viewing molecular mechanisms of ageing through a lens - 2005

Ageing Research Reviews1 (2002) 465–479

Review

Viewing molecular mechanisms of ageingthrough a lens

John J. Harding∗Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, Oxford, OX2 6AW, UK

Received 16 January 2002; accepted 17 January 2002

Abstract

Many late-life diseases are conformational diseases in tissues where there are unfolded or mis-folded proteins which can form aggregates. These diseases have other common features in theiraetiology. Cataract is one such disease and post-translational modifications of proteins in the lensduring cataract formation are described as a possible guide to the changes in other age-relatedconditions. Delineation of common pathways in these diseases could lead to common treatmentregimes, and in this respect, there are promising results for aspirin-like drugs in Alzheimer’s dis-ease, cataract, myocardial infarction, stroke and various cancers. © 2002 Elsevier Science IrelandLtd. All rights reserved.

Keywords: Ageing; Cataract; Conformational disease;�-Crystallin; Post-translational modification of proteins;Unfolding

1. Introduction

In ageing and in at least some age-related diseases, there is post-translational modificationof proteins, unfolding of proteins and eventually aggregation of proteins. Diseases featur-ing unfolded or misfolded proteins have been called the conformational diseases (Carrelland Lomas, 1997), and many of these occur late in life (Table 1). In cataract, which mostlyoccurs late in life, the conformational changes were identified 30 years ago and theircauses have been the subject of much research since. This review summarises some of thatresearch and explores the relationship between cataract and other age-related diseases to seewhether the common features derive from a common aetiology. Researchers may benefitfrom cross-fertilisation between different disease areas.

∗ Tel.: +44-1865-248996; fax:+44-1865-794508.E-mail address: [email protected] (J.J. Harding).

1568-1637/02/$ – see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.PII: S1568-1637(02)00012-0

466 J.J. Harding / Ageing Research Reviews 1 (2002) 465–479

Table 1Some conformational diseases

Amyloid diseasesAlzheimer’sSystemic amyloidosisDown’s syndrome etc.

Prion diseasesCJDvCJDBSEKuru

Huntington’s diseaseAlexander’s diseaseCataract

2. The lens

All tissues are unique but some are more unique than others. The lens is very peculiar.It is transparent, devoid of a blood supply, and at its centre are cells present before birthcontaining proteins not replaced since. It is suspended between the aqueous humour, whichprovides its nutrients, and the vitreous body (Fig. 1). It is contained within a basementmembrane, the lens capsule (Fig. 2). Inside the anterior surface of the lens, there is asingle layer of epithelial cells which divide and move to the equator and elongate to formthe long, thin fibre cells that fills most of the lens. During elongation the cells lose all

Fig. 1. The eye showing the lens between the aqueous humour and vitreous body.

J.J. Harding / Ageing Research Reviews 1 (2002) 465–479 467

Fig. 2. The lens showing the single layer of epithelial cells with the rest of the lens filled with fibre cells. Originaldrawing by John Cronin.

organelles which otherwise would scatter light and compromise lens transparency. Theloss of protein synthesis machinery means that most of the lens cells retain their proteinfor life, that is more than 70 years for the average cataract patient. Most of the proteinpresent is structural protein called crystallins (�, � and �) but these cells also containenzyme activity. Surely, the lens has something to tell us about how to keep enzymeshappy.

3. Conformational changes to proteins in cataract

Cataract is the most common cause of blindness worldwide, accounting for four mil-lion newly blind each year, in India alone (Minassian and Mehra, 1990). A typical humancataract is shown in Fig. 3. An early change identified in human cataract is the unfold-ing of proteins demonstrated by the increased susceptibility to tryptic digestion and theincreased reactivity of the thiol groups (Fig. 4; Harding, 1972). At that time, only oneother possible conformational disease had been identified (Jacob et al., 1968) but now thereare many (Table 1). It is possible that what we have learnt about the unfolding proteinof the lens could help with other conformational diseases, unless the unique character-istics of the lens mean it has no relevance to other systems. Certainly findings on theextremely long-lived proteins of the lens give an insight into what happens to other long-lived proteins.

Over the years different authors have proposed a variety of possible causes of the con-formational changes in lens (Table 2). These are all simple chemical changes to the protein,many representing different types of adduct formation. All are non-enzymic and therefore,are not specific to particular proteins, although some are more susceptible than others. Allhave been identified in human cataracts. Glycation and carbamylation are described in moredetail in Tables 3 and 4.


Fig. 3. A typical human cataract.

