance of the log plot (Fig. 1, inset) is similar to that obtained previously (Heys et al., 2004); however, the curve is displaced to higher values. That is, fresh lenses were generally stiffer than frozen lenses of a similar age. Despite this the magnitude of the increase in stiffness with age was similar to that documented earlier; that is, a ~500-fold increase in stiffness taking place over a time span of 20–60 years. The data are plotted both in linear and log formats; the non-log plot emphasizes the marked increase in lens stiffness after age 50.

The comparison of the two data sets suggested that freezing/thawing may affect lens stiffness values and this feature was investigated using pig lenses. Porcine lenses are readily available in quantity and are close to human lenses in terms of size and water content, although pigs are thought not to accommodate. The study revealed that freezing of lenses did influence the stiffness measurements and, furthermore, that stiffness was dependent on the time that the lenses were stored frozen prior to analysis (Fig. 2). The reason for this is not clear, but may be related to the size of ice crystals.

image

Figure 2. Effect of freezing on lens stiffness. Porcine lenses were analysed fresh (T = 0), and following freezing for different times at –80 °C. Stiffness was measured in the centre of individual lenses using dynamic mechanical analysis (DMA) (n = 4 lenses at each time-point, mean value ± standard deviation).

Download figure to PowerPoint

We attempted to correlate lens stiffness with various parameters in an effort to determine the biochemical basis for the marked changes in the physical properties of human lenses with age. A clear change in shape of the lens nucleus occurs when accommodation (focusing) takes place (Brown, 1974; Koretz et al., 1997; Dubbelman et al., 2003). Therefore, accommodation presumably involves alterations to the shapes of the fibre cells in the lens centre, which, in turn, would necessitate some redistribution of cytoplasm and a degree of membrane fluidity. We first examined lipid composition of lens cores from young and old lenses using mass spectrometry. The shotgun lipidomics approach (Mitchell et al., 2004) revealed no major changes, although cholesterol did increase and the proportion of phosphatidylcholines did decrease slightly with age (data not shown); however, phosphatidylcholines are a very minor component of fibre cell membranes in humans (Huang et al., 2005).

In contrast, as depicted in Fig. 3A, aging was associated with significant and progressive insolubility of proteins in the lens core. Most, but not all, of the decrease in soluble protein could be accounted for by the appearance of insoluble protein that could be solubilized in 8 m urea. Some crystallins in older lenses remained insoluble even in this strong deaggregating agent and we have preliminary evidence that this may reflect strong interactions between these crystallins, which may be denatured, and the fibre cell membranes. In these experiments, we extracted the lenses using buffer at pH 7 to, as closely as possible, mimic the conditions in the lens. Protease inhibitors were included to eliminate any effect of proteolysis on crystallin solubility. Soluble protein measurements were confirmed by amino acid analysis of six selected fractions.

image

Figure 3. (A) Change in the solubility of proteins in human lenses as a function of age. Values for individual lens nuclei are shown. Soluble (inline image) : insoluble protein (inline image) was solubilized in 8M urea. (B) Changes in the content of soluble α-crystallin (inline image) and high molecular weight protein (inline image) in the lens nucleus as a function of age. Individual human lenses were extracted with buffer at pH 7 and an aliquot separated by gel filtration high performance liquid chromatography (HPLC).

Download figure to PowerPoint

Concomitant with the overall loss of soluble proteins there was an almost linear, age-dependent decline in the concentration of α-crystallin in the soluble protein fraction (Fig. 3B), such that by age 40–50, very little, or no soluble α-crystallin remained in the lens nuclei. This result is in agreement with others (Roy & Spector, 1976a; McFall-Ngai et al., 1985). In some older lenses a small peak was present in the α-crystallin region that was included in the plot shown in Fig. 3B, although sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel tryptic digestion revealed that the peak consisted largely of β-crystallins. The most likely explanation for the loss of soluble α-crystallin is that it had acted as a chaperone and had bound to other crystallins in the lens as these polypeptides denatured, or to other cellular components, for example, cytoskeletal proteins (Der Perng et al., 2004; Xi et al., 2006). Many studies have examined the behaviour of this small heat shock protein in vitro, and it appears that α-crystallin binds to non-native ‘molten globule’ conformations of substrate proteins as they unfold (Treweek et al., 2000; McHaourab et al., 2002), leading to the formation of large soluble protein aggregates. In agreement with this supposition, a high molecular weight (HMW) peak was detected in the gel filtration high performance liquid chromatography (HPLC) profile of older lenses (Fig. 4) and it increased in amount with age up to age 30, and then declined (Fig. 3B). A likely explanation is that after age 30, the HMW peak becomes incorporated into the insoluble protein. α-Crystallin was found to be a major component of the HMW peaks and insoluble proteins as judged by SDS-PAGE. The identification was confirmed with in-gel tryptic digestion and Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (Supplementary Fig. S1). This finding is in agreement with other similar studies (Roy & Spector, 1976b).

