Altered Composition of Liver Proteasome Assemblies Contributes to Enhanced Proteasome Activity in the Exceptionally Long-Lived Naked Mole-Rat

The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life

The naked truth:a comprehensive clarification and classification of current 'myths' in naked mole-rat biology

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions

Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer\u27s disease

Amyloid beta (Aβ) is implicated in Alzheimer\u27s disease (AD) as an integral component of both neural toxicity and plaque formation. Brains of the longest-lived rodents, naked mole-rats (NMRs) approximately 32 years of age, had levels of Aβ similar to those of the 3xTg-AD mouse model of AD. Interestingly, there was no evidence of extracellular plaques, nor was there an age-related increase in Aβ levels in the individuals examined (2-20+ years). The NMR Aβ peptide showed greater homology to the human sequence than to the mouse sequence, differing by only 1 amino acid from the former. This subtle difference led to interspecies differences in aggregation propensity but not neurotoxicity; NMR Aβ was less prone to aggregation than human Aβ. Nevertheless, both NMR and human Aβ were equally toxic to mouse hippocampal neurons, suggesting that Aβ neurotoxicity and aggregation properties were not coupled. Understanding how NMRs acquire and tolerate high levels of Aβ with no plaque formation could provide useful insights into AD, and may elucidate protective mechanisms that delay AD progression

Levels of markers of an inflammatory response were higher in naked mole-rat than in mice.

<p>We quantitated protein levels in Western blot analyses of liver tissue lysates probed with anti NFκB and TNFα antibodies. Both NFκB and TNFα protein levels were more than two-fold higher (p≤0.01) in naked mole-rats. Samples from three different individuals of each species were used and the experiment was repeated with lysates from different animals several times to verify the outcome. The blots shown are representative of these experiments. Actin was used as a loading control and lysates from mouse spleen tissue (MsSp) represented a positive control for these immune-related markers.</p

Analysis of proteasome subunit composition showed that naked mole-rats had higher protein content of 19S and immunoproteasome subunits than mice.

<p>(A) Representative Western blots with PVDF-transferred proteins were probed with antibodies specific for 20S, 26S and immunoproteasome subunits. Different content of various subunits revealed an upregulation of particular proteasome subassemblies. Samples from three different animals from each species were used per experiment and the experiment was repeated with samples from different animals at least one additional time to verify the outcome. The blots are representative of these sets. Actin was used as a loading control for our analyses. For immunoproteasome subunits, lysates from mouse spleen tissue (MsSp) were also used as a positive control. (B) Quantitation of Western blots grouped by 20S, 19S or immunoproteasome. Not only did naked mole-rats have higher content of constitutive non-catalytic subunits, but they also tended to have more immunoproteasome components (β2i, β5i, PA28α) than did mice. Naked mole-rats also had increased protein content of two critical 19S subunits (RPT5, RPN10). Values represent the mean ± SE with significant p-values highlighted in the figure.</p

The largest species differences observed were the contribution of the cytosolic fraction for both ChT-L and T-L activities.

<p>Percent contribution of the total activity was calculated using the values of specific activities presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035890#pone-0035890-g004" target="_blank">Figure 4</a>. In both species proteasome activity was highest in the microsomal fraction, but the microsomal contribution to the total activity within the lysate was greater in mouse samples (76%) than in naked mole-rat samples (50%) for ChT-L and this difference in % contribution was even greater for TL-activity (87%, 53% respectively). Nuclear fractions, regardless of the catalytic activity, only contributed 7% or less to the total activity in both species. PGPH activity showed a similar distribution within the subcellular fractions in both species. In sharp contrast the cytosolic fraction of ChT-L activity of naked mole-rats showed more than double (46%) the proportionate contribution to that of mice (18%) and this species difference was even greater for T-L activity (32% to 7%) in revealing that distributional differences in the observed total activity between species could be explained by interspecific differences in cytosolic activity.</p

Chymotrypsin- and trypsin-like activities, but not the caspase-like activity were higher in the whole cell lysates from naked mole-rat than in mouse lysates.

<p>In each assay 50 µg of whole cell liver lysates from physiologically age-matched young mice (4 mo) and naked mole rats (2 yr) were used. The samples were incubated with 100 µM of substrate specific for the type of active center of the proteasome being measured. A saturating concentration of proteasome inhibitor N-(benzyl-oxycarbonyl) leucinyl-leucinal (MG132), determined by titration, was added to parallel samples. The difference of the fluorescence released with and without inhibitor was used as a measure of the specific peptidolytic activity of proteasome. Hatched lines indicate the amount of non-specific protease activity in excess of net specific proteasome activity. Values are means ± SE. Significant p-values are indicated in the figure.</p

Data from: Sustained high levels of neuregulin-1 in the longest-lived rodents; a key determinant of rodent longevity

Naked mole-rats (Heterocephalus glaber), the longest-lived rodents, live 7-10 times longer than similarly–sized mice and exhibit normal activities for ∼75% of their lives. Little is known about the mechanisms that allow them to delay the aging process and live so long. Neuregulin-1 (NRG-1) signaling is critical for normal brain function during both development and adulthood. We hypothesized that long-lived species will maintain higher levels of NRG-1 and that this contributes to their sustained brain function and concomitant maintenance of normal activity. We monitored the levels of NRG-1 and its receptor ErbB4 in H. glaber at different ages ranging from 1 day to 26 years and found that levels for NRG-1 and ErbB4 were sustained throughout development and adulthood. In addition, we compared seven rodent species with widely divergent (4-32y) maximum lifespan potential (MLSP) and found that at a physiologically-equivalent age, the longer-lived rodents had higher levels of NRG-1 and ErbB4. Moreover, phylogenetic independent contrast analyses revealed that this significant strong correlation between MLSP and NRG-1 levels was independent of phylogeny. These results suggest that NRG-1 is an important factor contributing to divergent species MLSP through its role in maintaining neuronal integrity