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Reactive oxygen species (ROS), inevitable byproducts of aerobic metabolism, are known to cause oxidative damage to cells and molecules. This, in turn, is widely accepted as a pivotal determinant of both lifespan and health span. While studies in a wide range of species support the role of ROS in many age-related diseases, its role in aging per se is questioned. Comparative data from a wide range of endotherms offer equivocal support for this theory, with many exceptions and inconclusive findings as to whether or not oxidative stress is either a correlate or a determinant of maximum species lifespan. Available data do not support the premise that metabolic rate and in vivo ROS production are determinants of lifespan, or that superior antioxidant defense contributes to species longevity. Rather, published studies often show either a negative associate or lack of correlation with species longevity. Furthermore, many long-living species such as birds, bats and mole-rats exhibit high levels of oxidative damage even at young ages. Similarly genetic manipulations altering expression of key antioxidants do not necessarily show an impact on lifespan, even though oxidative damage levels may be affected. While it is possible that these multiple exceptions to straightforward predictions of the free radical theory of aging all reflect species-specific, “private” mechanisms of aging, the preponderance of contrary data nevertheless present a challenge to this august theory. Therefore, contrary to accepted dogma, the role of oxidative stress as a determinant of longevity is still open to question.

Fibroblasts from long-lived mutant mice are resistant to many forms of lethal injury as well as to the metabolic effects of rotenone and low-glucose medium. Here we evaluated fibroblasts from young adult naked mole-rats (NMR; Heterocephalus glaber), a rodent species in which maximal longevity exceeds 28 years. Compared to mouse cells, NMR cells were resistant to cadmium, methyl methanesulfonate, paraquat, heat, and low-glucose medium, consistent with the idea that cellular resistance to stress may contribute to disease resistance and longevity. Surprisingly, NMR cells were more sensitive than mouse cells to H(2)O(2), ultraviolet (UV) light, and rotenone. NMR cells, like cells from Snell dwarf mice, were more sensitive to tunicamycin and thapsigargin, which interfere with the function of the endoplasmic reticulum (ER stress). The sensitivity of both Snell dwarf and NMR cells to ER stress suggests that alterations in the unfolded protein response might modulate cell survival and aging rate.

Phospholipids containing highly polyunsaturated fatty acids are particularly prone to peroxidation and membrane composition may therefore influence longevity. Phospholipid molecules, in particular those containing docosahexaenoic acid (DHA), from the skeletal muscle, heart, liver and liver mitochondria were identified and quantified using mass-spectrometry shotgun lipidomics in two similar-sized rodents that show an approximately 9-fold difference in maximum lifespan. The naked mole rat is the longest-living rodent known with a maximum lifespan of >28 years. Total phospholipid distribution is similar in tissues of both species; DHA is only found in phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS), and DHA is relatively more concentrated in PE than PC. Naked mole-rats have fewer molecular species of both PC and PE than do mice. DHA-containing phospholipids represent 27-57% of all phospholipids in mice but only 2-6% in naked mole-rats. Furthermore, while mice have small amounts of di-polyunsaturated PC and PE, these are lacking in naked mole-rats. Vinyl ether-linked phospholipids (plasmalogens) are higher in naked mole-rat tissues than in mice. The lower level of DHA-containing phospholipids suggests a lower susceptibility to peroxidative damage in membranes of naked mole-rats compared to mice. Whereas the high level of plasmalogens might enhance membrane antioxidant protection in naked mole-rats compared to mice. Both characteristics possibly contribute to the exceptional longevity of naked mole-rats and may indicate a special role for peroxisomes in this extended longevity.

Maximum life span differences among animal species exceed life span variation achieved by experimental manipulation by orders of magnitude. The differences in the characteristic maximum life span of species was initially proposed to be due to variation in mass-specific rate of metabolism. This is called the rate-of-living theory of aging and lies at the base of the oxidative-stress theory of aging, currently the most generally accepted explanation of aging. However, the rate-of-living theory of aging while helpful is not completely adequate in explaining the maximum life span. Recently, it has been discovered that the fatty acid composition of cell membranes varies systematically between species, and this underlies the variation in their metabolic rate. When combined with the fact that 1) the products of lipid peroxidation are powerful reactive molecular species, and 2) that fatty acids differ dramatically in their susceptibility to peroxidation, membrane fatty acid composition provides a mechanistic explanation of the variation in maximum life span among animal species. When the connection between metabolic rate and life span was first proposed a century ago, it was not known that membrane composition varies between species. Many of the exceptions to the rate-of-living theory appear explicable when the particular membrane fatty acid composition is considered for each case. Here we review the links between metabolic rate and maximum life span of mammals and birds as well as the linking role of membrane fatty acid composition in determining the maximum life span. The more limited information for ectothermic animals and treatments that extend life span (e.g., caloric restriction) are also reviewed.

The naked mole rat (NMR; Heterocephalus glaber) is the longest-living rodent known [maximum lifespan potential (MLSP): >28 yr] and is a unique model of successful aging showing attenuated declines in most physiological function. This study addresses age-related changes in endothelial function and production of reactive oxygen species in NMR arteries and vessels of shorter-living Fischer 344 rats (MLSP: ∼3 yr). Rats exhibit a significant age-dependent decline in acetylcholine-induced responses in carotid arteries over a 2-yr age range. In contrast, over a 10-yr age range nitric oxide (NO)-mediated relaxation responses to acetylcholine and to the NO donor S-nitrosopencillamine (SNAP) were unaltered in NMRs. Cellular superoxide anion (O2•−) and H2O2 production significantly increased with age in rat arteries, whereas they did not change substantially with age in NMR vessels. Indicators of apoptotic cell death (DNA fragmentation rate, caspase 3/7 activity) were significantly enhanced (∼250–300%) in arteries of 2-yr-old rats. In contrast, vessels from 12-yr-old NMRs exhibited only a ∼50% increase in apoptotic cell death. In the hearts of NMRs (2 to 26 yr old), expression of endothelial NO synthase, antioxidant enzymes (Cu,Zn-SOD, Mn-SOD, catalase, and glutathione peroxidase), the NAD(P)H oxidase subunit gp91 phox, and mitochondrial proteins (COX-IV, ATP synthase, and porin, an indicator of mitochondrial mass) did not change significantly with age. Thus long-living NMRs can maintain a youthful vascular function and cellular oxidant-antioxidant phenotype relatively longer and are better protected against aging-induced oxidative stress than shorter-living rats.

Oxygen affinity and other hematological parameters in strictly subterranean mole-rats, Cryptomys hottentotus (subspecies pretoriae) were measured immediately upon capture and after 14-21 days in captivity. The pH, hematocrit, hemoglobin (Hb) concentration, blood oxygen content, 2,3 bisphosphoglycerate (2,3 BPG) concentration and oxygen dissociation curves (ODC), as well as tonometric measurements, were determined using whole blood. Additionally ODCs were also determined for stripped hemolysates of individual animals. Compared to other mammals, blood of freshly caught animals had low pH (7.32+/-0.22), elevated hematocrits (48.4+/-3.8 %) and significantly lower P50 values for whole blood (21.1+/-1.6 mm Hg at pH 7.4) than those reported for other similar-sized fossorial and terrestrial mammals. Blood carbon dioxide content (22.4+/-3.9 mMol L(-1)), hemoglobin concentration (1.9+/-0.15 mMol L(-1)), oxygen content (164.8+/-26 mL L(-1)), bicarbonate concentrations (22.5+/-3.5 mMol L(-1)) were within the range of values reported for similar-sized mammals. We conclude that high blood-oxygen affinity, low body temperature and possibly also high hematocrit enable C. h. pretoriae to maintain an adequate oxygen supply to the tissues in a potentially hypoxic burrow atmospheres, but that the blood of this species shows no exceptional CO2 sensitivity or buffering capacity.

Aging is the greatest risk factor for developing neurodegenerative diseases, which are associated with diminished neurotransmission as well as neuronal structure and function. However, several traits seemingly evolved to avert or delay age-related deterioration in the brain of the longest-lived rodent, the naked mole-rat (NMR). The NMR remarkably also exhibits negligible senescence, maintaining an extended healthspan for ~75 % of its life span. Using a proteomic approach, statistically significant changes with age in expression and/or phosphorylation levels of proteins associated with neurite outgrowth and neurotransmission were identified in the brain of the NMR and include: cofilin-1; collapsin response mediator protein 2; actin depolymerizing factor; spectrin alpha chain; septin-7; syntaxin-binding protein 1; synapsin-2 isoform IIB; and dynamin 1. We hypothesize that such changes may contribute to the extended lifespan and healthspan of the NMR.

Life‐history theory concerns the trade‐offs that mold the patterns of investment by animals between reproduction, growth, and survival. It is widely recognized that physiology plays a role in the mediation of life‐history trade‐offs, but the details remain obscure. As life‐history theory concerns aspects of investment in the soma that influence survival, understanding the physiological basis of life histories is related, but not identical, to understanding the process of aging. One idea from the field of aging that has gained considerable traction in the area of life histories is that life‐history trade‐offs may be mediated by free radical production and oxidative stress. We outline here developments in this field and summarize a number of important unresolved issues that may guide future research efforts. The issues are as follows. First, different tissues and macromolecular targets of oxidative stress respond differently during reproduction. The functional significance of these changes, however, remains uncertain. Consequently there is a need for studies that link oxidative stress measurements to functional outcomes, such as survival. Second, measurements of oxidative stress are often highly invasive or terminal. Terminal studies of oxidative stress in wild animals, where detailed life‐history information is available, cannot generally be performed without compromising the aims of the studies that generated the life‐history data. There is a need therefore for novel non‐invasive measurements of multi‐tissue oxidative stress. Third, laboratory studies provide unrivaled opportunities for experimental manipulation but may fail to expose the physiology underpinning life‐history effects, because of the benign laboratory environment. Fourth, the idea that oxidative stress might underlie life‐history trade‐offs does not make specific enough predictions that are amenable to testing. Moreover, there is a paucity of good alternative theoretical models on which contrasting predictions might be based. Fifth, there is an enormous diversity of life‐history variation to test the idea that oxidative stress may be a key mediator. So far we have only scratched the surface. Broadening the scope may reveal new strategies linked to the processes of oxidative damage and repair. Finally, understanding the trade‐offs in life histories and understanding the process of aging are related but not identical questions. Scientists inhabiting these two spheres of activity seldom collide, yet they have much to learn from each other.