image

Figure 4. Representative gel filtration high performance liquid chromatography (HPLC) profiles of the soluble proteins from human lens nuclei extracted at pH 7. (a) 20 years old, (b) 74 years old. α, α-crystallin; βL, β low crystallin; βH, β high crystallin; γ, γ-crystallin; HMW, high molecular weight protein.

Download figure to PowerPoint

Because the time of disappearance of α-crystallin coincided with the increase in lens stiffness, we hypothesized that soluble α-crystallin may be involved in maintaining the flexibility of the lens tissue. In order to examine this in greater detail, we established a model system using intact pig lenses. Porcine lenses were heated at various temperatures and then removed at time intervals and the stiffness measured. Stiffness of the lens nuclei increased with time in a temperature-dependent manner (0.42 Pa min−1 at 43 °C; 31.3 Pa min−1 at 50 °C; 88.1 Pa min−1 at 55 °C; 185 Pa min−1 at 60 °C; and 882 Pa min−1 at 70 °C).

The time course for the increase in stiffness of the lens core during incubation at 50 °C is shown in Fig. 5A. In this experimental system the temperature in the middle of the lens, as determined using a thermocouple, reached 50 °C within 5 min. Thermo gravimetric analysis and differential scanning calorimetry confirmed that the lens did not change in hydration during the course of the experiment. A comparison of the lens stiffness profile with that of soluble α-crystallin content was revealing. After an initial lag period of 2 h, there was an almost linear increase in lens stiffness. By comparison, soluble α-crystallin had decreased by 54% in the first 2 h and to less than 10% of the original value by 4 h at 50 °C (Fig. 5A). This disappearance is in accord with it acting as a small heat shock protein and its chaperone function becoming more apparent at higher temperatures (Raman et al., 1995; Reddy et al., 2000). In agreement with this, an HMW peak appeared in the gel filtration HPLC profiles of heated lenses and there was also a significant conversion of soluble into insoluble protein over the course of the incubation (Fig. 5B). This pattern of loss in soluble α-crystallin, appearance of HMW protein followed by its disappearance, and conversion of soluble into insoluble protein mimics closely that seen in the human lens as a function of age. No changes were detected in the lipid composition following heating. These data suggest that heating of intact pig lenses can reproduce the pattern of changes observed in human lenses with age.

image

Figure 5. Response of intact pig lenses to heat. (A) Stiffness in the centre of porcine lenses following incubation at 50 °C (inline image). Stiffness readings were taken in the centre of individual lenses and correlated with measurements of the α-crystallin (inline image) and high molecular weight (HMW) (inline image) contents in the soluble protein fraction as assessed using peak areas in the gel filtration high performance liquid chromatography (HPLC) profile. At each time-point three lenses were taken for measurement of stiffness as for Figure 1. Each point is the mean value of 27 separate measurements ± standard deviation. (B) Change in the solubility of proteins in porcine lenses following incubation at 50 °C. Values for individual lens nuclei (n = 3 ± standard deviation) are shown. Soluble protein (inline image) : insoluble protein (inline image) was solubilized in 8 m urea.

