Procedimientos Específicos

The possible role of mitochondria in ageing is by now clear given the link to free radicals and oxidative damage to other cell structures as discussed above. One proposal is that the primary abnormality is within the mitochondria itself (Shigenaga at a/. 1994), leading into a vicious cycle whereby a

dysfunctional MRC leads to enhanced free radical production, ovenwhelming host defences and causing a further decline in respiratory capacity. Some take this further and have proposed that the central site of irreversible injury is the mtDNA itself rather than the biomembranes of the cell (Fleming at a/. 1982).

This is an attractive hypothesis for several reasons. Firstly mtDNA is situated close to a major site of free radical production and lacks the protective and

repair mechanisms of genomic DNA. Secondly significant damage to mtDNA would be expected to impair MRC function, which in turn leads to further free radical production and potential oxidative damage both to the mtDNA and to the MRC proteins (Zhang et al. 1990). Finally cellular ATP is fundamental to many

physiological functions. Thus a vicious cycle would be established leading to a fall in the production of cellular ATP, loss of energy dependant cellular functions and ultimately cell death. In this section I shall review in some detail the evidence for accumulated mtDNA damage and a fall in MRC function with ageing.

3.4.1 Mitochondrial DNA and ageing

3.4.1.1 Oxidative damage to mtDNA and the "common" deletion

In accordance with the mitochondrial theory of ageing there is evidence that mtDNA is more susceptible to oxidative damage with increasing age than nuclear DNA. Using similar study methods to those on nuclear DNA, the increase of OHSdG in mtDNA with advancing age appears considerable, with a 70 year old human brain displaying a 15-fold excess over that found in infants (Mecocci at si. 1993). The same is true in post-mortem heart samples

(Hayakawa at ai. 1992) and the increase exponential as might be predicted.

This is merely evidence of oxidation to mtDNA bases, but there is no doubt that the prevalence of identifiable mtDNA mutations also increases with age. Large scale sequence changes such as deletions have been easiest to detect, initially using southern blotting and more recently using PCR based methods for particular known sequence changes such as the "common" 4,977bp deletion associated with KSS and Pearson's syndrome. Applied to various post mortem tissues in subjects ranging from fetal/neonatal to over 90 years old, numerous studies have demonstrated that the common deletion starts to be easily detectable in most adult human tissue during middle age and increases both in prevalence and quantity with advancing years e.g. (Linnane at al. 1990;

has been confirmed in muscle and skin samples from living donors without known mitochondrial disease (Hsieh et al. 1994; Yang at al. 1994).

In keeping with the mitochondrial hypothesis those tissues most susceptible to oxidative stress, namely brain and muscle, have more common deletion than others with lower metabolic rates such as liver, kidney or spleen (Cortopassi at al. 1992). Combined information from these studies suggests

that in brain and muscle by 30 years of age 50% of us have sufficient mtDNA with the common deletion to be detectable by 30 PCR cycles and by 50 years this is nearly 100%. In skin and other tissues prevalence is lower, first appearing in the 60's in 20% of subjects, 50% in the 70's and found in over 80% of us who reach 80 years.

Absolute quantification of levels is difficult, and no method infallible. Most studies that attempt this can at best only offer estimates. Scinitillation counting of radioactively labelled PCR products, compared to those of a standard curve constructed from known proportions of deleted/complete mtDNA proposes a maximum of 0.1% deleted mtDNA or 1/1000 molecules in the >70 year old age group (Cooper at al. 1992b), approximating to an average of 1

deleted mtDNA molecule per cell. This is where the mitochondrial theory starts to have problems. Even allowing for the fact that some deletions may cluster together in a single cell, it is hard to imagine such a low level could really contribute significantly to functional decline.

3.4.1.2 Other mtDNA mutations

One explanation for this problem is that the common deletion represents only the "tip of the iceberg" being only one manifestation of much more extensive mtDNA damage. There are fewer studies addressing this but those that do are in support. Increasing levels of tandem duplications (Lee at al.

1994), other deletions (Baumer at al. 1994) and a 3243 point mutation (Zhang

atal. 1993) have all been demonstrated in human tissues with increasing donor

together with possibly thousands of different as yet undetected mutations the total mutant load might be considerable, and could be capable of affecting MRC function.