Sympatric speciation (SS), i.e., speciation within a freely breeding population or in contiguous populations, was first proposed by Darwin [Darwin C (1859) On the Origins of Species by Means of Natural Selection] and is still controversial despite theoretical support [Gavrilets S (2004) Fitness Landscapes and the Origin of Species (MPB-41)] and mounting empirical evidence. Speciation of subterranean mammals generally, including the genus Spalax, was considered hitherto allopatric, whereby new species arise primarily through geographic isolation. Here we show in Spalax a case of genome-wide divergence analysis in mammals, demonstrating that SS in continuous populations, with gene flow, encompasses multiple widespread genomic adaptive complexes, associated with the sharply divergent ecologies. The two abutting soil populations of S. galili in northern Israel habituate the ancestral Senonian chalk population and abutting derivative Plio-Pleistocene basalt population. Population divergence originated ∼0.2-0.4 Mya based on both nuclear and mitochondrial genome analyses. Population structure analysis displayed two distinctly divergent clusters of chalk and basalt populations. Natural selection has acted on 300+ genes across the genome, diverging Spalax chalk and basalt soil populations. Gene ontology enrichment analysis highlights strong but differential soil population adaptive complexes: in basalt, sensory perception, musculature, metabolism, and energetics, and in chalk, nutrition and neurogenetics are outstanding. Population differentiation of chemoreceptor genes suggests intersoil population's mate and habitat choice substantiating SS. Importantly, distinctions in protein degradation may also contribute to SS. Natural selection and natural genetic engineering [Shapiro JA (2011) Evolution: A View From the 21st Century] overrule gene flow, evolving divergent ecological adaptive complexes. Sharp ecological divergences abound in nature; therefore, SS appears to be an important mode of speciation as first envisaged by Darwin [Darwin C (1859) On the Origins of Species by Means of Natural Selection].

The naked mole-rat (NMR) is the longest-lived rodent, with a maximum lifespan of >31 years. Unlike every other mammal studied to date, this species withstands cardiovascular structural and functional changes for at least 75% of its lifespan. Due to the intersection of oxidative stress, aging, and cardiovascular disease, we questioned if NMRs were more resistant to oxidative stress-induced cardiac dysfunction compared to short-lived mice. Echocardiography showed that 7 days after a 20 mg/kg dose of doxorubicin (DOX), mice had a 25% decline in fractional shortening (36 ± 1% to 27 ± 2%). In contrast, the fractional shortening of NMRs was unchanged with DOX treatment (27 ± 1%). Previously we observed that while basal cardiac function is low, NMRs have a robust cardiac reserve, displaying a 1.7 fold increase in fractional shortening under exercise-like conditions. DOX-treated NMRs had a significant reduction in their dobutamine response, signifying a diminished cardiac reserve. Intriguingly, we found no changes in phosphorylation or expression of myofilament proteins in the NMR heart with DOX treatment. Mice on the other hand, increased the phosphorylation of cardiac myosin binding protein-C and switched expression from predominantly α-myosin heavy chain to the β-isoform. Electron microscopy showed that DOX caused marked mitochondrial swelling and loss of cristae as well as massive cardiac myofibrillar disarray in mice. Conversely, DOX-treated NMRs had only slight alterations to myofilament structure. NMRs additionally had twofold higher levels of glutathione in their hearts, indicating a high antioxidant capacity. These findings reveal that long-lived NMRs are less susceptible to oxidative stress-induced cardiac dysfunction than mice. The NMR’s low basal cardiac function, unique regulation of myofilament proteins, and high glutathione levels may all be integral in the species’ ability to withstand oxidative damage and preserve cardiac function well into its third decade of life.

The ubiquitin-proteasome system (UPS) is responsible for the recycling of misfolded proteins. Dysfunction of the UPS has been implicated in the pathophysiology of multiple heart disorders, including heart failure and reperfusion injury, but the basic science of cardiac UPS function remains unclear. An attractive mode of inquiry into the cardiac proteasome is the long-lived naked mole rat (NMR), which maintains intact cardiac reserve and diastolic function exceptionally late into its lifespan; equivalent to a 90 year old human with a 30 year old’s heart. In this study, we investigated whether the long-lived and healthful NMR had upregulated aspects of UPS function in comparison to the short-lived well-characterized mouse. NMR hearts have more than twofold (p

Traditionally scientists have attempted to understand the biology of aging through engineering tractable systems, employing methods such as transgenesis and environmental or nutritional manipulation. An alternative approach relies on the phenomena of natural, extreme biology. Numerous examples exist of species that persist under conditions that promote pathology in humans and considerable insight has been garnered by uncovering the molecular mechanisms mediating this "evolutionary experimentation". Here we focus on a few well-studied naturally long-lived species to evaluate how natural selection has permitted an attenuated aging process without the physiological decline that plagues short lived species. Animals with exceptional endocrine and metabolic systems, as well as animals that tolerate oxidative stress are proposed as good models for studying the mechanisms of longevity. Cumulatively, this review will highlight some advantages and shortcomings of using a comparative approach to study aging.

The preternaturally long-lived naked mole-rat, like other long-lived species and experimental models of extended longevity, is resistant to both endogenous (e.g., reactive oxygen species) and environmental stressors and also resists age-related diseases such as cancer, cardiovascular disease, and neurodegeneration. The mechanisms behind the universal resilience of longer-lived organisms to stress, however, remain elusive. We hypothesize that this resilience is linked to the activity of a highly conserved transcription factor, nuclear factor erythroid 2-related factor (Nrf2). Nrf2 regulates the transcription of several hundred cytoprotective molecules, including antioxidants, detoxicants, and molecular chaperones (heat shock proteins). Nrf2 itself is tightly regulated by mechanisms that either promote its activity or increase its degradation. We used a comparative approach and examined Nrf2-signaling activity in naked mole-rats and nine other rodent species with varying maximum lifespan potential (MLSP). We found that constitutive Nrf2-signaling activity was positively correlated (P = 0.0285) with MLSP and that this activity was also manifested in high levels of downstream gene expression and activity. Surprisingly, we found that species longevity was not linked to the protein levels of Nrf2 itself, but rather showed a significant (P < 0.01) negative relationship with the regulators Kelch-like ECH-Associated Protein 1 (Keap1) and β-transducin repeat-containing protein (βTrCP), which target Nrf2 for degradation. These findings highlight the use of a comparative biology approach for the identification of evolved mechanisms that contribute to health span, aging, and longevity.

There is an increasing demand for exploration of the transcriptomes of multiple species with extraordinary traits such as the naked-mole rat (NMR). The NMR is remarkable because of its longevity and resistance to developing cancer. It is of scientific interest to understand the molecular mechanisms that impart these traits, and RNA-sequencing experiments with comparator species can correlate transcriptome dynamics with these phenotypes. Comparing transcriptome differences requires a homology mapping of each transcript in one species to transcript(s) within the other. Such mappings are necessary, especially if one species does not have well-annotated genome available. Current approaches for this type of analysis typically identify the best match for each transcript, but the best match analysis ignores the inherent risks of mismatch when there are multiple candidate transcripts with similar homology scores. We present a method that treats the set of homologs from a novel species as a cluster corresponding to a single gene in the reference species, and we compare the cluster-based approach to a conventional best-match analysis in both simulated data and a case study with NMR and mouse tissues. We demonstrate that the cluster-based approach has superior power to detect differential expression.

The lateral geniculate nucleus (LG) is an important subcortical nucleus in the visual system. It receives primary projections from the retina and relays these to central visual structures. Although there are studies on the retina and visual cortex of animals with regressed vision, little is known about the LG in such animals. The strictly subterranean naked mole-rat (Heterocephalus glaber) has markedly reduced visual acuity with concomitant pronounced changes in the visual cortex. We used a novel method to reveal myelinated neural fibers in a histological study assessing if the LG shows regressive changes commensurate with the level of reliance on vision by this rodent. Myelin detection here relies on significant differences in visible light reflection between neural fibers and the gray matter. Moreover, this simple method does not interfere with further staining for additional analyses. This method reveals that the contribution of the LG to brain volume in the naked mole-rat is less than a third of that of the rat. This shows that the retinogeniculocortical system in the naked mole-rat is considerably smaller than that of rodents that rely heavily on their visual system, but is nevertheless less regressed than that of the extensively studied blind mole-rat; this may facilitate limited responses to visual stimuli.

The naked mole-rat (NMR) is a mouse-sized rodent with a maximum longevity of >31 years. The species exhibits low basal heart rate (256 bpm) and cardiac output (7 ml/min) for its body size, as well as low fractional shortening (28%) for a rodent. However unlike other well-studied mammals, the NMR maintains cardiac reserve and diastolic function for at least 75% of its maximum lifespan - at ages equivalent to 90 year old humans. We questioned if this low basal cardiac function was due to NMR myofilament composition and function. NMR ventricles are comprised primarily of the β-myosin heavy chain isoform, which is associated with slowed myocardial contraction and increased efficiency. This is in stark contrast to mouse ventricles, which express predominately the α-isoform, and switch to the β-isoform upon experimental induction of heart failure. Compared to mice, NMR myofilament proteins such as cardiac troponin I and cardiac myosin binding protein-C display lower levels of phosphorylation. Such levels are indicative of decreased activation of myofilament proteins and may relate to the species’ low basal cardiac function. Both the NMR’s predominance of β-myosin heavy chain and the low basal level of myofilament phosphorylation present a phenotype much closer to that seen in human ventricles than in those of mice. Intriguingly, maximal force developed by skinned NMR cardiomyocytes is not significantly different to that of mouse cardiomyocytes (NMR: 70.9 ± 9.3mN/mm2 vs. mouse: 87.7 ± 0.6 mN/mm2). This is likely a reflection of the NMR’s ability to enhance cardiac function to the level of a mouse when stimulated, as is evident when both species are treated in vivo with dobutamine (3 μg/g i.p.). Such low basal cardiac function may put less overall strain on the heart over time and could be critical to the NMR’s ability to maintain cardiac function with age.