Fig. 4. Reactivity of thiol groups from normal and cataractous lenses. Lenses were treated with iodoacetate.Unchanged thiol groups were determined at zero time and intervals thereafter. Data from Harding (1972).


Table 2Possible causes of the conformational change in human cataract

GlycationCarbamylationDeamidationAddition of glutathioneAddition of corticosteroidsC-terminal degradationN-terminal degradationProtein–protein disulphide formationRacemisation of aspartic acidOxidation of methionine

Table 3Glycation

Non-enzymic reaction of sugars with proteinsNot specificIncreased in diabetesDiabetes is a risk factor for cataractGlycation causes unfolding of proteinsGlycation causes inactivation of enzymesGlycation causes aggregationAspirin and ibuprofen decrease glycation

Table 4Carbamylation

Reaction of isocyanate with proteinsIsocyanate is derived from equilibrium with ureaUrea levels are raised in renal failure and severe diarrhoea, which are risk factors for cataractAspirin and ibuprofen decrease carbamylation

4. Glycation

Glycation, the non-enzymic reaction of sugars with protein, occurs normally but to anincreased extent in ageing, in cataract and in diabetes, where it is associated with a variety ofcomplications. Cerami et al. (1987) suggested that glycation was the major cause of tissueageing, but it is one of the growing groups of post-translational modifications that couldplay a role in ageing tissues. Carbamylation is the non-enzymic reaction of isocyanate withproteins. The isocyanate is derived from urea, which again is normally present but occurs atincreased levels in renal failure and in severe diarrhoea, and both these conditions are riskfactors for cataract (Harding, 1991). The common sugars and isocyanate react preferentiallywith the amino groups on proteins, as do corticosteroids. In doing so, they decrease thenumber of positive charges on the protein surface. Other modifications in Table 2 alsohave the effect of making proteins more anodic, and an increasingly anodic tendency hasbeen reported for many proteins during ageing (Robinson and Rudd, 1974). The glycation

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reactions are shown in Fig. 5. The early reactions are very simple: the formation of a Schiffbase between the protein amino group and the aldehyde function of glucose in the openchain form, but later reactions producing yellow, fluorescent moieties are more complicated.Some of the late products form cross-links making the proteins more intractable.

In addition to identifying products of glycation and other non-enzymic modifications inageing human lens and especially in cataract, it has been shown in vitro that glycation canreproduce many other features of cataract. For example, glycation of lens protein causesconformational change, yellowing, increased fluorescence, and aggregation (Beswick andHarding, 1987; Raza and Harding, 1991).

Post-translational modifications like glycation have been studied mostly using structuralproteins because they tend to be long-lived and are available in relatively large amounts. Thelens cells are packed with�-, �- and�-crystallins which provide the high refractive indexnecessary to focus images on to the retina at the back of the eye (Fig. 1). These are toughproteins suited to their long lives, and the�- and�-crystallins have compact structures. Theamino acid sequences of many of these crystallins are known and the full X-ray structurehas been determined for�B2- and�B-crystallins (Bax et al., 1990; Wistow et al., 1983).

Although, the emphasis has been on structural proteins, non-enzymic post-translationalmodifications are not specific and so other proteins including enzymes, receptors, channelproteins and hormones will be modified and attention is turning to the effects on functionsof these specialised proteins. Modification of enzymes by sugars, isocyanate, and corticos-teroids inactivates them (Hook and Harding, 1998). An example is shown in Fig. 6. Theinactivation occurs readily at almost physiological concentrations of sugar, and it occursto all enzymes we have tested to date. For example, 5 mM fructose inactivated seven dif-ferent enzymes completely within 6 days (Hook and Harding, 1998). Pyruvate kinase wasinactivated in 4 h. In contrast, experiments on changes to structural proteins have used con-centrations of sugar from 20 to 100 mM (Beswick and Harding, 1987) and time periods upto 41 days (Stevens et al., 1978).

Fig. 6. Inactivation of malate dehydrogenase by 5 mM glucose (from Heath et al., 1996).


Fig. 7. Protection of malate dehydrogenase by 5�g/ml �-crystallin (from Heath et al., 1996).

5. �-Crystallin as a molecular chaperone

Up to 1992,�-crystallin was regarded purely as a structural protein, then Horwitz (1992)showed that it protected proteins against heat-induced aggregation providing the first evi-dence that it had chaperone-like properties. Since then it has been shown to protect againstinactivation of enzymes induced by glycation (Ganea and Harding, 1995), carbamylation(Ganea and Harding, 1996) and reaction with corticosteroids (Hook and Harding, 1996),as well as against reduction-induced aggregation of insulin (Farahbakhsh et al., 1995).It provided full protection against inactivation of several enzymes at low concentrations(Fig. 7).

