Anson Pierce

High oxidative damage levels in the longest‐living rodent, the naked mole‐rat

Oxidative stress is reputed to be a significant contributor to the aging process and a key factor affecting species longevity. The tremendous natural variation in maximum species lifespan may be due to interspecific differences in reactive oxygen species generation, antioxidant defenses and/or levels of accrued oxidative damage to cellular macromolecules (such as DNA, lipids and proteins). The present study tests if the exceptional longevity of the longest living (> 28.3 years) rodent species known, the naked mole‐rat (NMR, Heterocephalus glaber), is associated with attenuated levels of oxidative stress. We compare antioxidant defenses (reduced glutathione, GSH), redox status (GSH/GSSG), as well as lipid (malondialdehyde and isoprostanes), DNA (8‐OHdG), and protein (carbonyls) oxidation levels in urine and various tissues from both mole‐rats and similar‐sized mice. Significantly lower GSH and GSH/GSSG in mole‐rats indicate poorer antioxidant capacity and a surprisingly more pro‐oxidative cellular environment, manifested by 10‐fold higher levels of in vivo lipid peroxidation. Furthermore, mole‐rats exhibit greater levels of accrued oxidative damage to lipids (twofold), DNA (~two to eight times) and proteins (1.5 to 2‐fold) than physiologically age‐matched mice, and equal to that of same‐aged mice. Given that NMRs live an order of magnitude longer than predicted based on their body size, our findings strongly suggest that mechanisms other than attenuated oxidative stress explain the impressive longevity of this species.

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach.

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/microg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.

The long lifespan of two bat species is correlated with resistance to protein oxidation and enhanced protein homeostasis

Altered structure, and hence function, of cellular macromolecules caused by oxidation can contribute to loss of physiological function with age. Here, we tested whether the lifespan of bats, which generally live far longer than predicted by their size, could be explained by reduced protein damage relative to short‐lived mice. We show significantly lower protein oxidation (carbonylation) in Mexican free‐tailed bats (Tadarida brasiliensis) relative to mice, and a trend for lower oxidation in samples from cave myotis bats (Myotis velifer) relative to mice. Both species of bat show in vivo and in vitro resistance to protein oxidation under conditions of acute oxidative stress. These bat species also show low levels of protein ubiquitination in total protein lysates along with reduced proteasome activity, suggesting diminished protein damage and removal in bats. Lastly, we show that bat‐derived protein fractions are resistant to urea‐induced protein unfolding relative to the level of unfolding detected in fractions from mice. Together, these data suggest that long lifespan in some bat species might be regulated by very efficient maintenance of protein homeostasis.—Salmon, A. B., Leonard, S., Masamsetti, V., Pierce, A., Podlutsky, A. J., Podlutskaya, N., Richardson, A., Austad, S. N., Chaudhuri, A. R. The long lifespan of two bat species is correlated with resistance to protein oxidation and enhanced protein homeostasis. FASEB J. 23, 2317–2326 (2009)

Proteomic Screening of Glycoproteins in Human Plasma for Frailty Biomarkers

The application of proteomics methodology for analyzing human blood samples is of increasing importance as a noninvasive method for understanding, detecting, and monitoring disease. In particular, glycoproteomic analysis may be useful in the study of age-related diseases and syndromes, such as frailty. This study demonstrates the use of methodology for isolating plasma glycoproteins using lectins, comparing the glycoproteome by frailty status using two-dimensional polyacrylamide gel electrophoresis and identifying glycoproteins using mass spectrometry. In a pilot study, we found seven glycoproteins to differ by at least twofold in prefrail compared with nonfrail older adults, including haptoglobin, transferrin, and fibrinogen, consistent with known inflammatory and hematologic changes associated with frailty. Enzyme-linked immunosorbent assay analysis found that plasma transferrin concentration was increased in frail and prefrail older adults compared with nonfrail, confirming our proteomic findings. This work provides evidence for using a reproducible methodology for conducting clinical proteomic comparative studies of age-related diseases.

A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2-4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1(+/0)) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1(+/0) mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1(+/0) cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1(+/0) cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

Detection and quantification of protein disulfides in biological tissues a fluorescence-based proteomic approach.

While most of the amino acids in proteins are potential targets for oxidation, the thiol group in cysteine is one of the most reactive amino acid side chains. The thiol group can be oxidized to several states, including the disulfide bond. Despite the known sensitivity of cysteine to oxidation and the physiological importance of the thiol group to protein structure and function, little information is available on the oxidative modification of cysteine residues in proteins because of the lack of reproducible and sensitive assays to measure cysteine oxidation in the proteome. We have developed a fluorescence-based assay that allows one to quantify both the global level of protein disulfides in the cellular proteome as well as the disulfide content of individual proteins. This fluorescence-based assay is able to detect an increase in global protein disulfide levels after oxidative stress in vitro or in vivo. Using this assay, we show that the global protein disulfide levels increase significantly with age in liver cytosolic proteins, and we identified 11 proteins that show a more than twofold increase in disulfide content with age. Thus, the fluorescence-based assay we have developed allows one to quantify changes in the oxidation of cysteine residues to disulfides in the proteome of a cell or tissue.

mTOR regulates chaperone networks and cognitive outcomes in mice modeling Alzheimer's disease

Background:Alzheimer’s disease (AD) is anage-dependent neurodegenerative condition that has in common with other neurodegenerations the accumulation and aggregation of misfolded proteins, which in the case of AD is represented by a wide range of aggregates ofamyloid-ß (Aß) peptides. The target of rapamycin (TOR) pathway is a majorsignaling hub that integrates nutrient/growth factor availability with cell metabolism. Reduced activity of the TOR pathway extends invertebrate lifespan, and, in mice, pharmacologic reduction of TOR signaling during adulthood extends life and strongly inhibits mTOR function in brain. We recently showed that systemic, longterm inhibition of mTOR by rapamycin lowers Aß levels and preserves learning and memory in transgenic mice modeling AD.Methods: To determine the mechanisms by which inhibition of mTOR modulates histopathological and cognitive outcomes in AD we performed proteomic and gene expression studies in brain tissues of control orrapamycin-treated PDAPP transgenic mice. Results: A large proportion of protein supregulated in rapamycin-treated transgenic brains weremembers of the chaperone family of proteins, of which many are induced by cellular stress. Heat shock proteins and chaperones promote proteostasis by assisting in the proper folding of proteins or in their targeting for degradation. The master transcriptional regulator of heat shock proteins is heat shock factor 1 (HSF1), which is negatively regulated by TOR. HSF1 is required for lifespan extension and conversely, its overexpression is sufficient to extend longevity in Celegans. Consistent with the observed up regulation of heat shock proteins in rapamycin-treated transgenic brains, we found that phosphorylation of HSF1 at Ser326, which is associated with increased transcriptional activity, was significantly elevated in rapamycin-treated brain tissues. In contrast, phosphorylation of HSF1 at Ser330, which inhibits transcriptional activity, was unchanged by rapamycin treatment. Examination of brain sections byimmunohistochemistry revealed that activated HSF1 was preferentially increased and was Tran located to the nucleus of granular cells in hippocampal dentate gyrus. Conclusions:Maintenance of proteostasis may be sufficient to prevent Aß accumulation and improve cognitive outcomes in mice modeling AD. We will report on the requirement for enhanced proteostasis for the reduction in Aß levels and the preservation of cognitive function in PDAPP transgenic animals producing high levels of amyloid.