[Frontiers in Bioscience 3, d25-43, January 1, 1998]
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HOW DO INTRACELLULAR PROTEOLYTIC SYSTEMS CHANGE WITH AGE?

Ana Maria Cuervo and J. Fred Dice

Department of Physiology Tufts University School of Medicine, Boston, USA

Received 12/5/97 Accepted 12/9/97

5. TISSUE-SPECIFIC CHANGES IN PROTEOLYTIC SYSTEMS WITH AGE

We discuss here four representative examples of changes in protein turnover that are organ-dependent. The age-related modifications in the activity of several protein degradation pathways in those tissues are summarized in table 3.

Table 3. Changes in several protein degradation pathways in specific cell types with age
LYSOSOMAL

Tissue

Ubiquitin/ Proteasome

Calpains

Macroautophagy

Microautophagy

Hsc73-mediated

Central nervous system

No change

Increase

Decrease

No data

No data

No data

Skeletal muscle

No change

No change

No data

No data

No change

Liver

No change

No change

Decrease

No data

Decrease

Eye lens

Decrease

No change

No data

No data

Decrease

5.1. Central nervous system

The central nervous system is one of the tissues that shows the most dramatic changes in protein degradation with age. Age-related morphological and neurochemical changes in brain cells have been very well-characterized, and many of them can be reproduced after exposure to different protease inhibitors (156, 157). Consequently, reduced protease activities or enhanced protease inhibitor activities might be involved in brain aging.

Several age-related changes in the activity of brain proteases have been reported (see table 4). In addition to changes in levels and activity of brain proteases with age, alterations in their substrate sensitivity to proteolytic attack need to be considered. For example, there are many reports of increased levels and activities of cathepsin D and calpain in the aged brain, but such changes do not correlate with an increased protein degradation, since proteolysis declines with age (169). In the same way, the accumulation of lipofuscin-pigment in lysosomes is not due to a decrease in cathepsin B activity with age (170). These discrepancies and others summarized in table 4 may be due to the different substrate proteins used (reviewed in ref. 169).

Table 4. Age-related changes in protein degradation in the nervous system

CHANGES IN PROTEASE ACTIVITY

REFERENCE

  • Increase of calpain activity in spinal cord

158

  • Increase in calpastatin levels in brain

159

  • Increase in calpain and cathepsin D activity in brain

160

  • Decrease in calpain levels but increase in calpain activity in Alzheimer’s disease patients

110,161

  • Increase in levels of cathepsin D in brain

162

  • Increase of brain calpain activity depends on the cerebral area analyzed

163

  • Increase in cathepsin D, E, and B activities and decrease in cathepsin L activity in brain

162

CHANGES IN SUBSTRATE SUSCEPTIBILITY TO PROTEASES

  • Microtubule-associated proteins in brain are more susceptible to cathepsin D-like proteases

164

  • Phosphorylation and carbonyl modification of neurofilament proteins reduce their proteolytic susceptibility

161, 165, 166

  • Age-related modifications of synaptosomal membrane proteins inhibits their proteolysis

167

  • Binding of aluminun to neurofilament proteins inhibits their proteolysis

161

  • Peroxidation of myelin membrane proteins increases their susceptibility to proteolysis

168

The lysosomal/endosomal and calpain systems seem to be the most affected by age in neuronal tissues. Thus, as described above, lysosomes undergo marked morphological changes in senescent cells and participate in neurodegenerative processes. As a result of aging or metabolic and oxidative stress, indigestible or incompletely degraded material accumulates within the endosomal-lysosomal system and produces a heterogeneous group of residual bodies containing lipofuscin (171-173). Although those aggregates can also be caused by treatment with lysosomotropic inhibitors (174), their accumulation in aging does not appear to be due to reduced protease activity but to changes in proteolytic susceptibility of the substrate proteins. An increased expression of some lysosomal proteases (cathepsin D, B and E) (175) together with a decreased stability of the lysosomal membrane (176) have also been described in senescent brain.

In Alzheimer's disease, one of the most common neurodegenerative diseases, proteins of the cell membrane and cytoskeleton are abnormally processed and accumulate in the brain. It is now believed that lysosomal alterations are important in the pathogenesis of this disease and in the accumulation of amyloidogenic peptides (177-179). Acid hydrolases accumulate in atrophic and degenerating neurons, and their release to the extracellular space may contribute to senile plaque formation (179). The typical amyloid deposits in Alzheimer's disease are normally produced in an acidic compartment by noncysteine proteases, and then they are eliminated by lysosomal cysteine proteases (mainly cathepsins B and L) (177, 180-182). Treatment with general cysteine proteinase inhibitors cause accumulation of potentially amyloidogenic precursor protein fragments (183, 184).