Download figure to PowerPoint

Individual human lenses also increased in stiffness when they were heated at 50 °C and this was accompanied by a conversion of soluble into insoluble protein in the lens core (Fig. 6). At the end of the 8-h incubation period, insoluble protein had increased compared to the contralateral (unheated) lens by 18% (45 years old) and 13% (55 years old).

image

Figure 6. Change in the stiffness of three human lenses (aged 36, 45 and 55) following incubation at 50 °C. Lens sections were incubated in the chamber of the dynamic mechanical analysis (DMA) instrument and stiffness measurements were made at 0, 4 and 8 h.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

The temperature of the eye can vary significantly depending on the ambient temperature. Lenses of rabbits that were housed at 4 °C dropped from 35 °C to 29 °C (Schwartz, 1965). On the other hand, the lenses of rabbits that were placed in sunlight at 40 °C reached 41 °C within 10 min. Similarly when a monkey was exposed to the sun with an outside temperature of 49 °C, its lens temperature rose rapidly to 42 °C (Al-Ghadyan & Cotlier, 1986). The increase in eye temperature seems to result from a combination of absorption of solar radiation (e.g. by the pigment epithelium of the iris) and exposure of the eye to a warmer environment.

One of the major crystallins of the mammalian lens, α-crystallin, is a member of the small heat shock protein family (Horwitz, 1992). It has been proposed that one function of this protein could be to chaperone other crystallins as they denature over time. This protein denaturation occurs as a direct consequence of the growth pattern of the lens. The lens grows continuously throughout life by the addition of lens cells to the lens that was present at birth. Thus, no lens cells are lost as we grow older and the lens that existed at birth becomes the centre (nucleus) of the adult lens. As there is little or no protein turnover in the nucleus, the structural proteins are present for as long as the individual. A raft of post-translational modifications have been documented in aged human lens crystallins, including racemization (Masters et al., 1977), truncation (Takemoto, 1995; Lampi et al., 1998) and modification by small reactive chemicals derived from the decomposition of ultraviolet filters (Korlimbinis & Truscott, 2006) and ascorbate/monosaccharides (Cheng et al., 2004). Such processes may ultimately compromise the solubility of these proteins. Deamidation is quantitatively the most prevalent post-translational modification (Wilmarth et al., 2006) and the percentage of deamidation is greater in the insoluble proteins, suggesting that the introduction of negative charges may play a role in the crystallin denaturation over time.

It has been proposed that one of the roles of α-crystallin is to bind to lens proteins as they denature, and in the process, HMW soluble aggregates are produced (Rao et al., 1995; Derham & Harding, 1999). These aggregates may over time become insoluble. In support of this hypothesis, it was demonstrated in our study that nuclei of human lenses above the age of 40 contain no soluble α-crystallin; presumably all of it has been used to chaperone other proteins as they unfolded. The effects of such biochemical changes on the physical properties of the lens have not been investigated previously.

Accommodation is the change in the focus of the eye. In higher primates, it occurs as a result of a change in the shape of the lens. When close focus is required, the lens becomes more rounded as the ciliary muscles in the eye contract forwards and the resting tension on the lens is released. It is clear that the success of this strategy relies on the natural elasticity of the lens. Techniques, such as slit lamp photography, have clearly demonstrated that it is the nucleus of the lens that changes shape when the tension on the lens is released (Brown, 1974; Koretz et al., 1984; Koretz & Handelman, 1988). Therefore, the discovery that the human lens nucleus becomes massively stiffer with age could provide an explanation for the gradual loss of accommodative ability with age, which results ultimately in presbyopia by age 45–50.

In this study, we measured stiffness in human lenses that had been removed from the eye as soon as possible, and not frozen prior to analysis. This procedure avoided potentially confounding variables such as freezing/thawing and time of storage. Storage can affect the properties of lenses quite markedly (Augusteyn et al., 2006) and we demonstrated that the time of freezing could indeed affect the values obtained for lens stiffness. Despite these caveats, the same trend in stiffness with age was obtained although the fresh lenses were on average slightly stiffer at each age than frozen ones that have been measured previously (Heys et al., 2004). Following DMA, the lens nuclei were examined for a variety of biochemical parameters to discover if some of these could be correlated to the increase in lens stiffness.

It is likely that the shapes of individual fibre cells in the lens centre must change, if the nucleus as a whole is to become more spherical. Therefore, the fluidity of the cell membranes could significantly affect this ability to accommodate. Fluidity is determined largely by the lipid composition of the membranes (Stubbs & Smith, 1984) and age-related changes to human lens lipids have been reported (Borchman et al., 1994). Our data, however, revealed little change in the lipid composition of the lens nucleus with age and no significant changes were observed after age 50 whereas the stiffness of lenses continued to increase substantially. This finding suggests that other factors may be of greater importance.

