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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:435-443 (2006)
© 2006 The Gerontological Society of America

The Effect of Aging on the Chaperone Concentrations in the Hepatic, Endoplasmic Reticulum of Male Rats: The Possible Role of Protein Misfolding Due to the Loss of Chaperones in the Decline in Physiological Function Seen With Age

Richard R. Erickson, Lisa M. Dunning and Jordan L. Holtzman

Departments of 1 Pharmacology and 2 Medicine, and 3 Division of Environmental Health Sciences, University of Minnesota, Minneapolis.
4 Research and 5 Medicine Services, Veterans Affairs Medical Center, Minneapolis, Minnesota.

Address correspondence to Jordan L. Holtzman, MD, PhD, 4710 Girard Ave. S., Minneapolis, MN 55419. E-mail: holtz003{at}umn.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The endoplasmic reticulum (ER) chaperones are highly conserved proteins that catalyze the posttranslational processing of all secretory and membrane proteins. Our studies suggest that chaperone declines are one of the two central defects in Alzheimer's disease. We propose that similar declines in other organ systems underlie the physiological deficits of aging. Rats were maintained in a colony from age 21 days to death. Animals were killed at regular intervals, and hepatic, ER chaperone contents were determined by immunoblotting. ERp55, ERp57, ERp72, BiP, and calnexin constitutive levels declined 30%–50% with age. Calreticulin was unaffected. BiP (also known as GRP78), ERp55, and ERp57 showed marked swings with peaks occurring in midwinter and midsummer. This cyclics declined 73% with age. Considering the role of the ER chaperones in membrane and secretory protein posttranslational processing, these data support the concept that their loss could lead to many of the physiological declines associated with aging.

AGING is a unique characteristic of almost all eukaryotic cells. In vertebrates it is associated with a slow decline in all physiological processes, including a loss of muscle mass, a failure of the immune system, decreases in the maximal synthesis and release of peptide hormones (such as insulin and growth hormone), a decline in renal function, and decreases in cognitive skills. A number of hypotheses have been proposed as the basis for these declines. Among them are suggestions that they are due to the long-term accumulation of lethal mutations, cellular damage from oxidant injury (1,2), and declines in telomerase activity leading to faulty cellular replication (3). Although there is no question that each of these processes plays an important role in the physiological declines observed in elderly persons, none of them completely explains the characteristic patterns of decline associated with aging. A major problem is that such factors as accumulation of mutations and decreases in telomerase activity would have their most profound effects on replicating cells, yet much of the decline is seen in terminally differentiated tissues. Furthermore, cells which do replicate throughout life—such as enterocyte, epidermal, and blood stem cells—do not lose teleromase activity. Similarly, the successful cloning of viable, healthy, mammalian individuals by using the nuclei from adults of several species would suggest that multiple mutations do not have a major role in aging (4). If these nuclei had an excess of deleterious mutations, they could not direct the development of healthy adults.

It has also been suggested that the "Hayflick phenomenon" underlies aging (5,6). A number of years ago, Hayflick observed that during the in vitro cultivation of fibroblasts, they would undergo only a limited number of replications. Furthermore, cells derived from elderly persons underwent fewer replications than did those from younger individuals. This phenomenon has been attributed to the accumulation of the cell cycle suppressor proteins P53 and P16 rather than to a loss of the telomeres (7). Yet, although the "Hayflick phenomenon" (like the telomerase hypothesis) may underlie the limited capacity of elderly persons to repair tissue injury, a major problem with this model is that much of the aging process is associated with terminally differentiated cells, such as neurons. Furthermore, many cell types, such as enterocyte, epidermal, and blood stem cells, replicate thousands of times throughout life. For example, enterocyte stem cells replicate every 4 days (8). For an individual who lives 100 years, these cells will undergo a total of 9000 replications.