The maximum lifespan of naked mole-rats (NMRs; Heterocephalus glaber) is greater than that of any other rodent. These hystricognaths survive in captivity >28 years, eight-times longer than similar-sized mice. The present study tested if NMRs possess superior antioxidant defenses compared to mice and if age-related interspecies changes in antioxidants were evident. Activities of Cu/Zn superoxide dismutase (Cu/Zn, SOD), Mn SOD, catalase and cellular glutathione peroxidase (cGPx) were measured in livers of physiologically equivalent age-matched NMRs (30, 75 and 130 months) and CB6F1 mice (4, 12 and 18 months). In mice, Mn SOD activity increased with age, while the activity of catalase and cGPx declined. None of the antioxidants changed with age in mole-rats. cGPx activity of NMRs was 70-times lower (p < 0.0001) than in mice, and resembled that of cGPx knock-out animals. NMRs may partially compensate for the lower cGPx when compared to mice, by having moderately higher activities of the other antioxidants. It is nonetheless unlikely that antioxidant defenses are responsible for the eight-fold longevity difference between these two species. Maintenance of constant antioxidant defenses with age in NMRs concurs with previous physiological data, suggesting delayed aging in this species.

Genome-wide analysis demonstrates extensive genomic adaptive complexes involved in sympatric speciation between blind mole rats (Spalax galili) in abutting populations living in basalt and chalk soils. Among the gene ontology (GO) enrichment, musculature and metabolism stood out in basalt dwellers while nutrition and neurogenetics were highlighted in chalk residents. Measurements of mechanisms regulating protein homeostasis inspired by these GO terms suggest that at the proteomic level there is also a habitat/soil-type driven divergence with the basalt residents exhibiting higher proteasome activity whereas elevated levels of markers of autophagy are evident in the chalk inhabitants.

Aging is characterized by declines in all physiological processes and concomitant changes in body composition. Age-related changes in metabolism, body composition and gastrointestinal function were investigated in naked mole-rats (Heterocephalus glaber), rodents that exhibit extended longevity. Maximum lifespan of these 40 g rodents (>27 year) is approximately 9 times greater than predicted allometrically. We investigated changes in basal metabolic rate (BMR), body composition and intestinal glucose transport in 1, 5, 10 and 20-year-old male individuals. Body composition was measured using dual X-ray absorptiometry and activity of sodium glucose co-transporters (SGLT1) determined using everted gut sleeves. One-year-olds had lower body mass than other age cohorts, as they had not attained full adult form. Among the 5, 10, and 20-year-olds, no age-related changes in body mass, BMR, percentage body fat, fat-free mass or bone mineral density were found. SGLT1 activity declined moderately (

Mammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages.

Tau protein is primarily expressed in neuronal axons and modulates microtubule stability. Tau phosphorylation, aggregation, and subcellular mislocalization coincide with neurodegeneration in numerous diseases, including Alzheimer's disease (AD). During AD pathogenesis, tau misprocessing accompanies As accumulation; however, AD animal models, despite elevated As, fail to develop tauopathy. To assess whether lack of tau pathology is linked to short life span common to most AD models, we examined tau processing in extraordinarily long-lived, mouse-sized naked mole-rats (NMRs; approximately 32 years), which express appreciable levels of As throughout life. We found that NMRs, like other mammals, display highest tau phosphorylation during brain development. Although tau phosphorylation decreases with aging, unexpectedly adult NMRs have higher levels than transgenic mice overexpressing mutant human tau. However, in sharp contrast with the somatodendritic accumulation of misprocessed tau in the transgenic mice, NMRs maintain axonal tau localization. Intriguingly, the adult NMR tau protein is 88 kDa, much larger than 45-68 kDa tau expressed in other mammals. We propose that this 88 kDa tau protein may offer exceptional microtubule stability and neuroprotection against lifelong, elevated As.

It has been 20 years since the Orentreich Foundation for the Advancement of Science, under the leadership Dr. Norman Orentreich, first reported that low methionine (Met) ingestion by rats extends lifespan (Orentreich et al., 1993). Since then, several studies have replicated the effects of dietary methionine restricted (MR) in delaying age-related diseases (Richie et al., 1994; Miller et al., 2005; Ables et al., 2012; Sanchez-Roman and Barja, 2013). We report the abstracts from the First International Mini-Symposium on Methionine Restriction and Lifespan held in Tarrytown, NY, September 2013. The goals were (1) to gather researchers with an interest in MR and lifespan, (2) to exchange knowledge, (3) to generate ideas for future investigations, and (4) to strengthen relationships within this community. The presentations highlighted the importance of research on cysteine, growth hormone (GH), and ATF4 in the paradigm of aging. In addition, the effects of dietary restriction or MR in the kidneys, liver, bones, and the adipose tissue were discussed. The symposium also emphasized the value of other species, e.g., the naked mole rat, Brandt's bat, and Drosophila, in aging research. Overall, the symposium consolidated scientists with similar research interests and provided opportunities to conduct future collaborative studies (Figure 3).

The extraordinarily long-lived mouse-sized naked mole-rat (NMR) shows an attenuated age-related decline in diastolic function compared to mice and humans, despite having a maximum lifespan of >31 years. Using echocardiography, we have examined the left ventricles (LVs) of young (2-5 years), middle-aged (8-10 years) and old (16-22 years) NMRs (n=10/cohort). Young NMRs displayed low LV contractility, as measured by fractional shortening (~28%), which was not significantly changed with age. LV wall thicknesses were significantly greater in young NMRs when compared with physiologically age-matched mice, and NMR LV wall thickness did not decline with age as has been reported for mice. Dobutamine stress echocardiography (3 μg/g body weight i.p.) elicited similar percent changes in fractional shortening, ejection fraction, and cardiac output in each cohort, indicating that cardiac reserve in the NMR is maintained with age. This is in contrast to reports to age-related declines in cardiac reserve in both mice and...

Proteostasis is an integral component of healthy aging. In most metazoans, protein quality declines during aging, resulting in accrual of damaged or self-aggregating cytotoxic proteins, linked to several age-associated diseases (e.g, Alzheimer’s Disease, Parkinson’s Disease) and pathology (e.g.,sarcopenia). The mouse-sized naked mole-rat [NMRs] lives ~5 times longer than expected based on body size, and despite detected high levels of oxidative damage even at a young age, maintain good health for most of their long lives. Like other long-lived animal models, both in vivo and in vitro studies reveal that NMRs are resistant to a broad spectrum of environmental stressors. Collectively these findings suggest that NMRs possess efficient mechanisms to maintain protein quality. Our research has previously shown that this is attributed in part to altered proteasome forms and subcellular location. However, changes in proteasome-related molecular chaperone activity that assists in the transport of damaged proteins ...

Oxidative damage to terminally differentiated neurons is regarded as a leading cause of Alzheimer’s disease (AD; Bonda et al., 2010). AD is characterized by the progressive loss of synapses and/or neurons and worsening deficits to cognitive function. Oxidative stress may exacerbate accumulation of oxidative damage by downregulating antioxidants and degradation processes. In AD, these altered functions all contribute to the accumulation of Aβ (for review see Bonda et al., 2010; Riederer et al., 2011). A decline in hippocampal volume is considered a reliable predictor for mild and moderate dementia. Oxidative damage in the hippocampus is evident in the preclinical (PCAD) stages of AD (Lovell et al., 2011). Indeed, the hippocampus is commonly regarded as the initiation site of neuronal loss or damage in AD, spreading to the cortex and eventually affecting the entire brain (Gosche et al., 2002). In contrast, the cerebellum appears relatively impervious to these forms of insults until the late stages of the disease (Rapoport et al., 2000). It is not known whether the human hippocampus is more protected against oxidative damage or more pro-oxidative and/or has poorer antioxidant defenses than other regions, thus contributing to its increased vulnerability. Nevertheless, antioxidants are routinely proposed as a therapeutic approach to combat the pathological changes in AD (Joseph et al., 2003; Pocernich et al., 2011). Amyloid-β (Aβ) is formed from a sequential cleavage of amyloid precursor protein (APP), a ubiquitous membrane protein thought to be involved in synapse formation and neural plasticity (Priller et al., 2006). Accumulation of Aβ can be caused by overproduction as a result of increased activity of the β- and γ-secretases, such as in the familial form of AD (for review see Gotz et al., 2011), or by impairments to mechanisms for its removal, as was recently shown in a human study (Mawuenyega et al., 2010). Aβ degradation is orchestrated by various mechanisms including insulin-degrading enzyme (IDE), autophagy, and the ubiquitin-proteasome pathway (UPS; Kurochkin and Goto, 1994; Oddo, 2008; Caccamo et al., 2010). Manipulating these levels in AD mouse models resulted in a correlation with Aβ levels in either direction, depending on the modification (Farris et al., 2003; Miller et al., 2003; Leissring et al., 2003; Spilman et al., 2010; Majumder et al., 2011; Medina et al., 2011). Long-lived naked mole rats (Heterocephalus glaber; NMRs), show high levels of oxidative damage to visceral tissues even at a young age (Andziak and Buffenstein, 2006; Andziak et al., 2006). Surprisingly, this does not appear to affect their aging negatively. NMRs are not only able to live an additional 20 years with these high levels of oxidative damage but also show delayed and attenuated age-related declines in physiological function, molecular markers, and behavior for at least 75% of their 32-year maximal life span (Buffenstein, 2008). Surprisingly, Aβ levels were found to be high in young (2–9 years) NMR brain, and similar to those shown in 3xTg-AD mice (Edrey et al., 2013). Nevertheless, these young NMRs have the potential to live an additional ~20–30 years in captivity and maintain seemingly normal cognitive functions. NMRs are eusocial subterranean mammals and live in large colonies. They rely on spatial orientation to navigate through a maze of underground burrows and exhibit complex social interactions and conspecific communication. In humans (and other mammals), many of these functions are known to be regulated by the hippocampus, a brain region considered the epicenter of AD. The unexpected observation of high levels of Aβ in young NMRs led us to ask two questions: 1) Are these levels dispersed evenly across the brain? 2) are these high levels the result of increased production of Aβ or lower efficiency in degradation? We hypothesized that, in young NMRs, the hippocampus would be spared from high levels of Aβ because of either low production or increased degradation. We therefore compared Aβ metabolism in cortex, hippocampus, and cerebellum of young NMRs and investigated a possible relationship among Aβ distribution, levels of heat shock proteins (Hsps), and redox status in these brain regions.