5.2. Liver and cultured fibroblasts

In spite of the many differences between hepatocytes and fibroblasts, the parallelism of most of the results regarding age-related changes in intracellular protein degradation lead us to consider them together in this section. Many of the age-related changes in the degradative systems described in section 4.2. were obtained using rat liver or human fibroblasts in culture, and these results are summarized in table 5.

Table 5. Comparison of the age-related changes in protein degradation in rat liver and cultured human fibroblasts

PROTEIN DEGRADATION

LIVER

FIBROBLASTS

CHANGE

REFERENCE

CHANGE

REFERENCE

  • Total protein degradation

Decrease

113

Decrease

185

  • Proteolytic susceptibility

Increase

186

Increase

187

  • Oxidized and modified proteins

Increase

188

Increase

189

PROTEOLYTIC SYSTEMS

Cytosolic

  • Proteasome

  • Proteasome levels

No change

86

No change

190

  • Degradation of

ubiquitinated proteins

No change

191

No change

91

  • Calpain activity

No data

Increase

110

Lysosomal

  • Elimination of autophagic
  • vacuoles

Decrease

141

Decrease

140

  • Half-life of microinjected
  • proteins

Increase

195

Increase

20

  • Hsc73-mediated

Decrease

Cuervo & Dice, unpublished

Decrease

20

  • Number and sizes of
  • lysosomes

Increase

192

Increase

114

  • Activity of cathepsins

Increase

193

Increase

194

The liver is one of the organs in which a decrease in total protein degradation with age has been well-established (113). The most affected proteolytic system in liver from old animals is the lysosomal system. Lysosomes are responsible for more than 80 % of protein degradation in liver (196, 197). Characteristic age-related changes in the morphology and function of the macroautophagy/lysosomal system have been described in hepatic cells: there is an increase in number and size of lysosomes in liver cells with age (192); the activity of lysosomal enzymes increases with age in parenchymal and non-parenchymal cells of the liver (193); there is a decrease in the formation of autophagic vacuoles and in the degradation of their content (141); the half-lives of proteins microinjected in hepatic cells increase with the age of the donor animal (table 2; 195); and the selective uptake of cytosolic proteins by lysosomes from rat liver decreases with age (figure 5B; Cuervo and Dice, unpublished results). This decrease in the activity of the hsc73-mediated lysosomal pathway in rat liver with age is considered to be one of the major proteolytic pathways responsible for the decrease in total protein breakdown in liver.

As described previously, human fibroblasts in culture have been extensively used for studies of aging (see section 3). In spite of the controversy in accepting senescence in culture as a good model of aging in vivo, fibroblasts in culture show very similar alterations in protein degradation pathways as does liver from aged rats. table 5 summarizes these changes in proteolytic pathways.

5.3. Skeletal muscle

In contrast to the nervous system, liver, and fibroblasts, where lysosomes appear to play a key role in alterations of protein degradation associated with aging, the ubiquitin-dependent system is mainly responsible for protein degradation in skeletal muscle, especially in many muscle-wasting conditions (198, 199). During aging there is a progressive loss of muscle mass (200) that originates because of an imbalance between synthesis and degradation of proteins (reviewed in ref. 201). However, when basal rates of skeletal muscle protein turnover are analyzed, there is a significant reduction with age in both synthesis and degradation. Studies analyzing rates of protein synthesis by [14C]tyrosine incorporation into muscle, and degradation by urinary excretion of 3-methylhistidine, related to myofibrillar protein breakdown, have demonstrated that both processes significantly decrease in skeletal muscle of rats with age (202). The protein synthesis/degradation imbalance in senescence is only evident after induction of a catabolic state such as in starvation, denervation atrophy, cancer, acidosis or trauma, and reflect a failure to rapidly restore muscle protein after those circumstances (203). Those authors also demonstrated that muscle wasting resulted from increased proteolysis in normal rats, but it is caused by a decrease in protein synthesis in senescent rats. Under catabolic conditions only a small increase of cathepsin D and milli-calpain have been observed, but it does not modify total protein breakdown (204).

5.4. Eye lens

Another tissue where age-related changes in the activity of proteolytic systems has been studied is the eye lens. In the lens the proteolytic removal of damaged proteins may play an important role in maintaining the lens transparency (90, 205-207). Normal lenses have the ability to increase ubiquitin conjugation activity in response to oxidative stress, but that response is impaired in senescent lenses (92, 97). No changes in levels of ubiquitin conjugating enzymes in lenses were detected with age, but changes in their ability to respond to oxidative stress were found (97). The age-related decrease in the ability to mount a ubiquitin-dependent response upon oxidation may contribute to the accumulation of damaged proteins in the old lenses.

Other proteolytic systems also decline with age in lens. Studies in cultured epithelial lens cells revealed that, after successive passages, there was a decrease in the proteolytic response to serum removal. This degradative defect mainly affects proteolysis of long-lived proteins suggesting that the degradative defect may be related with the hsc73-mediated lysosomal pathway of protein degradation (208).