Finally, another problem with some of these models, such as the role of oxidative stress and random mutations as the underlying biochemical processes leading to the declines associated with aging, is that the characteristic effects of aging are not random processes, but rather appear to be a programmed series of events in which specific changes occur at set times and in specific organ systems. This is exemplified by the overt physical changes associated with aging. For example, gray hair is one of the most conspicuous manifestations of aging. Yet, this frequently does not develop over the entire body nor in a diffuse pattern; rather, it may initially appear as "graying" at the temples. This loss of pigmentation specifically results from a decreased capacity of the melanocytes to synthesize tyrosinase, the enzyme which catalyzes the oxidation and polymerization of tyrosine to form melanin. Clearly this decline could be due to decreases in the transcription of the gene. But an alternative possibility is that it is due to a decreased capacity of the endoplasmic reticulum (ER) of the melanocytes to catalyze the posttranslational processing of this enzyme. In particular, tyrosinase has six N-glycosylation sites; failure to conjugate any one of these sites leads to misfolding and recycling through the ER-associated degradation pathway (ERAD) (9–12). The general application of this paradigm to other systems which undergo declines with age is supported by our recent studies on the likely biochemical defects underlying the deposition of plaque in patients with Alzheimer's disease (13).

This disease is characterized by the development of dementia in association with the deposition of plaque in the neocortex. A major component of plaque is a "garbage" protein, ß-amyloid (abeta) (14). This protein is produced in everyone during the normal posttranslational processing of a member of a family of growth factors, amyloid precursor protein-like proteins (14–16). One conundrum associated with Alzheimer's disease is that, although abeta is produced in everyone, plaque is observed in the brains of only elderly persons. Furthermore, work from a number of laboratories has demonstrated that in plaque the abeta is present as the naked peptide (17–20). In contrast, in our studies of cerebrospinal fluid from normal individuals, we have found that it is present as a complex with two ER chaperones, ERp57 and calreticulin (13). Based on work from other laboratories, these chaperones appear to bind to proteins only after N-glycosylation, suggesting that abeta is probably N-glycosylated at ASN27 (21,22). The discrepancy between our observations and the mass spectral studies of abeta in plaque suggests that plaque formation is due to a failure of the ER in neurons to catalyze the normal, posttranslational processing of the abeta. Our studies further suggest that plaque may be only a marker for a more generalized defect in ER function and that the dementia characteristic of Alzheimer's disease is due to a failure of the ER to process the nascent membrane proteins which form the synapses that are required for the initiation, consolidation, and retrieval of memory (13,23,24).

On the basis of these studies, we propose a new paradigm in which the aging process could result from a generalized decline in the capacity of the ER to catalyze the posttranslational processing of nascent secretory and membrane proteins. Considering the crucial role that this system serves in cell function and survival, decreases in this activity would have profound effects on the ability of elderly persons to function. This catalytic system in the ER is one of the two major posttranslational, protein-processing systems found in the eukaryotic cell; the other being that associated with the cytoskeleton in the cytosol. The third protein-processing system in the cell, the synthetic apparatus in the mitochondria, serves only a minor role. Prokaryotes do not have an ER, but they have a homologous system which is present in the periplasmic space of eubacteria and which also catalyzes the posttranslational processing of secretory and membrane proteins (25).

The posttranslational processing in the ER is catalyzed by two major sets of proteins. The first are the ER chaperones (25,26). They arrange nascent peptides into their native configurations, catalyze the formation of protein disulfides, participate in protein transfer to the golgi, monitor protein quality in the ER, and maintain the function of some secretory and plasma membrane proteins. Many of them are homologous to the heat shock proteins (HSPs) found in the cytosol. Others, such as ERp55, ERp57, and ERp72, are members of the thioredoxin, glutaredoxin, protein disulfide isomerase superfamily (25,26). Another pair of chaperones characteristic of the ER in all eukaryotes are members of the calreticulin/calnexin family. A second group of proteins are in the complexes that catalyzes the glycosylation of nascent proteins. In particular, those catalyzing N-glycosylation of the ß-amide of asparagines are crucial for cell survival. There are two complexes catalyzing this step; the first catalyzes the synthesis of the carbohydrate complex attached to a polyterpene cofactor, dolichol phosphate, and the other transfers this complex from the dolichol phosphate to the protein (27).