This study documents growth and dual-energy X-ray absorptiometry (DXA)-determined peak bone mass profiles in the rabbit model, and tests the hypothesis that rabbits show patterns of bone accretion similar to humans and thus may serve as a viable model for human bone physiology. It is also shown that dietary Ca intake affects peak bone mass and the temporal pattern of its attainment. Groups of weaned animals were administered two nutritionally complete but calcium-altered diets (0.5% or 1.0% Ca). We evaluated growth, bone mass accretion, and Ca metabolism from 20 to 56 weeks of age in both the 1.0% Ca and 0.5% Ca groups of rabbits. For each monthly period, we monitored body mass, naso-tail length, food consumption, and fecal output. In addition, we collected blood and 24 h urine samples for biochemical analyses, and measured bone mass variables of the lumbar spine with DXA. The 1.0% Ca group had a lower apparent fractional absorption of Ca and higher urinary Ca excretion, but retained more Ca than the 0.5% Ca group during the growth phase. Furthermore, the 1.0% Ca group had lower parathyroid hormone (PTH) and bone biochemical marker concentrations throughout the study than the 0.5% Ca group. The lower levels of PTH and bone markers of resorption and formation, may have resulted in a reduction in skeletal remodeling, and this physiological mechanism may have contributed to the 10% increase in peak bone mineral density of the lumbar spine in the 1.0% Ca group of rabbits.

Eastern grey kangaroos (Macropus giganteus) are generally regarded as mesic inhabitants. Even though access to drinking water in permanent stock watering troughs is commonly available, these animals are still found in only low densities in arid pastoral areas. We hypothesised that the differential success of red and grey kangaroos in the arid zone may be due to higher energy requirements of M. giganteus with a concomitant need for increased food, rather than limitations imposed by inadequate water access. We set out to test this by indirectly measuring energy expenditure through the monitoring of heart rate by radio telemetry in semi-free-ranging eastern grey and red kangaroos (Macropus rufus). Radio telemetry measurements of heart rate were calibrated against oxygen consumption and were used in the assessment of energy expenditure of animals maintained in an 8-ha enclosure in the arid zone of southeast Australia. Heart rate provided a reliable estimate of oxygen consumption. This well-correlated relationship was curvilinear and was established for each individual. Behavioural observations revealed that both kangaroo species spent most of the day in low energy demanding activities. M. rufus were more active at night whilst M. giganteus were more active in the early mornings and late afternoons. Like other marsupials, both species had low field metabolic rates (FMRs). However, M. giganteus in keeping with their mesic history had higher FMRs than the more arid-adapted M. rufus, particularly during water restriction. Body temperature telemeters revealed a further species difference in that under hot conditions when water is freely available, M. rufus exhibits a higher and more labile daytime body temperature than M. giganteus. During the hottest part of the day M. giganteus maintain body temperature, relying upon increased evaporative cooling mechanisms, such as licking. Indeed, only when access to drinking water was restricted was thermolability evident in M. giganteus. Differences in behaviour and concomitant energy expenditure may thus contribute substantially to the divergent distribution and abundance of these two kangaroo species.

The lungs of the naked mole-rat Heterocephalus glaber were morphologically (qualitatively) and morphometrically (quantitatively) investigated in adult captive in-burrow body-temperature-maintained (CBTMA) and captive cold-exposed animals (CCEA). The observations were compared with those of field (burrow-dwelling) animals (FA) examined in an earlier study. The lungs of the captive animals were morphologically (qualitatively) similar to those of the field mole-rats. Conspicuous paedomorphic features, i.e. a preponderant double pulmonary capillary arrangement, incomplete differentiation of alveolar pneumocytes, and extension of a cuboidal epithelium to the immediate vicinity of the alveoli, were observed. The prevalent pulmonary underdevelopment was attributed to the relatively low metabolic rate of the poikilothermic naked mole-rat, a species that has evolved in a thermally stable environment. Measurable differences and similarities were found between the lungs of the captive naked mole-rats themselves (i.e. the CBTMA and the CCEA) and between the captive and the field ones. Genetic factors may help explain the conserved pulmonary structural features while environmental (gaseous) change from a hypoxic–hypercapneic (an oxygen deficient–high carbon dioxide concentration) to a normoxic–normocapnic atmosphere and the thermal shift respectively may have enforced pulmonary transformations between the CBTMA and the FA and between the CCEA and the CBTMA. Paradoxically, the CCEA had a significantly lower mass-specific total morphometric pulmonary diffusing capacity of 0.024 compared with that of 0.102 mlO2.s−1.mbar−1.kg−1 in the CBTMA. The observed morphometric differences indicate that the lung of the naked mole-rat, though morphologically unchanged, is intrinsically tractable to environmental shifts in its habitat. Studies on captive naked mole-rats should not be indiscriminately taken to be representative of the species.

The mean corpuscular fragility (MCF) of erythrocytes may reflect phylogenetic characteristics as well as an animal's ability to respond to the osmotic challenges associated with cyclic dehydration and rehydration. This type of ecophysiological stress is commonly encountered by animals living in arid regions and low MCF may contribute to their ability to survive and thrive in these xeric habitats. The eastern grey kangaroo has only in recent times extended its range into the arid zone, and is considered a more mesic inhabitant than the red kangaroo. We therefore compared the ability of eastern grey kangaroos and red kangaroos to handle prolonged periods of water restriction, as well as the MCF of the erythrocytes of free-ranging red, eastern grey and western grey kangaroos found at the Fowlers Gap field station. In addition, the MCF of free-ranging sheep inhabiting the same pastures were used as an experimental control; they are phylogenetically unrelated yet are subjected to the same acclimatisation stresses. While red kangaroos exhibited greater tolerance of dehydration compared to eastern grey kangaroos, the MCF of all three kangaroo species was similar and more resilient to osmotic stresses (MCF, 130 mosmol/kg) than erythrocytes of sheep (MCF, 220 mosmol/kg). The MCF did not change with water restriction, however, the erythrocytes of long-term captive populations fed a comparatively better quality diet were more resistant to osmotic shock than the free-ranging animals. Phylogenetic commonality rather than ecophysiological responses to life in the arid zone appeared to influence MCF. The MCF values of sheep corresponded to that of other ovines; similarly the MCF of kangaroos concurred regardless of their preferred habitats. ecological history and differential success in the arid zone.

High sugar consumption and diabetes increase the risk of developing Alzheimer's disease (AD) by unknown mechanisms. Using an animal model of AD, here we show that high sucrose intake induces obesity with changes in central and peripheral insulin signaling. These pre-diabetic changes are associated with an increase in amyloid-β production and deposition. Moreover, high sucrose ingestion exacerbates tau phosphorylation by increasing Cdk5 activity. Mechanistically, the sucrose-mediated increase in AD-like pathology results from hyperactive mammalian target of rapamycin (mTOR), a key nutrient sensor important in regulating energy homeostasis. Specifically, we show that rapamycin, an mTOR inhibitor, prevents the detrimental effects of sucrose in the brain without altering changes in peripheral insulin resistance. Overall, our data suggest that high sucrose intake and dysregulated insulin signaling, which are known to contribute to the occurrence of diabetes, increase the risk of developing AD by upregulating brain mTOR signaling. Therefore, early interventions to modulate mTOR activity in individuals at high risk of developing diabetes may decrease their AD susceptibility.