In contrast to the cell membrane lipids, the structural proteins in the lens were found to undergo large changes. α-Crystallin in the nucleus disappeared by age 50, as has been reported previously (Roy & Spector, 1976a; McFall-Ngai et al., 1985). HMW proteins in the soluble protein fraction increased up to age 30–40 then gradually declined (Fig. 3B). Although there was some degree of scatter, there was a steady increase in the amount of insoluble protein at the expense of soluble protein. Using our method of extraction at pH 7, approximately half of the total soluble protein in the lens had become insoluble by age 50 (Fig. 3A). Others have reported variable yields of soluble and insoluble protein depending on the buffers and conditions used (Coghlan & Augusteyn, 1977; Li et al., 1986; Bours et al., 1987) although a consistent finding is an age-dependent increase in insoluble protein. The overall decrease in soluble protein content and α-crystallin with age parallels the increase in lens stiffness up to age 50. Of particular note, after 50 when the soluble α-crystallin has all been incorporated into HMW and insoluble protein, lens stiffness increases dramatically (Fig. 1).

In order to see if we could reproduce some of the age-related changes in a model system, we used intact pig lenses and heated them at different temperatures. Porcine lenses were chosen because they closely resemble human lenses in terms of protein content and size, although pigs are not thought to accommodate. When pig lenses were incubated at temperatures ranging from 43 to 70 °C, there was a marked increase in stiffening of both nuclear and cortical regions, with a rate that depended on the temperature. Fifty degree Celsius was chosen for subsequent experiments; however, all features were observed at the other temperatures.

Stiffness was found to increase very little in the first 2 h, but afterwards increased linearly with time (Fig. 5A). In this initial lag period, there was a small decrease in soluble protein; however, the content of soluble α-crystallin had decreased by approximately one-half, and the amount of HMW protein had doubled. Most soluble α-crystallin had disappeared after 4 h at 50 °C and, after this time, the content of HMW protein also decreased. After 8 h of incubation, the changes in soluble/insoluble as well as α-crystallin and HMW protein appeared to be largely complete. As α-crystallin was a major component of the HMW and insoluble protein fractions (as determined by SDS-PAGE), these data may indicate that in intact lenses, α-crystallin becomes converted into HMW protein, which in turn becomes insoluble.

This pattern of change in the proteins of heated porcine lenses over time was found to closely mimic that observed in human lenses with age. This was true for the α-crystallin, soluble and insoluble protein, as well as the appearance and gradual disappearance of the HMW peak (Figs 3 and 5A). In addition, SDS-PAGE revealed that the composition of the HMW peaks in both cases was similar with α-crystallin being the major component (see Supplementary Fig. S1). As these changes were associated with a pronounced increase in lens stiffness in both human and porcine lenses, two conclusions can be drawn. First, the major age-related changes observed to human lens proteins in this study and by others (Roy & Spector, 1976a; McFall-Ngai et al., 1985) could be explained largely on the basis of denaturation of crystallins, and that this can be induced by heat. Second, these protein changes may contribute to the marked increase in stiffness of the lens nucleus that is observed with age.

Our working hypothesis is that heat-induced denaturation of crystallins takes place in vivo during our lifetime under conditions where the temperature is lower than that used in our model studies, but where the time frame is much longer. If lens stiffening in humans was indeed largely the result of lifetime ocular exposure to heat, and if such stiffening was the underlying cause for presbyopia, one could expect to see a relationship between the ambient temperature and the age of onset of presbyopia. This would be predicted because of known variations in eye temperature in response to external temperature fluctuations (Al-Ghadyan & Cotlier, 1986; Sliney, 1986). Just such a relationship has been observed (Miranda, 1979), where the age of onset of presbyopia in different regions of the world decreased linearly as the ambient temperature increased.

One implication that can be derived from our data is that the high concentration of α-crystallin, found in young lenses (Roy & Spector, 1976a; McFall-Ngai et al., 1985), assists in maintaining lens flexibility and, therefore, may play a functional role in the lens other than simply being a structural protein that contributes to refractive index. An hypothesis that α-crystallin could act to delay opacification of the lens by acting as a chaperone was advanced previously on the basis of experiments using isolated lens proteins. In this study with α-crystallin present, HMW protein was formed by heating a mixture of crystallins at 55 °C; however, if α-crystallin were selectively removed, then exposure to higher temperatures leads to the formation of insoluble protein (Rao et al., 1995).