In light of the critical role played by the ER chaperones and N-glycosylation system in cell function and survival and the apparent defect of this system in Alzheimer's disease (13), we have in the current study examined whether with age there is a decline in the levels of several ER chaperones. Such a decline could account for the decreased capacity of elderly individuals to produce the membrane proteins necessary for normal physiological function. We find that several of them known to be critical for survival do show significant declines with age. These studies are consistent with the concept that a major factor in the physiological deficits seen with aging could be due to a decreased capacity of the ER to process newly synthesized membrane and secretory proteins.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
The Animal Model
The animals used in these studies were male, specific pathogen-free, Sprague-Dawley rats purchased from Harlan Laboratories (Madison, WI). They were obtained as a single batch of 400 animals at age 21 days. For the remainder of their lives they were housed in a windowless, controlled environment, "state-of-the-art," barrier facility with a constant temperature of 22°C ± 2° and a 12-hour light/dark cycle. The humidity was maintained at 50% ± 20%. The animals were housed two to a cage and had free access to food and water. Each cage containing study animals had an air filter cover. An additional 40 animals were housed in cages without filters. These animals were not part of the study population, but rather were maintained as "sentinel animals" to determine whether there had been any breaks in the sterile conditions. The sentinel animals were replaced at regular intervals with new batches of young, specific pathogen-free rats. These animals did not show an increase in mortality indicating that the colony was pathogen free.

All animals were removed from their cages once a week when the cages were cleaned and every 4–6 weeks when the animals were weighed. These procedures were performed under clean conditions with everyone entering the animal room wearing sterile gloves, gowns, and masks. The animals were fed a diet based on the AIN76A recommendations with the exception that the carbohydrate was supplied as 40% starch and 25% sucrose (BioServe, Frenchtown, NJ). This change in the carbohydrate composition was a result of our preference to provide the diet as pellets instead of as a powder and to reduce the incidence of obesity which is associated with high sucrose diets. Potassium citrate (6.5%) was added to prevent vascular disease (28).

The primary goal of our studies was to determine the effect of aging on the activity of the hepatic, microsomal cytochrome P450s. For the first 18 months, six animals were killed at 6-week intervals and microsomes were prepared by differential centrifugation (29). For the remaining 12 months, six animals were killed at 3-month intervals. We performed a large number of incubations to determine the effect of aging on several P450-dependent metabolic activities. Microsomal suspensions were then frozen at –70°C for the future determination of the contents of the various forms of P450. We were able to obtain tissue samples from 120 (or 30%) of the initial 400 study animals. The remainder died of natural causes.

At the end of the study, we determined the microsomal protein contents and the contents of BiP (also known as GRP78), calnexin, calreticulin, ERp55, ERp57, and ERp72 in the stored microsomal suspensions. Calnexin is bound to the ER membrane, but the luminal portion of this chaperone is highly homologous to calreticulin (30). Because on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) they have markedly different Mr values, we have used antibodies against calreticulin to determine both of these chaperones in a single gel (31,32). BiP and antibodies to it were purchased from StressGen (Victoria, BC, Canada). Antibodies to the other chaperones were prepared in our laboratory as previously described (32).

Analytical Procedures
At the end of the study the total microsomal protein contents were determined by the bicinchoninic acid (BCA) assay in all of the samples. We next determined the chaperone contents in these samples by immunoblotting (13,31–33). In this procedure the proteins were first separated by SDS–PAGE using the method of Laemmli (34) in which the samples were heated to 55°C for 5 minutes in the presence of SDS (1%), glycerol (5%), and mercaptoethanol (2.5%). After the electrophoresis, we transblotted the proteins onto polyvinylidene fluoride (PVDF) membranes by the method of Towbin and colleagues (35) and reacted the blots with the various specific antibodies followed by the appropriate goat anti-immunoglobulin antibodies coupled to alkaline phosphatase. The indicator dye was a combination of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad, Richmond, CA). Because this stain tends to fade with exposure to air, the blots were computer scanned within 24 hours of staining and then encased in clear, heat-sealed plastic. The densities were quantitated by NIH Image (version 1.60; PC Version Scion, Frederick, MD). A pooled microsomal suspension was included on each of the gels as a secondary standard. The contents of the chaperones in these microsomes were calibrated against a series of purified proteins. The immunoassays were run in triplicate and in the linear range for each of the individual chaperones.