Motor neurons form a specialized synapse with skeletal muscle known as the neuromuscular junction (NMJ), and degeneration of the NMJ has been implicated in disease and aging. Histone deacetylases mediate NMJ-regulated gene transcription and are involved in neurogenic muscle atrophy, although their role in age-related muscle atrophy is not known. HDAC4 and HDAC5 knockout mice are protected against surgical denervation, and pharmacological inhibition of histone deacetylases is protective in multiple models of neuromuscular disease. In this study, we examined the effect of butyrate, a histone deacetylase inhibitor, on muscle atrophy during sciatic nerve crush and age-related muscle atrophy. We demonstrate that butyrate increases histone acetylation in vivo and protects against the muscle loss induced by sciatic nerve crush and aging. Control-fed mice lost 22% of their gastrocnemius mass while the butyrate-fed mice lost only 11% one week after sciatic nerve crush surgery. Butyrate protects against the loss of cross-sectional area, increases catalase and MnSOD activity, and reduces oxidative damage during nerve crush. Consistently, butyrate protects against age-related muscle atrophy in mice by modulating antioxidant activity, reducing oxidative damage, and increasing mitochondrial biogenesis. We also report improved metabolism in old mice fed butyrate, including improved glucose tolerance and increased whole-body oxygen consumption. Future studies will determine the mechanism by which butyrate protects against age-related muscle atrophy., Synucleinopathies, including Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies, are age-related neurodegenerative disorders characterized by pathological a-synuclein inclusions. Synucleinopathies have common motor deficits and affect millions of patients worldwide. The A53T human a-synuclein mutation is linked to both sporadic and familial PD. Rapamycin, an mTOR inhibitor, reduces human a-synuclein accumulation and neurodegenerative phenotype in vitro and in vivo. Long-term feeding of a rapamycin diet extends mouse lifespan and the mechanisms are hypothesized to be mediated via delaying age-related diseases including PD. The aim of the study is to determine whether long-term feeding rapamycin diet at the dose that extends mouse lifespan attenuates motor deficits in neuronal A53T a-synuclein transgenic mice. A diet containing microencapsulated rapamycin (14 ppm in diet; 2.25 mg/kg body weight/day) or the microencapsulation polymer was fed to age-matched wild-type and A53T mice from 13 weeks of age. After 24 weeks of treatment, rapamycin improved performance on forepaw stepping adjustment test, accelerating rotarod test and pole test in both genders. Rapamycin also increased front stride length in male A53T mice. Total human a-synuclein levels in midbrain, striatum, brain stem, cerebellum, and spinal cord were not altered by rapamycin. Oxidative stress plays an important role in pathogenesis of neurodegenerative diseases. The lipid peroxidation product 4-hydroxynonenal has been detected in the postmortem brain of PD patients and is believed to contribute to the neurotoxic effects of a-synuclein. Levels of 4-hydroxynonenal protein adducts were significantly decreased in midbrain, striatum, and spinal cord of both genders of A53T mice as well as brain stem and cerebellum of female A53T mice. In conclusion, long-term rapamycin treatment attenuated motor deficits and reduced 4-hydroxynonenal in the central nervous system regions that subserve motor function and motor coordination in the A53T mice., The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signal transduction cascade called the unfolded protein response (UPR). The most immediate response is the attenuation of protein synthesis, initiated by autophosphorylation of PKR-like ER kinase (PERK). Recently, our group reported that calcineurin (CN) strengthened the UPR by binding to PERK and enhancing its autophosphorylation (Bollo et al. PLoSOne 5 (8): e11925). Here, we report that CN-A beta protects astrocytes from ER stress, likely by enhancing autophosphorylation of PERK. First, we found that levels of phosphorylated-PERK and CN-A beta were significantly increased in astrocytes within 1 hour of oxygen and glucose deprivation (OGD). Second, overexpression of CN-A beta significantly increased the viability of wild-type astrocytes during OGD (1 hr), but not that of PERK−/ − astrocytes. Third, co-immunoprecipitation showed that CN-A beta preferentially interacted with PERK in ER-stressed astrocytes. Fourth, experiments with recombinant proteins demonstrated that PERK autophosphorylation and oligomerization were increased in the presence of CN-A beta. Finally, rapamycin-induced dimerization of CFP-FRB-cytochrome5 (ER anchor) and YFP-FKBP-CN-A beta inside human astrocytes increased PERK phosphorylation. Taken together, we suggest a novel physiological function of the classic phosphatase CN-A beta is to bind PERK and enhance the early UPR., The subventricular zone (SVZ) is the largest reservoir of neural stem cells (NSCs) in the adult brain. Neurogenesis is reduced during aging and this decline in NSC function is thought to contribute to reduced brain function. Here, we propose immune activation and cellular senescence as contributors to age-related declines in NSC function. We observed a sharp decrease in neurogenesis in mice between the ages of 6 and 12 months. Neuroblast number was also reduced with age. We observed the appearance of β-galactosidase-positive cells, a marker of cellular senescence concomitant with reductions in neurogenesis. Senescent cells secrete factors into the extracellular environment, a phenomenon known as the senescence associated secretory phenotype (SASP). The SASP has been associated with upregulated inflammation and immune activation due to proinflammatory factors. Microglia are the prominent immune cells in the brain. Microglial activation results in the release of proinflammatory cytokines. There was no change in the number or percent of microglia cells relative to other cells in the SVZ during aging. However, both morphological changes and increased CD68 expression were observed during aging indicative of microglia becoming more activated. Interestingly, a short-term diet of encapsulated rapamycin, an inhibitor of the mTOR pathway, resulted in reduced cellular senescence in the aged brain compared to the young and an overall increase in NSC proliferation. Our results suggest that during aging there is increased inflammation in the SVZ, which may be an important contributor to declines in neural stem cell function., Increased mitochondrial reactive oxygen species (ROS) is believed to play a key role in cognitive decline associated with aging and Alzheimer's disease, but the mechanism remains unclear. Inflammasomes are multiprotein oligomers that recognize damage-associated molecular patterns (DAMPs) to activate caspase-1, thereby allowing for cleavage and subsequent release of proinflammatory cytokines IL-1β and IL-18. Among the inflammasomes, the NLRP3 inflammasome plays a key role in sensing oxidative stress such as increased mitochondrial ROS. We are interested in studying the role of mitochondrial ROS in cognitive decline associated with aging and Alzheimer's disease using mouse models exposed to paraquat, a mitochondrial ROS inducer. Our results indicate that exposure to paraquat results in exacerbated cognitive decline in both WT mice and APP/PS1 mice. To determine whether paraquat may regulate expression of the NLRP3 inflammasome to impair cognition, we measured the relative mRNA levels of ASC and Nlrp3, two major components of the NLRP3 inflammasome, in brains from paraquat-exposed animals by qPCR. We found that paraquat-exposed WT mice had higher levels of both ASC and Nlrp3 mRNA than control WT mice. Paraquat-exposed APP/PS1 mice also had increased levels of ASC and Nlrp3 mRNA compared with control APP/PS1 mice. Our results further showed that paraquat treatment increased ASC and Nlrp3 mRNA levels in primary astrocytes. Thus, our data suggest that increased expression of NLRP3 inflammasome may play an important role in mediating cognitive decline induced by mitochondrial ROS., The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and metabolism. mTOR assembles with Raptor or Rictor to form mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively. Reduced activity of the TOR pathway extends invertebrate lifespan, and, in mice, pharmacologic reduction of mTOR signaling during adulthood, as well as germline reduction of mTOR or S6K1 extending lifespan. In other models of extended longevity, selective reduction of function in the nervous system is sufficient to extend lifespan. The role of mTORC1 signaling from the nervous system in the control of lifespan and metabolism in mammals, however, has not yet been determined. Because neuronal-specific Raptor null mice die perinatally, to explore this question we generated mice expressing a tamoxifen-inducible Cre recombinase in neurons in combination with individual homozygous floxed alleles of genes in the mTORC1 pathway (mTORfl/fl, Raptorfl/fl, and S6K1fl/fl). Cre recombinase was partially active without tamoxifen stimulation, resulting in the genetic ablation of 20–40% of the target floxed alleles during development. A greater than 20% neuronal knockdown of any of the target genes in the mTORC1 pathway reduced fat content in both sexes. Knockdown of mTOR or Raptor in neurons also significantly reduced body size as well as lower fasting and resting blood glucose levels. These data agree with previously reported data for the germline knockout of S6K1, which also exhibited reduced fat mass, but unlike the germline S6K1−/ − animals, body weight in neuronal-specific S6K1 knockdown mice was unchanged. Only neuronal knockdown of mTOR increased oxygen consumption. Lower glucose and increased lactate in blood were observed in WT and neuronal S6K1 knockdown mice, but not in neuronal mTOR and Raptor knockdown mice challenged with treadmill exercise, indicating altered muscle metabolism as a consequence of neuronal reduction of mTOR or Raptor. Remarkably, S6K1 knockout mice ran farther on the treadmill than WT mice before they reached exhaustion. In contrast, neuronal knockdown of mTOR and Raptor resulted in decreased exercise capacity. Survival to weaning age was reduced but adult mortality was unchanged for Raptor or S6K1 neuronal knockdown mice. Conversely, neuronal mTOR knockdown mice were weaned at the expected Mendelian rates but were short lived, with less than 10% surviving beyond 300 days., Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the degeneration of nigral dopaminergic neurons leading to motor dysfunction. Several lines of evidence implicate mitochondrial dysfunction, oxidative stress, and protein aggregation in the pathology of PD. Recent evidence, including evidence from our laboratory, suggests that shifts in dopamine and/or aldehyde metabolism lead to aldehyde accumulation, which may contribute to parkinsonism. Aldehyde cytotoxicity may be a result of their long half-life and their ability to cross cell membranes both locally and distant. Aldehyde dehydrogenase (Aldh) is the primary pathway for the detoxification of aldehydes. Mice with mutations for two midbrain isoforms of Aldh, cytosolic Aldh1a1 and mitochondrial Aldh2, exhibit age-dependent neurochemical and behavioral deficits, and respond to levodopa treatment. Aldehyde scavengers such as N-benzylhydroxylamine and hydralazine have been shown to provide cytoprotective effects both in vitro and in vivo. Thus, the detoxification or removal of aldehydes could prove to be effective in PD therapy. The goal of this study is to investigate the protective properties of the FDA-approved anti-hypertensive drug as an aldehyde trapping agent to suppress the parkinsonian phenotype observed in Aldh1a1−/ − X Aldh2−/ − mice. We found that chronic oral administration of hydralazine in drinking water to Aldh1a1−/ − X Aldh2−/ − mice ameliorated both behavioral and neurochemical deficits. In continuance of this investigation, primary cultures isolated from the mesencephalon of Aldh1a1 and/or Aldh2 deficient mice will be subjected to Complex I inhibitors, modeling environmental exposure in PD. Aldehyde load and neuronal death will be assessed in the absence and presence of hydralazine treatment., Alzheimer's disease (AD) is the most prevalent neurodegenerative disease. The majority of AD cases develop sporadically with unknown etiology. Over the past decade, a strong link between type 2 diabetes (T2D) and AD has been established; however, the mechanisms responsible for this relationship remain unknown. The mammalian target of rapamycin (mTOR) plays a key role in maintaining protein homeostasis, regulating energy metabolism, and diabetic insulin resistance. We hypothesized that mTOR may serve as a mechanistic link between AD and T2D. To explore this potential association, we supplemented the drinking water of 3xTg-AD mice, a transgenic mouse