The stoichiometry of protection for several enzymes was consistent with the�-crystallinaggregate (700 kDa) protecting one or two molecules of enzyme in a cavity (Hook andHarding, 1998) but it has been difficult to show stable chaperone complexes and it may bethat the protection is dynamic (Lindner et al., 2001). Nevertheless, cryo-electron microscopyshows a structure with a cavity (Haley et al., 1998).

It was thought that�-crystallin was exclusively a lens protein but it is in fact presentin many tissues, and is found at increased levels in various neurodegenerative diseases(Table 5), as would be expected of a stress protein. Note the overlap in the diseases listed

Table 5Diseases with raised levels of�-crystallin (Derham and Harding, 1999)

Alexander’s diseaseParkinson’s diseaseHuntington’s diseaseScrapie-infected brain of hamsters and miceCreuzfeldt–Jacob diseasePick’s diseaseIschaemic heart diseaseStrokeAlzheimer’s disease


in Tables 1 and 5. The R120G mutation of the�B-crystallin gene causes a desmin-relatedmyopathy (Vicart et al., 1998), probably due to lost chaperone function.

6. Common pathways in cataractogenesis

Although, individual cataracts may differ in appearance, there is good evidence thatcataract proceeds along common pathways in many human cataracts, and indeed in experi-mental cataracts. Changes found in human cataract are shown in Fig. 8 linked by arrowsto mark speculative causal pathways. Risk factors for cataract are arranged around the topof the figure. Most of the risk factors can be related to post-translational modifications ofproteins identified in human cataract. The modified proteins unfold and the unfolded proteinitself leads on to other problems including aggregation, which is directly responsible foropacification of the lens.

Similar schemes can be drawn for changes in experimental cataract, for example diabeticcataract in rats (Fig. 9). Again, there are a variety of changes that can be linked into possiblecausal pathways. This scheme was drawn up taking the sequences of observed changes into

Fig. 8. Scheme of changes seen in human cataract (Harding, 1991). G6P: glucose 6-phosphate; MDA: malondi-aldehyde.


Fig. 9. Scheme of changes seen in diabetic cataract in rats (Harding, 1991). F3P: fructose 3-phosphate; G6P:glucose 6-phosphate; S3P: sorbitol 3-phosphate; aa: amino acid; 3DG: 3-deoxyglucosone;�: �-crystallin; PD:polyol dehydrogenase; ppp: pentose phosphate pathway.

Fig. 10. Scheme of changes seen in Philly cataract in mouse (Harding, 1993). GSH: glutathione.


account, something that is more difficult in human disease. As in human cataract, thereappears to be a central role for post-translational modification, in this case glycation byglucose and its metabolites present at high levels in the diabetic lens.

A final scheme is for an inherited cataract in mouse (Clark and Carper, 1987), Phillycataract (Fig. 10). Various studies of lens biochemistry in Philly cataract revealed the usualarray of changes to cations, pumping, enzymes, glutathione, dry weight and protein aggre-gation seen in other experimental cataracts and in human cataract but in this model the initialevent is known, being the deletion of 12 nucleotides from the gene for one lens polypeptide,�B2-crystallin. The mutant protein with four missing amino acids is unable to fold normally.So in this model it is clear that one misfolded protein can lead to damage to all the othersystems. In human cataract where changes to protein have been unambiguously identified,it is probable that the conformational changes of many proteins, and the inactivation ofenzymes, lead to the otherwise bewildering set of changes reported in the literature.

The big question now is whether the bewildering set of biochemical, physical andmorphological changes seen in other late life diseases can be explained in a similar way.

7. Common pathways in other age-related diseases?

There are so many changes reported in other age-related diseases that it appears foolhardyto try and draw up common pathways as such, but there are certainly some common changes.Increased glycation was found in amyloid plaques, and in tau protein of neurofibrillary

Fig. 11. Post-translational modification of proteins, cataract and amyloid diseases.


tangles in Alzheimer’s disease (Vitek et al., 1994; Yan et al., 1994). The tangles contain tauprotein which is abnormally glycosylated, phosphorylated and possibly truncated (Brionet al., 1991; Wang et al., 1996). Excess glycation is also found in proteins, in tissues affectedby diabetic complications many of which correspond to accelerated ageing. Glycation andmost other post-translational modifications in disease alter the charge balance on the surfaceof proteins and this seems to be important in cataract and Alzheimer’s disease. Racemisationof aspartyl residues occurs not only in ageing and cataract but also in the�-amyloid proteinof senile plaques of Alzheimer’s disease (Finch and Cohen, 1997).