Data Analysis
Each data point was the average ± standard error of the mean of the contents of each chaperone in the hepatic microsomes obtained from each of six animals. The graphs were plotted and the regression parameters were determined with SigmaPlot (Jandel, San Francisco, CA). The effect of age on the chaperone contents of the microsomes was determined by either a first- or second-order linear regression versus time. The appropriate order was determined by maximizing the variance due to the regression parameters (R2) with increasing orders of the SigmaPlot regression equation. Three of the chaperones showed markedly cyclic variation in their contents. The constitutive levels of these three were determined by linear regression of the data points observed at each of the troughs. For the other three chaperones, which did not show any cyclic variation, the data were regressed through all of the data points. The regression analyses are indicated by the dotted lines on each of the graphs. The quality of the fit was determined by examining the R2 attributed to the regression parameters. All the regressions of the basal values had a high R2 value (>0.84).


   RESULTS
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 Results
Discussion
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Validation of the Animal Model
The mortality was minimal until the age of about 500 days (Figure 1). After that, the rate showed a Gompertz effect of a doubling of the rate every 96.1 days (Figure 2). Humans show a similar doubling of mortality with every decade of life. The Gompertz effect is presumably characteristic of a population that is dying of multiple causes rather than from some programmed process, such as that seen in many invertebrate and fish populations, in which the adults die immediately after breeding. A similar failure to show a Gompertz effect is seen in mammalian populations in which the animals, such as the Fisher 344 rat, have a high incidence of early tumor development in both sexes (36,37). The excellent fit of the mortality data to a Gompertz plot would suggest that the aging of these outbred, study animals is comparable to that seen in humans.


Figure 01
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  		Figure 1. Life table analysis of rat survival

 

Figure 02
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  		Figure 2. Gompertz analysis of the mortality rate as a function of age

 
The animals reached and maintained an average maximum weight of about 600 g (Figure 3). But after the age of 750 days their average weights declined to 475 g. This decline was not due to the preferential survival of lighter animals, as at each time point the average weight of the animals that survived to the end of the study was the same as the average weight of the total population. The average adult liver weight was 17.5 g and remained there (Figure 4). The mg of microsomal protein per g liver showed no significant change with age (Figure 5).


Figure 03
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  		Figure 3. Rat body weight as a function of age

 

Figure 04
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  		Figure 4. Rat liver weights as a function of age

 

Figure 05
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  		Figure 5. Effect of age on the microsomal protein per g liver

 
Effect of Aging on the Specific Content of the Chaperones in Hepatic Microsomes
When we examined the effect of aging on the microsomal contents of the six chaperones, we found that they showed markedly different patterns (Figures 6–11GoGoGoGoGo; Table 1). Three of the chaperones—ERp55 (Figure 6; Table 1), ERp57 (Figure 7; Table 1), and BiP (Figure 8; Table 1)—demonstrated a cyclic variation, whereas the other three did not. Both the basal levels of these three chaperones, as well as the height of the cyclic variations, declined with age.


Figure 06
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  		Figure 6. Age effects on hepatic endoplasmic reticulum ERp55

 

Figure 07
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  		Figure 7. Age effects on hepatic endoplasmic reticulum ERp57

 

Figure 08
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  		Figure 8. Changes in the specific content of BiP (also known as GRP78) with age

 

Figure 09
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  		Figure 9. Changes in the specific content of endoplasmic reticulum (ER)p72 with age

 

Figure 10
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  		Figure 10. Changes in the specific content of calreticulin with age

 

Figure 11
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  		Figure 11. Changes in the specific content of calnexin with age

 

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  		Table 1. Effect of Age and Cyclic Variation on the Concentrations of the Various Endoplasmic Reticulum (ER) Chaperones.