model of AD, with 20% sucrose. A second cohort of mice was given sucrose in conjunction with an mTOR inhibitor, rapamycin. All were compared to age and genotype-matched mice on control diet. We found that the sucrose treatment significantly increased fat mass, impaired glucose tolerance, and altered urine concentration all of which are consistent with physiological changes that occur in T2D. Additionally, sucrose exacerbated AD-like cognitive impairment and Aß and tau brain pathology. Impressively, rapamycin mitigated the sucrose-induced increase in pathology and cognitive impairment. Our results provide compelling in vivo evidence that diabetes-associated AD progression occurs through an mTOR-related mechanism. Furthermore, our results indicate that treating diabetic patients with mTOR modulators may decrease their risk of developing AD., To investigate the effects of pigment epithelium-derived factor (PEDF) peptides on muscle regeneration, a rat myonecrosis model of a single injection of bupivacaine into the soleus muscle was employed. PEDF peptides were delivered by bolus injection of alginate-based sustained release regiment. The muscle fiber proliferation was assayed by the incorporation of BrdU in the proliferating nuclei. The soleus muscle specimens were also stained for satellite cell marker, Pax7, so as to investigate the muscle regeneration activity. Results suggested that the administration enhances the proliferative activities of muscle fibers and/or satellite cells, which in turn may promote the muscle to regenerate. There were higher percentages of muscle fibers containing centrally located nuclei in animals treated with the PEDF peptides than with vehicle. Moreover, on average, diameters of muscle fibers from animals treated with PEDF peptides were larger than those from animals in the blank or bolus control group. These indicated that PEDF peptides stimulate both the proliferation and maturation of muscle stem cells. Gene expression profiling and inhibitor assay on C2C12 cells revealed that the stimulation on muscle progenitor cells by PEDF is mediated by PI3KAKT and mitogen-activated protein kinases (MAPK) p38., Nuclear receptor interaction protein (NRIP) is a calcium-dependent calmodulin (CaM) binding protein (Ca+2/CaM). To investigate the functional role of NRIP in skeletal muscle, we first generated conventional NRIP knock out mice. Here, we demonstrate that NRIP-deficient mice lose muscle strength from the assays of in vitro muscle contraction test with neuromuscular blocker to rule out the nerve effect on muscle contraction; plus NRIP-deficient mice show the susceptibility to fatigue during repetitive contraction. Additionally, the exercise performances using rotarod and treadmill tests, NRIP-deficient mice are shown to be weaker than wild-type (WT) mice. We then investigated the mechanisms of NRIP involved in muscle strength and contraction; the results illustrate that NRIP regulates striated muscle function at least via calcineurin phosphatase dephosphorylating NFATc1 and CaMKII phosphorylation to increase slow myosin gene expression and the influx of internal stored Ca+2 from sarcoplasmic reticulum by measuring caffeine-induced maximal muscle force. To further investigate the NRIP role in regeneration potential, we then subjected NRIP-deficient and WT mice to unilateral cardiotoxin injection into the tibialis anterior. NRIP-deficient mice show lower centralized nuclei, a lower cell-surface recovery, a decreased area of degenerated tissues, and heterogenous myofiber size compared to weight. In conclusion, NRIP regulates skeletal muscle strength via calcineurin-NFATc1 and CaMKII phosphorylation to regulate muscle excitation/contraction; plus it also has function for regeneration capacity from injured muscles., As life expectancy rises in developed countries, sarcopenia and dynapenia pose significant threats for the aged, both physically and economically. Current research methods used to study age-related muscle wasting may include limitations, including model organisms that exhibit muscle growth characterized by a pre-defined growth plateau. The lack of model organisms for sarcopenia which exhibit continual growth potential with age has possibly limited advances in understanding this condition for two reasons: (1) the presence of a growth asymptote prevents the discovery of novel genes and/or regulatory pathways involved in the continuation of muscle fiber recruitment throughout adulthood; and (2) the inherent growth plateau may lead to sarcopenia itself, as sustained muscle growth throughout adulthood predicts the presence of myogenic precursor cells resistant to aging. Therefore, we propose a unique muscle growth model for the study of sarcopenia that takes advantage of natural continued muscle fiber recruitment. Evidence from a comparative approach utilizing the subfamily Danioninae suggests that the indeterminate growth paradigm of many teleosts arises from adult muscle stem cells with greater proliferative capacity, even in spite of smaller progenitor populations. We hypothesize that paired-box transcription factors, Pax3/7, are involved with this enhanced self-renewal and that prolonged expression of these factors may allow some fish species to escape, or at least forestall, sarcopenia/dynapenia. Future research efforts should focus on the experimental validation of these genes as key factors in indeterminate growth, both in the context of muscle stem cell proliferation and in the prevention of skeletal muscle senescence., Sarcopenia, the severe loss of skeletal muscle mass and function, is a serious health problem affecting aged individuals. Sarcopenic individuals are weak and frail, which predisposes them to disability and death. The mechanisms responsible for muscle wasting during aging are poorly understood. Muscle maintenance depends primarily on muscle stem cells (satellite cells) that exit quiescence during regeneration and replicate to generate daughter cells that either differentiates to maintain muscle or return to quiescence for self-renewal. Controversy surrounds whether the satellite cell pool is heterogeneous in regards to self-renewal, replication or differentiation. Furthermore, during aging it is unclear whether there is a decline in satellite cell number, function or whether cell fate is altered for a subpopulation of cells. To examine satellite cell heterogeneity in vivo, we developed a novel multicolor fluorescent lineage system for studying satellite cell clonal expansion in young and aged mouse skeletal muscle. Our system involves an inducible Pax7-Cre driver to express an avian retroviral receptor specifically in satellite cells, permitting retroviral-mediated gene expression of one of three distinct fluorescent proteins. This highly flexible system has advantages over current multicolor recombination-based systems as the timing of receptor expression and infection can be controlled. This improved multicolor lineage system will permit examination of satellite cell heterogeneity via detection of bias in fluorescence in daughter cells. Characterizing satellite cell behavior in vivo will permit us to examine possible defects in cell fates and alterations in subpopulations that may be responsible for the age-related decline in muscle function., Growth of proliferative cells is dependent on regulation of protein synthesis and subsequent cell division. In post-mitotic tissues such as cardiac and skeletal muscle, growth is accomplished by increased protein synthesis and the donation of nuclear DNA by stem cells. Regardless of proliferative capacity, growth and DNA synthesis are associated with increased protein synthesis. Our central hypothesis is that somatic maintenance, which we define as increased protein synthesis in the absence of growth, is associated with slowed aging. To pursue this central hypothesis, we simultaneously measure both protein synthesis and DNA synthesis using deuterium oxide. Using this approach, we have reproducibly measured DNA synthesis in laboratory rodent cardiac tissue. When comparing several long-lived rodent models, we have shown that the rate of new DNA synthesis in the heart is altered and, in some cases, this is an age-dependent or sex-dependent finding. The observed changes in DNA synthesis in cardiac tissue, which is predominantly populated by terminally differentiated, post-mitotic myocytes, suggests that cardiac muscle has either greater proliferative potential than is perhaps appreciated, or that the new DNA synthesized can be accounted for by donation from cardiac progenitor cells. We will present a comparative analysis of DNA synthesis in cardiac tissue from several long-lived models and the current results of ongoing experiments to identify the origin of the new DNA., Traditional aging models are short lived, a useful trait for tracking changes over the course of their lifespan. However, it is within the exceptional long-lived models that we can expect to find the mechanisms necessary to resist aging, as evolution has provided them. To this end, we are utilizing a range of marine bivalve mollusk species, with lifespans ranging from under a decade to over 500 years, in a comparative study to investigate the hypothesis that a long life requires superior proteome stability. These ages can be individually determined by counting growth rings in the shell. This experimental system provides a unique opportunity to study closely related organisms with vastly disparate longevities, including the longest lived animal. We are testing the long-lived bivalve's ability to maintain protein structure and function under various stressors, and identifying the macromolecules responsible for this protection. As protein homeostasis has been implicated in the aging process and age-related diseases, these macromolecules could have dramatic medical value. Preservation of protein function is measured by representative enzyme activity, such as GAPDH, when stressed in vitro. Stressors include both oxidative and unfolding agents. We demonstrate a remarkable persistence in enzyme activity in the long-lived bivalves despite egregious chemical insults. Results also indicate that C57Bl6 exhibit very poor protein stability in comparison to the bivalves. The influence of each species’ metabolite fraction on this stability is also investigated, and an attempt to rescue stability in the short-lived bivalves is attempted. Ultimately, stabilizing compounds will be identified via mass spectrometry. Demonstrating the protection of these proteins and identifying the macromolecules facilitating enhanced proteostasis in the longest lived animal species could be dramatically important in relation to various age-related protein diseases., Caspase-2 is a protein that is well known for its involvement in cellular apoptosis. However, its function in osteoclasts, cells that resorb bone, has not yet been described. In this work, the role of caspase-2 in osteoclast differentiation and apoptosis is evaluated. During the differentiation process from macrophage precursors, caspase-2 levels decrease so that protein expression is low in the mature osteoclast. Interestingly, when caspase-2 is knocked down in the RAW 264.7 macrophage cell line, osteoclast formation is increased as shown by higher levels of the osteoclast marker Cathepsin K. Furthermore, in cells derived from global Casp2 knockout mice, osteoclast differentiation is enhanced as demonstrated by increased osteoclast numbers and TRAP activity. With regard to osteoclast apoptosis, cells exposed to oxidative stressors such as H2O2 and rotenone show increased cleaved caspase-2 protein expression indicating early apoptosis mediated by caspase-2. Furthermore, additional data show that osteoclasts lacking caspase-2 and exposed to oxidative stressors are more resistant to apoptosis compared to wild-type cells. Together, these data suggest that caspase-2 may play a dual role in the osteoclast whereby it modulates osteoclast differentiation and may help regulate osteoclast apoptosis., Genetic manipulations allow controlled interventions into aging progression by changing the genetic composition of model organisms. Such manipulations can include the addition of the ‘anti-aging’ genes, the inactivation of the ‘pro-aging’ genes, or the replacement of the ‘pro-aging’ allelic variants of certain genes with their ‘anti-aging’ counterparts. The genome engineering approaches that can be used to add or replace genes are based on site-specific nucleases and DNA recombinases. Our results indicate that the latter approaches, which currently utilize primarily Flp/FRT and Cre/loxP systems, can accomplish the task both efficiently and precisely if the fine-tuned dual recombinase-mediated cassette exchange is used as a gene delivery/replacement tool. Under optimal conditions, the efficiency of dual RMCE catalyzed by the Flp–Cre pair can reach about 50% of the transfected cells. Dual RMCE depends on the pre-introduction of the recombination target sites into genome before the replacement reaction