Stress resistance, Proteostasis and Aging reviewed (2015)

Let's start with a brief literature review by Alper, Bronikowski and Harper 2015 (1). I have excerpted some particularly noteworthy passages.

CR does not lead to epigenetically stable programming, hence an ex vivo model of CR has been hard to come by:
Interestingly, cells grown from dietary models of life extension fail to show this correlation. More specifically, dermal fibroblasts from mice subjected to life-long caloric restriction (CR) or provided with a diet low in the essential amino acid methionine, were no more stress resistant to multiple cytotoxins relative to their normal-fed counterparts (Harper et al., 2006b). Caloric restriction is perhaps the most robust life-extending intervention known (Fontana and Partridge, 2015) while diets low in methionine have been repeatedly shown to increase longevity in both rats and mice (Perrone et al., 2013, Sun et al., 2009 and Miller et al., 2005). A clue to this apparent discrepancy comes from studies using conditioned media; or more specifically, cells exposed to media supplemented with serum collected from rodents undergoing CR are more stress resistant than are cells grown in the presence of normal media alone. This suggests the presence of specific circulating factors needed for the life extending effects of dietary restriction that are lost during the derivation and expansion of individual cell lines (de Cabo et al., 2003 and De Cabo et al., 2015).
Multi-stress resistance correlates with long lifespans but there are exceptions to the rule:

Nevertheless, there have been several studies using other genetic mouse models of longevity that have also failed to show a significant difference in stress resistance among long-lived versus control littermates. This includes cells grown from mice globally deficient in insulin receptor substrate-1 (Page et al., 2014), as well as whole animal studies using mice with a brain-specific deletion of the insulin-like growth factor-1 receptor (Kappeler et al., 2008). Moreover, in contrast to the findings typical of Snell dwarf mice, pharmacological induction of endoplasmic reticulum (ER)-stress using both tunicamycin and thapsigargin indicated that Snell dwarf mouse fibroblasts were significantly more sensitive to ER stress than their littermate controls (Sadighi Akha et al., 2011). Although it’s still not clear what may account for these observations, differential regulation of the somatotrophic axis has been suggested (Page et al., 2014, Kappeler et al., 2008 and Sadighi Akha et al., 2011) which may be confounded by the use of a specific genetic background (Mulvey et al., 2014).

Proteasomes & aging: a brief update
I've blogged briefly on this topic before. To recap, Salway et al. 2011 (3) did not find a correlation between 20/26S proteasomal activity in tissue homogenates from liver, heart & brain and species lifespan (n=15). More recently, Pickering et al. concluded the opposite (ref. 4, n=21) by using cultured fibroblasts. In addition they explored the molecular signaling behind these changes. For reasons beyond me, Pickering does not cite the Salway paper, which has been the most important and extensive work on this topic so far.

A comparison of the employed methods might be informative. Pickering et al. 2015 cultured fibroblasts from primates (n=21) and studied livers from a few long-lived mouse models (n=4). To detect proteasomal activity they used fibroblast homogenates directly and additionally ran an in-gel overlay assay with the chymotrypsin-like substrate Suc-LLVY-AMC. The largest difference was seen with ATP-independent 20S proteasome activity.
Salway et al. included mammals, a few birds and Snell mice. By and large they used the same protocol as above to study proteasome activity in their tissue homogenates. Yet if anything, they found the opposite. In livers a significant negative correlation between proteasome function and lifespan emerged.

Both papers measured proteasome activity in long-lived mice yet they are hardly comparable. Pickering studied liver proteasome activity from Rapamycin-, NDGA-, EST- (estradiol-derivative) treated and Snell dwarf mice. Although, Salway also used Snell dwarfs, sadly, they focused on brain and heart. Again the conclusions were opposed, yet both papers may be "right".

Both papers adjusted for phylogenetic relatedness and body mass, but the Pickering paper focused on the clade of primates to make 1. statistical analysis easier (I guess) and 2. to allow protein detection on conserved epitopes by western blot. The data by Pickering et al. was robust even after adjustment.

The first thing that jumps out is that Salway measured activity in tissue homogenates even including post-mitotic organs (brain, heart). This is a very meaningful readout. In contrast, no one knows if fibroblast stress resistance translates to other cell types or whole organisms (1), although, a few studies suggest it does (dwarf mice survive paraqaut quite well, for instance). What if fibroblasts are a poor model or display some cell culture artefact that makes them stress resistant or upregulates the proteasome? There seems to be more than one way to reconcile the two papers. First, fibroblasts may be mildly stressed in cell culture, whereas no effect would be seen at steady state, as in tissue extracts. In fact, a similar pattern is seen with autophagy. It is difficult to detect in situ, but stressed or starved dwarf mouse derived fibroblasts do show prolific autophagy. Second, it might be an effect related to immune function (culture conditions causing immunologic stimulation due to latent contamination?). Third, it might be a cell culture artifact, say an effect of passaging. Perhaps long-lived species maintain proteasome function during in vitro aging. Interestingly the cells were quite "old" at passage numbers of ~10, whereas in mouse experiments P3 is usually used.

What does upregulation of the immunproteasome do for these cells?
It is possible that an enhancement in immunoproteasome function would increase cellular proteostatic capacity and turnover of oxidized or otherwise damaged proteins. Alternatively, enhancement in immunoproteasome may lead to an increase in MHC class I cell surface antigen presentation and in this way augment immune defenses to viral infection and cancer.

Mitochondria, stress resistance & lifespan
All papers on lipid composition and lifespan tend to draw my attention. This one (2) was mentioned in a recent review and is new to me, but what does it add? Not much, and this may be the reason why I never heard of it before. Tropical birds tend to be longer-lived than their temperate counterparts. They also have more plasmalogens, less cardiolipin (and hence inner mt membrane?) and less mit lipid (fewer or smaller mt?).

See also:
as well as (5)

 1. Exp Gerontol. 2015 Sep 3. pii: S0531-5565(15)30041-3. doi: 10.1016/j.exger.2015.08.018. [Epub ahead of print]
Comparative cellular biogerontology: Where do we stand?
Alper SJ1, Bronikowski AM2, Harper JM3.

2. Physiol Biochem Zool. 2014 Mar-Apr;87(2):265-75. doi: 10.1086/674696. Epub 2014 Feb 20.
Linkages between mitochondrial lipids and life history in temperate and tropical birds.
Calhoon EA1, Jimenez AG, Harper JM, Jurkowitz MS, Williams JB.

3. Age (Dordr). 2011 Mar;33(1):33-47. doi: 10.1007/s11357-010-9157-5. Epub 2010 Jun 22. Enhanced protein repair and recycling are not correlated with longevity in 15 vertebrate endotherm species. Salway KD1, Page MM, Faure PA, Burness G, Stuart JA.

4. Lifespan of mice and primates correlates with immunoproteasome expression.
Pickering AM, Lehr M, Miller RA.
J Clin Invest. 2015 May;125(5):2059-68. doi: 10.1172/JCI80514. Epub 2015 Apr 13.

5. Liver mitochondrial membranes of long-lived species show a lower level of free radical production and a lower degree of unsaturation of fatty acids which, in turn, may contribute to slowing down the aging process (65). However, lifelong treatment with the beta-blocker drug atenolol decreases membrane fatty acid unsaturation and oxidative stress in heart and skeletal muscle mitochondria without changing mice longevity (66). In contrast, diets containing a low proportion of PUFAs and high amount of monounsaturated and saturated fats may maximize life span in animals maintained on CR (67).