 
The constitutive levels of ERp55 were best regressed as a second-order plot (Figure 6). The highest values were observed in the youngest animals, and showed a 51% decline as the animals reached maturity (dotted line, Figure 6; R2 = 0.8989). This is the pattern which would be predicted for a constituent which is critical for growth and proliferation. Because on reaching maturity the hepatocytes enter into a G0 phase, these cells have far less demand for new membrane components. Hence, it would be expected, as observed, that a chaperone involved in membrane synthesis would show a significant decline with age. After the age of 200 days, the ERp55 content showed a markedly slower, but steady decline.

The initial peak in the first winter was markedly blunted compared to that in the following summer. This may have been due to the high constitutive levels observed until the age of 200 days (Figure 6). Hence, the cells were already producing nearly maximal quantities of this chaperone. In contrast, in older animals the constitutive levels had declined, but, at least in the next cycle, they were still able to mount a vigorous stress response (Figure 6; Table 1). This cyclic response significantly decreased with age showing a 73% decline by 874 days. At the hazard of being repetitious, I believe that it is important to emphasize again that the microsomal protein and chaperone contents were all determined at the end of the study, hence the declines seen with age and the cyclic changes seen in these proteins were not due to interday analytical variations.

Because at no time after the age of 21 days were these animals exposed to any environmental stress, it is most likely that this periodicity had been imprinted into the animals' genetic, biological clock. Such a clock would have been synchronized with the periods of stress to which their ancestors were exposed in the wild. Similar cyclic variations have been reported in peptide and steroid hormones levels in studies of both animals (38) and middle-aged, normal, human volunteers (39).

ERp57, also known as GRP58, showed a similar pattern, but there were also marked differences (Figure 7; Table 1). First, the constitutive levels were best represented by a first-order regression. Second, the values actually increased as the animals reached maturity and then showed a 35% decline between the ages of 84 days and 874 days (dotted line, Figure 7; R2 = 0.84144). ERp57 also showed a circum semiannual rhythm (Figure 7). Again the peaks coincided with midwinter and midsummer (Figure 7). This cyclic response showed a 71% decline with age.

As demonstrated in yeast genetic studies, these two chaperones serve a critical role in cell function and survival. Yeast have several homologs of these chaperones. In genetic studies, when they were knocked out the yeast became dormant (40,41). This inhibition of growth could be partially reversed by transfection with the human gene for ERp55. These data would suggest that in eukaryotes these two chaperones are essential for life.

ERp55 and ERp57 also protect the cell from oxidant injury (25,42). These proteins were originally identified by their catalysis of the glutathione reduction of protein disulfides (42–44). This activity is termed thiol:protein disulfide oxidoreductase (TPDO). Along with glutaredoxin (thiolase) and methionine sulfoxide reductase, they are the primary enzymes which repair oxidant damage to proteins. Hence decreases in their concentrations would reduce the cell's capacity to survive oxidant injury. Such an increase in oxidant injury has been demonstrated in mouse liver by Rabek and colleagues (45). But, contrary to our studies, these authors did not see a decrease in protein disulfide isomerase (PDI), presumably ERp55.

A third chaperone, BiP, is a member of the HSP70 family and is also known as GRP78. It showed a pattern similar to ERp57 and ERp55 (Figure 8), with a 39% decline in the constitutive values with age (dotted line, Figure 8; R2 = 0.95855). It too showed some seasonal variation (Figure 8) with a 50% decrease in the cyclic response (Table 1).

A fourth chaperone, ERp72, which is also a member of the thioredoxin superfamily, showed a totally different pattern from the other two members of this superfamily, ERp55 and ERp57 (Figure 9; Table 1). Like ERp57, the young, mature animals (84–120 days) had the highest concentrations. Subsequently, ERp72 showed a 30% decline with age (dotted line, Figure 9; R2 = 0.8840). In contrast, it showed no seasonal variation. The exact function of this protein has not been clearly defined, but it is thought to be a chaperone and is a major, ER calcium binding protein, Calcium Binding Protein 2 (32,46).