The presence of so many diseases in Table 1 indicates that conformational change, or atleast incorrect conformation, is a common feature of all those diseases. Post-translationalmodification, conformational change and aggregation would make a good start to a commonpathway to age-related tissue damage (Fig. 11). The apparent protective effect of aspirin andrelated drugs against some age-related diseases provide another common link (see later).

8. Aspirin, ibuprofen, paracetamol and similar drugs

Evidence is accumulating that aspirin and similar drugs can prevent cataractous changes.Aspirin and ibuprofen decrease rates of glycation, carbamylation of lens proteins and pre-vented the cyanate-induced opacification of incubated rat lenses (Harding, 1991, 2001).Aspirin, paracetamol and ibuprofen protected rats against diabetic cataract (Swamy andAbraham, 1989; Blakytny and Harding, 1992). Aspirin and other NSAIDs prevent cataractin other experimental models (Harding, 2001). More importantly, evidence for a protectiveeffect is accumulating from human studies. The initial observations of Cotlier were followedby two case-control studies in England showing a protective association between cataracton the one hand and aspirin, paracetamol and ibuprofen on the other (van Heyningen andHarding, 1986; Harding et al., 1989). About 40% of cataract could be prevented if theprotective relationship is causal. The protective association was confirmed in studies fromIndia and the US (Mohan et al., 1989; West et al., 1987; Age-related Eye Disease Study,2001).

Confirmation of a major protective effect like this requires a randomised clinical trial ofaspirin-like drugs in patients with early cataracts, but for a variety of reasons this has nothappened. Earlier results from add-on studies to trials for myocardial infarction etc. mostlyreported negative results but had too few cataracts to answer the question (Harding, 2001).More recently, positive results have appeared. A re-analysis of the US physicians’ studyfound a significant protection by aspirin against one type of cataract and a marginally signif-icant 19% protection against cataract extraction (Christen et al., 1998). A lower incidenceof nuclear cataract over 5 years was found in subjects taking aspirin (Klein et al., 2001).

With accumulating evidence for protection against cataract, ischaemic heart disease(Antiplatelet Triallists’ Collaboration, 1988), stroke (Diener, 1999), Alzheimer’s disease(Anthony et al., 2000), colorectal cancer (Janne and Mayer, 2000) and prostate cancer(Nelson and Harris, 2000) by aspirin and related drugs it is important to consider the pos-sible mechanism for these protective actions. All these diseases are age-related and aremulti-factorial, so it is probably simplistic to expect to find a single mechanism to apply toall the diseases, and indeed to all the drugs. In cataract, the protective effect was associated


especially with aspirin, paracetamol and ibuprofen. Acetylation of vulnerable amino groupswas a reasonable explanation for protection of proteins by aspirin against post-translationalmodification by more noxious compounds, but could not be applied to the similar protectionby ibuprofen. In Alzheimer’s disease and cancer, many anti-inflammatories appear to beprotective. In vascular disease, the emphasis has been on aspirin. Aspirin has a transferableacetyl group not present on most of the other drugs, and the protection against myocardialinfarction has been explained purely by the well-known inactivation of cyclo-oxygenase byacetylation. The anti-inflammatory action has been invoked as the mechanism for protec-tion against cancer and laboratory models of cancer (Levi et al., 2001), but acetylation ofthe tumour suppressor p53 could be involved as enzymic acetylation occurs in its normalfunctioning. Other mechanisms are being considered (Paterson and Lawrence, 2001; Rigasand Shiff, 2000; Zhou et al., 2001). We may be missing the common protective mechanism,or it may be that the different properties of the drugs affect several pathways of importancein these diseases.

9. Other common features

The risks of cataract and of Alzheimer’s disease appear lower in women who have takenoestrogens (Klein et al., 1994; Harding, 1994; Kawas et al., 1997; Sohrabji and Miranda,1997). There has been no convincing mechanistic explanation for these epidemiologicalobservations.

10. Conclusions

Cataract is a conformational disease where the unfolding is probably caused by vari-ous post-translational modifications of the structural and other proteins. Conformationally-altered proteins are found in other late-life diseases, and may be formed in a similar way.Aspirin-like drugs may protect against a number of the late-life conformational diseases.�-Crystallin, a small heat-shock protein, may protect lens against age-related change andcould, with other heat-shock proteins, function similarly in other tissues.

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viewing molecular mechanisms of ageing through a lens - 2005

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