can be carried out. This dependence can be lifted if variants of site-specific recombinases are evolved to recognize pre-existing target-like sequences that flank a genome region of interest. Here, we present the results of the engineering of the hybrid Flp-TAL and Cre-TAL recombinases that recognize genomic sequences of interest. These task-specific variants of Flp and Cre recombinases can be paired to be used in the dual RMCE approach to replace the desired genome regions. We show that the engineered hybrid Flp-TAL and Cre-TAL recombinases can be used to mimic the replacement of the mutation that causes sickle-cell anemia in the model setting of CHO cells., Induced pluripotent stem (iPS) cells have the potential to improve health with age. IPS cells may be useful for biological teeth engineering. The critical cells in tooth development are neural crest-derived dental mesenchyme. Here, we utilize pharmacological molecules to assess iPS cells derived from the common marmoset (Callithrix jacchus) for the neural tube and neural crest lineage differentiation. Approach: To assess neural differentiation, we utilized an embryoid body generation assay in combination with a cocktail treatment of active small molecules: DMH1 (BMP inhibitor), SB431542 (activin/nodal/TGF-beta inhibitor), BIO (GSK-3 beta inhibitor), Y-27632 (ROCK/Rho inhibitor) and all-trans retinoic acid. Results: With 6 days of treatment with bFGF withdrawal and GSK-3 beta inhibitor, we found significant increases in the mRNA levels of neural markers NCAD (30-fold increase, P=0.0001) and ERBB3 (12-fold increase, P=0.0129). We found that bFGF addition had an inhibitory effect on NCAD/ERBB3 induction. When GSK-3 beta inhibitor (BIO) was included, NCAD mRNA levels further increased (from a 34- to 102-fold increase over baseline, P=0.0001), as was ERBB3 (34-fold, P=0.0035). bFGF withdrawal also increased FOXA2 levels (endoderm marker), but we report that inclusion of the GSK-3 beta inhibitor (BIO) ablated induction from a 15-fold increase to a 3-fold increase, P=0.0318). Conclusions: Results indicate that FGF inhibits neural differentiation, while GSK-3 beta inhibition promotes neuralization as indicated by NCAD and ERBB3 expression. We report that marmoset iPS cells respond to pharmacological inducers of neural differentiation. These studies will accelerate the development of the marmoset as a model for engineered teeth engraftment., The naked mole rat (Heterocephalus glaber; NMR) is the longest lived rodent, living >31 years. Unlike aged humans and mice that exhibit large declines in diastolic function, the NMR maintains both systolic and diastolic function for at least 66% of its long lifespan. We have further investigated NMR cardiac function using young (2–4 years) NMRs and have compared them to physiologically age-matched C57BL/6 mice (3–5 months). Surprisingly, we have found that NMRs have low heart rates compared to mice (256±8 vs. 704±11 bpm). NMR heart rates are also about half that predicted for their body size. NMRs display significantly lower cardiac fractional shortening (∼28%) than mice (∼39%). Invasive blood pressure measurements revealed that peak intraventricular pressure and ventricular contractility were much lower in the NMR (p, The concept that mitochondrial function declines during aging has been a basic tenet of the biology of aging for many years. However, it has been shown recently, first in invertebrates and later in mice, that lifespan is increased in response to genetic manipulations of some mitochondrial electron transport chain (ETC) complexes. For example, inhibition of complex IV (cytochrome c oxidase, COX), an essential transmembrane protein complex in the mitochondrial respiratory electron chain, has been shown to extend lifespan in worms and flies. Similarly, mice lacking the COX assembly protein Surf1 show increased longevity associated with decreased adiposity and enhanced insulin sensitivity, despite 50–70% reduction in COX activity. Here, we asked whether a mouse model of cytochrome c oxidase deficiency due to a mutation in the Sco2 gene, a copper chaperone that is required for the activity of COX, would have a similar metabolic phenotype as Surf1 −/ − mice. A complete knockout of the Sco2 gene in mice is embryonic lethal, however mice harboring a Sco2 knockout allele and a mutated Sco2 knockin allele (KI/KO) are viable, and have a 30–60% reduction in COX activity. We found that Sco2 KI/KO mice have increased fat mass associated with a reduction in whole white adipose tissue oxygen consumption. The Sco2 KI/KO mice have increased hepatosteatosis, elevated serum triglyceride (32%) and cholesterol levels (32%), and changes in circulating adipokine levels compared to wild-type controls. Interestingly, these alterations are associated with the development of insulin resistance in the Sco2 KI/KO mice. These findings counter to the metabolic phenotype of Surf1 −/− mice, illuminating the complex nature of mitochondrial dysfunction on physiology. Results from this study will further enhance our understanding of the role of complex IV in physiological outcomes due to mitochondrial dysfunction., Animal models of extended longevity have a shared characteristic of enhanced cytotoxin resistance. The longest lived rodent, the naked mole rat (∼31 years), is extremely resistant to cancer and a wide array of xenobiotics in vitro, and exhibits no age-related physiological or molecular declines during this extraordinarily long lifespan. We hypothesized that this profound broad-based stress resistance was due to enhanced signaling of the nuclear factor-erythroid 2-related factor-2 (Nrf2) cytoprotective pathway. The transcription factor Nrf2 is highly conserved in eukaryotes and ubiquitously expressed in all tissues. Nrf2 levels are regulated by kelch-like ECH-associated protein 1 (Keap1), which targets Nrf2 for ubiquitination and subsequent degradation via the proteasome. After a stressful insult (i.e. toxins, ROS), interactions between Nrf2 and Keap1 are inhibited and Nrf2 is able to translocate into the nucleus, bind to the antioxidant response element (ARE) and thereby activate the transcription of greater than 600 cytoprotective molecules, including those involved in detoxification, glutathione metabolism, molecular chaperones, and proteasome subunits. Commonly studied with regard to cancer, Nrf2 has also been shown to interact with p53 and p21, playing a role in modulation of the cell cycle and cancer progression. We found that Nrf2 signaling activity showed a positive correlation with maximum lifespan in rodents with varying longevity. Interestingly, this was largely due to inverse correlations with several negative regulators of Nrf2, including Keap1, which inhibited degradation of Nrf2. This Nrf2 signaling activity appears to be conserved with aging across 10 different rodent species and the type of regulation may have an impact on positive health span and longevity., In proliferative tissues, growth is accomplished with a doubling of cellular machinery by protein synthesis and subsequent cellular replication. In post-mitotic tissues, growth is accomplished by increased protein synthesis followed by DNA donation from stem cells to keep the cyto-nuclear domain constant. In both cases of growth, increased DNA synthesis is associated with increased protein synthesis. Our working hypothesis is that increased somatic maintenance, which we define as increased protein synthesis in the absence of growth, slows down aging. To this end, we have employed methods utilizing deuterium oxide (D2O) that simultaneously measure both protein synthesis and DNA synthesis in a variety of tissue types. We have repeatedly demonstrated DNA synthesis in skeletal muscle tissue in human and rodent models. Furthermore, in rodent models of slowed aging (caloric restricted, rapamycin treated, Snell, and crowded litter), we have demonstrated that this rate of new DNA synthesis in skeletal muscle can change based on the model or stage of life. The observable and changeable DNA synthesis in post-mitotic skeletal muscle indicates that skeletal muscle indeed has some replicative potential (as all tissues in mice are telomerase-positive) or that the new DNA comes from stem cells that have replicated and donated DNA to the myocyte. We will present changes in skeletal muscle DNA synthesis in models of slowed aging, and results of studies striving to determine the origin of the new DNA. It is hoped that these studies will provide further insight into how to maximize somatic maintenance for slowed aging., Over the last few decades, there was a 30% increase in the number of fathers over the age of 35 years. At this age, a father has the potential to transmit twice as many mutations to his child as a 20-year-old father. Paternal mutations continue to double approximately every 16 years, a phenomenon known as the paternal age effect. The driving force of the paternal age effect is mutagenesis. Base excision repair activity is essential for maintaining a low mutant frequency in the male germline. However, spermatogenic cells isolated from old mice display a 50% decrease in base excision repair activity, with a concomitant increase in mutant frequency, as compared to cells prepared from young mice. Reduced base excision repair activity appears to be mediated by reduced AP endonuclease 1 (APE1), a key base excision repair protein. Mice heterozygous for Ape1 show an increased germline mutant frequency as young adults, while APE1 transgenic mice are protected from age-dependent increases in spontaneous mutagenesis. Our objective is to delineate the mechanism/s mediating reduced APE1 abundance in spermatogenic cells with increasing age. In pachytene spermatocytes and round spermatids obtained from old mice, there is a significant 35 and 25% reduction, respectively, in APE1 abundance as compared to young mice. The age-related decrease in APE1 abundance is not accompanied by a reduction in Ape1 transcript abundance, thereby suggesting that post-transcriptional or post-translational regulation is involved. In somatic cells, p53 plays a role in regulating Ape1 abundance. There is a significant increase in p53 activation in spermatogenic cell populations obtained from old mice. APE1 expression is reduced by 40% in spermatogenic populations obtained from p53 null mice, relative to wild-type mice. Combined, these results indicate a strong relationship between APE1 abundance, germline mutagenesis, and p53 activation contributing to the paternal age effect., Nearly two decades have passed since the initial discovery that disruption of the mitochondrial electron transport chain can unexpectedly increase lifespan. Much research has been done to find the mechanisms behind this seeming paradox, but while many factors have been shown to be required, none has proven sufficient. The mitochondrial unfolded protein response (UPRmt) under the control of ATFS-1 has received much attention for its role in Mit mutant longevity, but it is not the only retrograde response induced by mitochondrial dysfunction. We show that the ATFS-1 pathway itself divaricates into separable pathways, one being the UPRmt and the other being the activation of SKN-1. Moreover, we have found a novel retrograde pathway independent of ATFS-1. This MAPK signaling cascade acts in a parallel and compensatory manner with the ATFS-1 network, such that if one pathway is deactivated, the other is turned up to compensate., Aging is associated with progressive loss of muscle mass, quality, and strength. Moderate (40–50%) calorie restriction is known to reduce the rate of muscle loss in animal models; however, too high of a calorie-restrictive diet can induce loss of bone mass. In this study, we tested the hypothesis that mild caloric restriction (CR) would still attenuate age-associated loss of muscle mass without any further deleterious effect on bone mass. To determine the long-term effect of mild CR (20% restriction of calorie without reducing vitamins and minerals relative to baseline intake of ad libitum (AL)) on musculoskeletal health during aging, 6-month-old C57BL/6 female mice were fed AIN93 diet AL or CR for 8 or 16 months. Mice were scanned with dual energy x-ray absorptiometry (DXA) at 6 months (baseline), 14 months (middle age), and 22 months (aging) and analyzed for the bone and muscle mass. Higher levels of muscle mass are maintained in both 14 and 22 months of age in 20% CR groups as compared to AL groups as measured by DXA, and wet weight at sacrifice. Better muscle strength is also maintained during aging in 20% CR groups as compared to AL groups as measured by rotarod performance test and endurance stress test. Interestingly, bone mass in different bone regions as measured by DXA was not reduced in 20% CR groups as compared to AL groups. These data indicate that 20% CR without compromising vitamin and minerals could be a natural, cost effective, and safe optimal dietary regimen that would improve muscle mass and strength without any deleterious effect on bone., Adipose