Another chaperone which has been found to be critical for cellular function is calreticulin. Unlike the others, it did not appear to decline with age (Figure 10). Rabek and colleagues (45) have also noted that calreticulin does not decline with age in mouse liver, although it did show increased oxidant injury. In contrast, its membrane-bound homolog, calnexin, showed a 29% decline, but no seasonal variation (Figure 11; R2 = 0.93979). Furthermore, like ERp57, it had low concentrations in young animals, reached a peak at 84 days, and then declined 32% by the age of 874 days.


   DISCUSSION
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 Materials and Methods
 Results
 Discussion
References
 
We feel that the declines we have observed in the constitutive levels of five major ER chaperones lend support to the concept that a major factor in the physiological deficits seen in elderly persons are due to a decreased capacity of the ER to catalyze the posttranslational processing of newly synthesized membrane and secretory proteins. These declines, when coupled with the reported declines in the activity of the N-glycosylation pathway (47,48), would suggest that these losses represent major factors in the aging process. Furthermore, these declines appear to be specific for the posttranslational system, because there was no decline with age in the total ER protein content (Figure 5). Some may speculate that a decline of 30%–50% in the chaperones would not have a significant effect on the ability of the cell to maintain the integrity of the plasma membrane, but this seems unlikely because, as discussed below, the ER system does appear to be capacity limited. Furthermore, in light of the critical role of the plasma membrane in maintaining cell function, any decrease in its functional capacity could be devastating.

The plasma membrane (and its associated proteins) is one of the fundamental structures that define all cellular organisms. It separates both prokaryotic and eukaryotic cells from the external environment and allows the cell to maintain metabolic processes which would otherwise be prohibited by the second law of thermodynamics. In fact, the evolution of this structure was probably the single most important event in the origin of life as we know it. Furthermore, the various receptor proteins, ion channels, and transport systems embedded in this membrane are crucial for the cell to communicate with its environment. Hence, any process which compromises the synthesis and function of the plasma membrane would have profound effects on cellular function and survival.

The three major metabolic pathways required for the synthesis and function of plasma membranes are lipid synthesis, synthesis of complex carbohydrates, and the posttranslational processing of nascent, membrane proteins. In all organisms this posttranslational processing is catalyzed by a diverse family of proteins, the chaperones (25,26). The ER chaperones are highly conserved proteins that catalyze the posttranslational processing of all secretory and membrane proteins. They arrange nascent peptides into their native configurations, catalyze the formation of protein disulfides, participate in protein transfer to the golgi, monitor protein quality in the ER, and maintain the function of some secretory and plasma membrane proteins (25,26). In eukaryotes, this processing also involves the formation of amide-bound carbohydrates to form N-glycosylated proteins (27). The failure of a cell to fold nascent proteins into a functional configuration activates various ER-associated, protein quality surveillance systems (12,49).

The chaperones play a major role in the folding of complex membrane proteins, such as the cystic fibrosis transconductance regulator (CFTR) and the nicotinic acetylcholine receptor (50,51). These are complex, intrinsic membrane proteins with several transmembrane loops. The insertion of each of the transmembrane portions involves a number of steps and is sensitive to the cellular environment (52). The role of the chaperone levels in this processing has been well-demonstrated in studies on the efficiency of folding of the nicotinic acetylcholine receptor in muscle cells. In freshly prepared chick myocytes it approaches 100% (53). In contrast, after culture the efficiency of folding declines to less than 20% with 80% of the nascent proteins being recycled through the ERAD system (12,54). This decline was partially reversed by transfection with the ER chaperone, calnexin (54).

If the ER cannot properly fold nascent proteins, the cell has evolved surveillance systems which prevent overloading of the ER with denatured proteins (55). One of the systems targets the protein for degradation, ERAD. ERAD identifies misfolded proteins and translocates them to the cytosol through the translocon, where they are ubiquinated and degraded by the proteosome (12,25,26,56).