In our experiments on intact lenses, after 2 h of heating at 50 °C, α-crystallin had decreased twofold and there was a corresponding increase in the HMW peak, yet, the stiffness measurements were largely unchanged. As α-crystallin disappeared further from the soluble fraction, and the amount of soluble and HMW protein also decreased, the stiffness of the lens began to increase (Fig. 5A). In human lenses there is a steady increase in lens stiffness up to age 50 as α-crystallin decreases and a more pronounced rate of increase after age 50, when there is no longer any soluble α-crystallin to chaperone proteins that denature (Figs 1 and 3B). While these data suggest that gross changes to the proteins in fibre cells in response to heat-induced denaturation may be implicated in presbyopia, there are likely to be other factors, for example, deamidation of glutamine and asparagine residues in crystallins over time (Wilmarth et al., 2006), that may also impact on the progression of this condition by promoting crystallin unfolding. The details of these processes remain to be elucidated.

It should be noted that Schachar and co-workers (Schachar & Anderson, 1995; Schachar et al., 1995) have postulated a quite different basis for accommodation, but this seems not to be supported by experimental evidence (Glasser et al., 2006). In addition, Schachar and Pierscionek (2007) recently published a brief article suggesting that lens hardness was not related to accommodative amplitude. Their assertion was based largely on atomic force microscopy (AFM) data of others (Ziebarth et al., 2007), where AFM was used to measure Young's modulus by placing the probe on the outside of intact monkey lenses. As such, this AFM procedure may provide information on the stiffness of the capsule, and possibly the very outermost cortex, but is unlikely to provide any meaningful data of the stiffness of the lens body. It is therefore unsurprising that Ziebarth et al. did not find a trend with age.

If, as our experiments suggest, temperature may contribute to presbyopia by altering the biochemical and physical properties of the lens, some lifestyle modifications could be made to limit the magnitude of these changes, and thus potentially delay the onset of presbyopia. First, it may be advisable to live in regions of the globe that are not subject to extremely high ambient temperatures (Miranda, 1979). Second, if one wishes to delay the uptake of reading glasses, one should perhaps limit exposure to activities, such as saunas, which are likely to significantly elevate the temperature of the lens.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Porcine eyes were acquired from Crownpork (Strathfield, NSW, Australia) and used within 24 h of death. Tris and EDTA were purchased from Sigma-Aldrich (St. Louis, MO, USA), sodium chloride from Amresco (Solon, OH, USA) and protease inhibitor tablets catalog no. 11836153001 from Roche (Indianapolis, IN, USA) Milli QTM (Millipore, Bedford, MA, USA) was used for the preparation of all solutions. Cryovials (2 mL) for incubation studies were purchased from Simport Plastics (Belloeil, Quebec, Canada). For all plotting of curves, SigmaPlot 8.0 (SPSS Inc.) was used. Amino acid analysis was undertaken at the Australian Proteome Analysis Facility.

Measurement of lens stiffness

Normal human lenses were collected from the Lions NSW Eye Bank at the Sydney Eye Hospital. Lenses were not frozen, but were stored on ice, or at 4 °C, until analysis, and were processed within 48 h of death. The work was approved by the human research ethics committees at the University of Wollongong and the University of Sydney.

Human and porcine lenses were sectioned equatorially. After being placed in the lens holder, a razor blade was used to cut through the lens. The instrument used for the measurement of stiffness was a Q800 dynamic mechanical analyser (TA Instruments, New Castle, DE, USA). The sample holder, DMA probe and the method of analysis were as described previously (Heys et al., 2004). In the experiments described here, stiffness measurements were carried out using the controlled force technique, and nine readings comprising a 3 × 3 1 mm grid pattern were taken in the centre of each lens section. An average value of these measurements was used to calculate the stiffness of the sample.

The effect of freezing on the stiffness of porcine lenses was carried out by storing lenses at –80 °C for varying periods of time. Each lens was allowed to thaw partially prior to sectioning in the lens holder. DMA was performed as outlined above.

Incubation in the DMA

The effect of heat on the stiffness of individual porcine and human lens sections was also examined directly in the DMA. An equatorial section approximately 2 mm thick was obtained by removing the anterior and posterior parts of the lens using a razor blade as described earlier. This lens section was then covered in a thin layer of silicon oil to prevent dehydration during the heating process. The sample was incubated at 50 °C in the DMA with the furnace closed. Measurements of stiffness of the lens centre were taken at time 0, 4 and 8 h using the controlled force technique and four to six measurements were taken from the centre of each lens section. An average value was used to calculate the stiffness at each time. At the end of the incubation the lens sections were removed for analysis of soluble and insoluble protein content.