3.4.1.3 Other work on mtDNA and ageing

One important paper by Hayashi et al. (1994a) concludes that whilst a

progressive biochemical defect in MRC function was seen with increasing donor age, as will be discussed in the next section, nuclear and not mitochondrial genes are responsible for the functional decline. Fusing enucleated fibroblasts from elderly donors with a transformed mtDNA-less cell {fPHeLa cells), thus

placing "elderly "mitochondria in a new nuclear environment, resulted in restoration of mitochondrial function to levels comparable to those of younger subjects. If validated in other laboratories this would effectively rule out a significant role for mtDNA mutations in ageing.

A second study against aspects of the mitochondrial/ oxidative damage theory of ageing has been performed by Moraes at al. (1995). This took the

basic principle that if oxidative damage plays a role in generating mtDNA damage in ageing, specifically the common deletion, then patients with mitochondrial diseases and consequent high levels of oxidative stress should also display higher levels of deleted mtDNA than would be expected for their age group. Quantitative PCR on muscle samples from healthy subjects confirmed an increase in the amount of deleted mtDNA with age, though the confidence limits were considerable with up to a thousand fold variation at any given age. Similar analysis on samples from patients with MERRF/ MELAS and OREO did not show any excess of the common deletion above that expected for the patients age-group. This implies that local oxidative stress is not a major factor influencing the increasing common deletion seen with age, though the sensitivity of the technique may be inadequate to draw firm conclusions.

3.4.2 MRC function and ageing 3.4.2.1 Complex I

Studies on mitochondria prepared from human muscle have shown a significant decline in NADH:ubiquinone oxidoreductase specific activity with age such that subjects in the 70 - 90 year age group had only 50% the activity of subjects in their 20's.(r = -0.667, p = 0.032; Cooper et al. 1992b). The same

trend was seen in polarographic studies with NAD-linked substrates (r = -0.767, p = 0.016). Similar findings have been reported by others in human muscle (Hsieh at al. 1994; Blin at al. 1994), and in ageing monkey brain mitochondria

(fronto-parietal cortex; Bowling at al. 1993). Consistent with these findings

assays of ATP production, again from monkey brain mitochondria (striatum), have also showed a negative correlation between NAD-linked ATP production and advancing age (Di Monte atal. 1993).

Others have failed to validate this data but either the numbers have been insufficient (Cardellach at al. 1993 - only 8 subjects) or the tissue studied not

appropriate. The normal life-span of platelets in the circulation for instance is only 7 - 1 0 days, so they might not be expected to show chronic accumulative damage such as proposed for ageing (Bravi at al. 1992).

Significant changes in complex II or III activity with age are not commonly reported.

3.4.2.2 Cytochrome oxidase/complex IV

Several groups have reported a correlation between the numbers of COX negative ragged red fibres and age in ante-mortem and post-mortem muscle samples. This is true in cardiac (Müller-Hôcker, 1989), diaphragmatic and skeletal limb muscles (Muller-Hocker, 1990; Rifai at al. 1995). In keeping

with this a fall in COX activity with increasing donor age has been demonstrated on mitochondria prepared from human muscle (Cooper at al. 1992b; Hsieh at

al. 1994), platelets (Van Zuylen at al. 1992), cultured fibroblasts (Hayashi at al.

decline in COX activity may have a critical role in the ageing process. Groups who did not observe any significant decline with age, as for complex I data, had smaller numbers which may account for this (e.g. Solmi et al. 1994).

3.4.3 Conclusions

There is considerable data then showing evidence of oxidative damage to many cell components with advancing age. I have not considered the data on antioxidant defences in relation to this. Although a huge amount of data exists, the results are neither consistent or helpful. Either increasing or decreasing antioxidant defences with age either can be interpreted as in favour of ongoing oxidative stress. An increase might represent a positive feedback system, whereas a fall might be considered indicative of a defence strategy overwhelmed by powerful attack. Moreover the putative enzymes involved are increasing in number such that any comprehensive review would be considerable in length, and not directly relevant to this work.

That mitochondria are involved somehow both in free radical production and in ageing seems well supported. The problem lies in identifying which, if any, of these changes depict primary pathogenetic mechanisms, and which are merely secondary markers of a more generalized functional decline. This question remains unsolved.