If enough misfolded proteins accumulate in the lumen of the ER, then the unfolded protein response (UPR) is initiated (49). The central element in this process is BiP. In the absence of unfolded proteins, BiP inactivates two protein kinases, PERK and IRE-1, and a negative protranscription factor, AFT-6. If sufficient concentrations of unfolded proteins accumulate, the BiP is drawn off of these by mass action, and they become activated. This leads to a downregulation of the transcription of membrane proteins and the increased transcription of certain of the ER chaperones (49).

If the concentration of misfolded proteins continues to increase, then the decline in free BiP within the lumen of the ER leads to cell death through both apoptosis and necrosis. The former is due to the activation of another protein kinase, PEEK, which activates a negative transcription factor, CHOP, and initiates apoptosis. The other mechanism for the initiation of cell death is the leakage of Ca2+ from the ER into the cytosol through the translocon.

The translocon is a pore in the ER membrane which facilitates the passage of newly synthesized peptides from the polyribosomes in the cytosol to the lumen of the ER (25,26,56). The free concentration of Ca2+ in the ER is three orders of magnitude greater than that in the cytosol. When newly synthesized proteins are being translocated from the ribosome in the cytosol to the lumen of the ER through the translocon, they block the leakage of Ca2+ into the cytosol (57). In contrast, when the cell is not actively synthesizing secretory or membrane proteins, BiP blocks the channel of the translocon to prevent the Ca2+ leakage (57). If the luminal, free concentration of BiP declines significantly due to the presence of high concentrations of misfolded proteins, then it comes off of the translocon and allows the leakage of Ca2+ into the cytosol. This increased cytosolic Ca2+ concentration activates various hydrolases and thereby initiates cell death through necrosis (29,58).

The presence of these various quality assurance pathways in the ER and the known marginal capacity of the ER to synthesized fully functional membrane proteins, together would suggest that our observations of significant declines in the ER chaperones with age could represent the central element in the loss of physiological capacity seen in elderly persons. Yet, the question then arises on what is the mechanism of what appears to be a fairly specific decline in the components of this system? In contrast, the constitutive levels of the HSP chaperones (such as HSC70), which catalyze normal protein folding in the cytosol, are unaffected by age (59,60) even though cells from elderly animals do show a decreased heat-induced response with a muted induction of the stress HSPs, such as HSP70 (59–61). In light of this high degree of specificity, we would speculate that, analogous to the "Hayflick phenomenon," it is due to the accumulation of some negative regulator factor which leads to the decreased transcription or translation of the ER chaperones and the components of the N-glycosylation pathway.

In recent years, a number of processes have been demonstrated to inactive either transcription or translation of genes with age. Among these are methylation of DNA and reduced acetylation of histones. Because they represent an "all-or-nothing" phenomena, neither of these processes would appear to be good reasons for the gradual declines in the ER chaperone contents that we observed in our studies. In contrast, two such regulatory systems which have been identified are the sRNAis and the microRNAs. The sequences for these RNAs are present in the noncoding regions of the DNA. They appear to prevent the translation of specific messenger RNAs. This family of nucleic acids may have evolved as a defense against viral replication. In analogy with the "Hayflick phenomenon," these RNAs could accumulate with age and gradually turn off the synthesis of the ER chaperones and the components of the N-glycosylation pathway.

Conclusion
Our current data, along with our recent findings on the biochemical basis for the deposition of plaque in Alzheimer's disease, clearly indicate that, even in the liver (an organ the functional capacity of which is well-conserved throughout life), there are significant and specific declines in the components of a fundamental, metabolic pathway which are necessary for normal cell functioning and survival. These data support a new paradigm for the aging process which postulates that the physiological declines associated with aging are due to a specific loss of the capacity of the ER to fold nascent proteins into their functional configurations.


   Acknowledgments
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These studies were support in part by funds from the General Research Service of the Department of Veterans Affairs.


   Footnotes
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Decision Editor: James R. Smith, PhD

Received August 4, 2005

Accepted October 14, 2005


   References
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