Incubation of porcine lenses

Intact lenses were dissected from fresh porcine eyes and suspended on a bed of folded Parafilm above a 500-µL reservoir of water (to avoid the possibility of dehydration) in sealed screw capped vials. Lenses were incubated individually at 50 °C and four lenses were removed at selected times. Three lenses were used for the analysis of stiffness (DMA) and for soluble and insoluble protein content and one lens for the analysis of water by differential scanning calorimetry and thermo gravimetric analysis.

Water soluble and insoluble protein fractions from porcine and human lenses

Human and porcine lenses were divided into nuclear and cortical regions using a 4.5-mm trephine. One millimeter from each end of the nuclear core was removed with a scalpel. Lens tissues were homogenized in 400 µL of buffer A (10 mm phosphate, pH 7.0 containing 0.1 mm ethylene glycol tetraacetic acid (EGTA) and a protease inhibitor (Roche), one tablet per 10 mL. The homogenate was spun at 100 000 g for 30 min at 4 °C in a TL-100 ultracentrifuge (Beckman, Fulletton, CA, USA). The supernatant was removed and the pellet was homogenized with buffer A for a further three times, to yield a total volume of ~1600 µL containing the water soluble protein. The insoluble pellet was then suspended in 400 µL of buffer B (10 mm Tris, pH 8.0 containing 8M urea), homogenized and spun at 20 000 g for 30 min at 4 °C. The pellet was re-extracted with 400 µL of Buffer B and the supernatants were combined to yield the water insoluble protein fraction. The Micro BCA assay (Pierce, Rockford, IL, USA) was used to determine protein content of the water-soluble and water-insoluble fractions. Amino acid analysis was undertaken at the APAF.

Hplc fractionation of water-soluble proteins

Water-soluble proteins were fractionated by size exclusion chromatography on two BioSep (Phenomenex, Torrance, CA, USA) S-3000 and S-4000 HPLC columns linked in tandem. Chromatography was performed in 10 mm Tris buffer pH 7.4 containing 2 mm EDTA and 50 mm NaCl with a flow rate of 0.5 mL min−1. Protein samples from human or porcine lenses (~1 mg) were separated using a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) with absorbance monitored at 280 nm. Peak areas for quantitation were determined using class VP software. Collected HPLC peaks were desalted using a Centricon (Millipore) prior to lyophilization and SDS-PAGE. Bands were identified using in-gel tryptic digestion followed by analysis of the peptides by MALDI mass spectrometry (Harrington et al., 2004) (see Supplementary Fig. S1).

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References

Roger J. W. Truscott is a National Health and Medical Research Council fellow. Support for this work was provided by the Australian Research Council (DP0666847) and the Ophthalmic Research Institute of Australia. Jane Deeley is thanked for lipid analysis, Peter Hains for assistance with MALDI and Mr Raj Devasahayam from Sydney Lions Eye bank for providing the human lenses.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. References
  • Al-Ghadyan AA, Cotlier E (1986) Rise in lens temperature on exposure to sunlight or high ambient temperature. Br. J. Ophthalmol. 70, 421426.
  • Augusteyn RC, Rosen AM, Borja D, Ziebarth NM, Parel JM (2006) Biometry of primate lenses during immersion in preservation media. Mol. Vis. 12, 740747.
  • Borchman D, Byrdwell WC, Yappert MC (1994) Regional and age-dependent differences in the phospholipid composition of human lens membranes. Invest. Ophthalmol. Vis. Sci. 35, 39383942.
  • Bours J, Fodisch HJ, Hockwin O (1987) Age-related changes in water and crystallin content of the fetal and adult human lens, demonstrated by a microsectioning technique. Ophthalmic Res. 19, 235239.
  • Brown NP (1974) The change in shape and internal form of the lens of the eye on accommodation. Exp. Eye Res. 15, 441459.
  • Bruckner R, Batschelet E, Hugenschmidt F (1986) The Basel longitudinal study on aging (1955–1978). Doc. Ophthalmol. 64, 235310.
  • Cheng R, Feng Q, Argirov OK, Ortwerth BJ (2004) Structure elucidation of a novel yellow chromophore from human lens protein. J. Biol. Chem. 279, 4544145449.
  • Coghlan SD, Augusteyn RC (1977) Changes in the distribution of proteins in the aging human lens. Exp. Eye Res. 25, 603611.
  • Coleman DJ (1970) Unified model for accommodative mechanism. Am. J. Ophthalmol. 69, 10631079.
  • Der Perng M, Wen SF, Van Den Ijssel P, Prescott AR, Quinlan RA (2004) Desmin aggregate formation by R120G αB-crystallin is caused by altered filament interactions and is dependent upon network status in cells. Mol. Biol. Cell 15, 23352346.
  • Derham BK, Harding JJ (1999) α-Crystallin as a molecular chaperone. Prog. Retin. Eye Res. 18, 463509.
  • Duane AJ (1912) Normal values of the accommodation of all ages. JAMA 59, 10101013.
  • Dubbelman M, Van der Heijde GL, Weeber HA, Vrensen GF (2003) Changes in the internal structure of the human crystalline lens with age and accommodation. Vision Res. 43, 23632375.
  • Eskeridge JB (1984) Review of ciliary muscle effort in presbyopia. Am. J. Optom. Physiol. Opt. 61, 133138.
  • Fisher R (1971) The elastic constants of the human lens. J. Physiol. 212, 147180.
  • Glasser A, Campbell MC (1999) Biometric, optical and physical changes in the isolated human crystalline lens with age in relation to presbyopia. Vision Res. 39, 19912015.
  • Glasser A, Wendt M, Ostrin L (2006) Accommodative changes in lens diameter in rhesus monkeys. Invest. Ophthalmol. Vis. Sci. 47, 278286.
  • Harrington V, McCall S, Huynh S, Srivastava K, Srivastava OP (2004) Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses. Mol. Vis. 10, 476489.
  • Helmholtz H (1855) The accommodation of the eyes. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 1, 170.
  • Heys KR, Cram SL, Truscott RJW (2004) Massive increase in the stiffness of the human lens nucleus with age: the basis for presbyopia? Mol. Vis. 10, 956963.
  • Horwitz J (1992) α-Crystallin can function as a molecular chaperone. Proc. Natl Acad. Sci. USA 89, 1044910453.
  • Huang L, Grami V, Marrero Y, Tang D, Yappert MC, Rasi V, Borchman D (2005) Human lens phospholipid changes with age and cataract. Invest. Ophthalmol. Vis. Sci. 46, 16821689.
  • Koretz JF, Cook CA, Kaufman PL (1997) Accommodation and presbyopia in the human eye. Changes in the anterior segment and crystalline lens with focus. Invest. Ophthalmol. Vis. Sci. 38, 569578.
  • Koretz JF, Handelman GH (1988) How the human eye focuses. Sci. Am. 259, 9299.
  • Koretz JF, Handelman GH, Brown NP (1984) Analysis of human crystalline lens curvature as a function of accommodative state and age. Vision Res. 24, 11411151.
  • Korlimbinis A, Truscott RJW (2006) Identification of 3-Hydroxykynurenine bound to proteins in the human lens. A possible role in age-related nuclear cataract. Biochemistry 45, 19501960.
  • Krag S, Olsen T, Andreassen TT (1997) Biomechanical characteristics of the human anterior lens capsule in relation to age. Invest. Ophthalmol. Vis. Sci. 38, 357363.
  • Lahm D, Lee LK, Bettelheim FA (1985) Age dependence of freezable and nonfreezable water content of normal human lenses. Invest. Ophthalmol. Vis. Sci. 26, 11621165.
  • Lampi KJ, Ma Z, Hanson SRA, Azuma M, Shih M, Shearer TR, Smith DL, Smith JB, David LL (1998) Age-related changes in human lens crystallins identified by two-dimensional electrophoresis and mass spectrometry. Exp. Eye Res. 67, 3143.
  • Li LK, Roy D, Spector A (1986) Changes in lens protein in concentric fractions from individual normal human lenses. Curr. Eye Res. 5, 127135.
  • Masters PM, Bada JL, Zigler J, Jr (1977) Aspartic acid racemisation in the human lens during ageing and in cataract formation. Nature 268, 7173.
  • McFall-Ngai MJ, Ding L-L, Takemoto LJ, Horwitz J (1985) Spatial and temporal mapping of the age-related changes in human lens crystallins. Exp. Eye Res. 41, 745758.
  • McHaourab HS, Dodson EK, Koteiche HA (2002) Mechanism of chaperone function in small heat shock proteins. Two-mode binding of the excited states of T4 lysozyme mutants by αA-crystallin. J. Biol. Chem. 277, 4055740566.
  • Miranda MN (1979) The geographic factor in the onset of presbyopia. Trans. Am. Ophthalmol. Soc. 77, 603621.
  • Mitchell TW, Turner N, Hulbert AJ, Else PL, Hawley JA, Lee JS, Bruce CR, Blanksby SJ (2004) Exercise alters the profile of phospholipid molecular species in rat skeletal muscle. J. Appl. Physiol. 97, 18231829.
  • Pau H, Kranz J (1991) The increasing sclerosis of the human lens with age and its relevance to accommodation and presbyopia. Graefes Arch. Clin. Exp. Ophthalmol. 229, 294296.
  • Raman B, Ramakrishna T, Rao CM (1995) Temperature dependent chaperone-like activity of alpha-crystallin. FEBS Lett. 365, 133136.
  • Rao PV, Huang QL, Horwitz J, Zigler JS, Jr (1995) Evidence that α-crystallin prevents non-specific protein aggregation in the intact eye lens. Biochim. Biophys. Acta 1245, 439447.
  • Reddy GB, Das KP, Petrash JM, Surewicz WK (2000) Temperature-dependent chaperone activity and structural properties of human αA- and αB-crystallins. J. Biol. Chem. 275, 45654570.
  • Roy D, Spector A (1976a) Absence of low-molecular-weight α-crystallin in nuclear region of old human lenses. Proc. Natl Acad. Sci. USA 73, 34843487.
  • Roy D, Spector A (1976b) High molecular weight protein from human lenses. Exp. Eye Res. 22, 273279.
  • Schachar RA, Anderson D (1995) The mechanism of ciliary muscle function. Ann. Ophthalmol. 27, 126132.
  • Schachar RA, Black TD, Kash RL (1995) The mechanism of accommodation and presbyopia in the primate. Ann. Ophthalmol. 27.
  • Schachar RA, Pierscionek B (2007) Lens hardness not related to the age-related decline of accommodative amplitude. Mol. Vis. 13, 10101011.
  • Schwartz B (1965) Environmental temperature and the ocular temperature gradient. Arch. Ophthalmol. 74, 237243.
  • Sliney D (1986) Physical factors in cataractogenesis: ambient ultraviolet radiation and temperature. Invest. Ophthalmol. Vis. Sci. 27, 781790.
  • Strenk SA, Strenk LM, Koretz JF (2005) The mechanism of presbyopia. Prog. Retin. Eye Res. 24, 379393.
  • Stubbs CD, Smith AD (1984) The modification of mammalian membrane polyunsaturated fatty acid composition in relation to membrane fluidity and function. Biochim. Biophys. Acta 779, 89137.
  • Takemoto L (1995) Age-dependent cleavage at the C-terminal region of lens beta B2 crystallin. Exp. Eye Res. 61, 743748.
  • Treweek TM, Lindner RA, Mariani M, Carver JA (2000) The small heat-shock chaperone protein, α-crystallin, does not recognise stable molten globule state of cytosolic proteins. Biochim. Biophys. Acta 1481, 175188.
  • Weeber HA, Eckert G, Soergel F, Meyer CH, Pechhold W, Van Der Heijde RG (2005) Dynamic mechanical properties of human lenses. Exp. Eye Res. 80, 425434.
  • Wilmarth PA, Tanner S, Dasari S, Nagalla SR, Riviere MA, Bafna V, Pevzner PA, David LL (2006) Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility? J. Proteome Res. 5, 25542564.
  • Xi J-h, Bai F, McGaha R, Andley UP (2006) α-Crystallin expression affects microtubule assembly and prevents their aggregation. FASEB J. 20, 846857.
  • Ziebarth NM, Wojcikiewicz EP, Manns F, Moy VT, Parel J (2007) Atomic force microscopy measurements of lens elasticity in monkey eyes. Mol. Vis. 13, 503510.