tissue has dynamic endocrine and secretory functions that play significant roles in the regulation of metabolism and health span. There is increasing evidence that different adipose depots may have different, and often contradictory, effects on this regulation. For example, visceral white adipose is largely thought to be detrimental to metabolism whereas subcutaneous white adipose has been shown to have several beneficial effects. Furthermore, aging is associated with a loss of subcutaneous adipose tissue, accumulation of fat in intra-abdominal visceral adipose tissue and ectopic accumulation of fat in other tissues such as muscle and liver. Adipocyte precursor cells (pre-adipocytes) play a significant role in the homeostatic regulation of adipose tissue. In this study, we tested whether differences in mitochondrial function among the pre-adipocyte pools of each adipose depot might be a determining factor in depot-specific functional differences. Pre-adipocytes from visceral and subcutaneous adipose depots were isolated and sub-cultured from young C57BL/6J mice. Using the Seahorse bioanalyzer, we found that mitochondrial respiration rate is high and reserve capacity is low in visceral depot-derived pre-adipocytes relative to those from subcutaneous depots. In addition, we found that pre-adipocytes from visceral depots are significantly more sensitive to oxidative stress in vitro than those from subcutaneous depots. These data then suggest that even modest increases in oxidative stress may dramatically alter the function of adipose precursor cells to inhibit adipogenesis and stimulate lipolysis in visceral depots, which could be a mechanism for increased adipose dysfunction with age. Future studies will address whether alleviation of oxidative stress can preserve healthy functional adipose tissue with age., Every year, millions of people are diagnosed with traumatic brain injury (TBI). Of those, many have repetitive TBI (rTBI). Athletes and soldiers are at particular risk for rTBI, and those with rTBI commonly exhibit psychological symptoms including depression, anxiety, poor impulse control, and suicidal ideation. Some also exhibit signs of neuro-degeneration. Currently, treatments to stem the long-term consequences of TBI have been unsuccessful. Astrocytes play a central role in neuronal support and are excellent targets for therapy after TBI. We previously showed that the purinergic agonist 2-methylthioadenosinediphosphate (2MeSADP) effectively reduces astrocyte edema following TBI in mice. We hypothesize that 2MeSADP can similarly reduce the potential long-term effects of repetitive TBI. Here, we report our preliminary findings of a novel rTBI mouse model. Three-month-old mice were subjected to five closed-skull cortical impacts over 5 days. One year after rTBI, behavior was analyzed using a variety of tests. We found that control rTBI mice exhibited more anxiety-like phenotypes compared to 2MeSADP and sham-treated mice in the open field test and the three chambered social anxiety test. Fewer control TBI mice were able to complete the vertical pole test, suggesting an impairment of motor coordination. Continuing experiments will involve histological analysis of brain tissue, and further elucidation of the behavioral phenotype in a second cohort of mice. To our knowledge, this is the first report of a long-term analysis of rTBI in mice., Autologous cell therapies have been proposed as the cure for a wide variety of human pathologies and injuries including diabetes, myocardial infarction, and spinal cord injuries. One obstacle that is often overlooked is that donor age could be a barrier to the derivation of induced pluripotent stem cells (iPSCs) from somatic cells. This is important, as the group for which cell therapy holds the most benefit would be the middle aged to elderly population. It has also been well documented that, during aging, mitochondrial mutations accumulate (due to ROS damage or low-fidelity replication) and oxidative capacity decreases. What, if any, effect these changes would have on the quality of iPSCs derived has yet to be addressed. It has also been shown that in a mouse model carrying a mutation that increases the accumulation of mtDNA mutations, a premature aging phenotype has been shown. Recently, publications have implicated the balance of mitochondrial oxidation to glycolysis as crucial to reprogramming efficiency and have reported that cells acquire high mitochondrial membrane potential during reprogramming. Conflicting reports that this is maintained and leads to increased oxidative capacity after redifferentiation led us to hypothesize that these age-related changes in mitochondrial metabolism play a controlling role in the efficiency of derivation of iPSCs. The current aim is the creation of a polycistronic, floxed retroviral vector to allow for reprogramming with a single virus, which will then be used to investigate effects of donor age on the quality of iPS cells, derived skin samples of marmosets of different ages., Participation in physical activity can effectively prevent muscle loss and functional decline that occurs with age. Thus, understanding the mechanistic basis for preservation of skeletal muscle health with exercise may provide clues for developing therapeutic tools to combat age-related disabilities. We have demonstrated that mesenchymal stem cells (MSCs) accumulate in the muscle of young mice in response to a single bout of eccentric exercise and indirectly facilitate muscle repair and vessel growth via paracrine factor release. The observation that mechanical strain can potently influence growth factor release from freshly isolated, muscle-derived MSCs (mMSCs) provided the impetus to utilize physiological preconditioning as a method to improve mMSC viability and function in the aged muscle microenvironment. In this study, mMSCs isolated from young mouse muscle were preconditioned (10% multiaxial strain, 5 hr) and immediately transplanted into the gastrocnemius/soleus complex of 24-month-old mice. Separate groups of mice receiving saline or non-strained mMSCs served as controls. Although satellite cell number and fiber size were unchanged in all groups, vessel size and number of NMJs were greater in mice injected with preconditioned mMSCs versus controls one week post-injection (P < 0.05), resulting in a trend toward an increase in muscle function. Additionally, in vitro experiments suggest that preconditioned mMSCs are able to overcome a stiff, collagen-rich microenvironment that mimics aged skeletal muscle, showing increased survival and proliferation as compared to non-strained mMSCs. Overall, MSC preconditioning prior to transplantation may provide a novel method to prevent or reverse age-related impairments in muscle health and function., It is known that an increase in caloric intake leads to an increase in plasma growth hormone (GH) level that subsequently induces secretion of insulin-like growth factor-1 (IGF-1) from the liver, what then leads to accelerated aging (Nature 2010;464:504). However, caloric restriction and a resulting decrease in plasma IGF-1 level have the opposite effect and extend lifespan. It is also known that adult tissues contain a population of pluripotent very small embryonic-like stem cells (VSELs) that play, as postulated, an important role in the rejuvenation of long-term hematopoietic stem cells (LT-HSCs) in bone marrow (BM) Leukemia 2011; doi:10.1038/leu.2011.73, Exp. Hematology 2011;39:225–237). As we observed previously, the number of these cells in murine BM decreases with age and VSELs are kept quiescent in BM and protected from premature depletion by the erasure of the somatic imprint in differentially methylated regions (DMRs) of some paternally imprinted genes involved in insulin/insulin growth factors signaling (IIS) such as, for example, Igf2-H19 and RasGRF1 (Leukemia 2009;23:2042). Hypothesis: To explain and connect these phenomena together, we hypothesized that prolonged insulin/insulin growth factor signaling (IIS) prematurely depletes VSELs from the adult tissues and in BM may negatively impact on a population of HSCs. Material and methods: The number of VSELs and HSCs in long-living murine strains with inborn low levels of circulating IGF-1 (Laron- and Ames-dwarfs) as well as in short-living mice with high levels of circulating IGF-1 (e.g. transgenic mice that overexpress bovine growth hormone; bGH) was evaluated by FACS. VSELs were isolated and the epigenetic status of genes regulating pluripotency (e.g. Oct 4) as well as imprinted genes regulating IIS was evaluated by employing bisulfate modification of DNA followed by sequencing and by COBRE assay. We also challenged long-living mice with low IGF-1 plasma levels by daily injections of recombinant GH or IGF-1. Results: We found that the number of VSELs and HSCs residing in BM inversely correlates with plasma GH/IGF-1 level. To support this, mice with low circulating plasma IGF-1 levels (Laron- and Ames-dwarf mice) have higher numbers of VSELs and HSCs in BM that, in contrast to age-matched normal litter mates, are maintained at high levels even into advanced age. The analysis of molecular signature of VSELs in these animals revealed prolonged retention of hypomethylation in the DMRs within the Igf2-H19 and RasGRF1 loci, which attenuates IIS signaling in these cells. The number of VSELs, however, decreased in these animals after prolonged treatment with GH or recombinant IGF-I. Conversely, mice with elevated IGF-I level in plasma due to expression of the GH transgene or normal wild-type mice injected for a sustained period with recombinant GH both exhibit significant decreases in the number of VSELs and HSCs in BM compared to control animals. These decreases were paralleled by epigenetic changes in Igf2-H19 and RasGRF1 loci in which DMRs became hypermethylated over time. These changes in methylation lead to increases in IGF-2 and RasGRF1 expression and may explain why GH transgenic mice have an increase in IIS that leads to a shortening of life span in these animals. Conclusions: Our data shed new light on the relationships between senescence, GH/IGF-1 level, prolonged IIS, and number of VSELs and LT-HSCs. Accordingly, we propose a new paradigm in which a decrease in IIS (e.g. due to caloric restriction that lowers plasma IGF-1 level) may delay the age-dependent elimination of VSELs from adult tissues. In contrast, chronic IIS (e.g. due to chronic high caloric intake and the resulting elevated GH and IGF-1 levels) prematurely depletes VSELs residing in adult organs, which for example in BM leads to a decrease in the number of LT-HSCs. This study also indicates that GH-based anti-aging therapies need careful re-evaluation of their potentially uncontrolled stimulation of VSELs in BM that may lead to development of hematological malignancies. In support of this, elevated GH and IIS lead to hematological malignancies, while, in contrast, Laron-dwarf mice and Laron-dwarf patients, which have low plasma IGF-1 levels, do not develop leukemia., Parkinson's disease is a neurodegenerative condition affecting every sixth person older than 70 years in the United States. Current therapeutic approaches provide succour from symptoms, but not a cure. Stem cells, with their propensity to regenerate the dopaminergic neurons (which are actually depleted in this condition) are offering new hope. Steady improvement in the ‘Off’/‘On’ unified Parkinson's disease rating scale (UPDRS), Hoehn & Yahr scores, Schwab and England scores, absence of tumorigenic growth on MRI as well as the absence of any AE's/SAE's following stereotactic transplantation of bone marrow derived mesenchymal stem cells (BM-MSC's) in the sub-lateral ventricular zone (in our earlier study) have infused tremendous optimism and galvanized us to undertake the present, open-labeled, non-randomized, single-center study to evaluate further on the positive outcomes on a larger scale. The current study plans to track the administered BM-MSCs, employing in vivo MRI imaging after labeling them with super paramagnetic iron oxide (SPIO), which might help us in confirming and quantifying the success of the transplantation. Also, it might provide early insights on possible mechanisms of any intercellular crosstalk between transplanted BM-MSCs and resident stem cells in the subventricular zone. Finally, this knowledge will help us modify, in future studies, the route of administration, optimize dosage, determine total duration/frequency of transplantation, thus enhancing the therapeutic efficacy. Three of the subjects have shown an 11% improvement in their UPDRS scores ‘Off’/‘On’. There is a marginal improvement in H&Y, as well as H&S scores, in at least five patients. The analysis report of the imaging studies is awaited., In mammals, the small intestines are lined by single-layered epithelium organized into self-renewing crypt-villus units. Dividing stem cells line the valley-like crypts, and these differentiate upward to become quiescent, functional cell types covering the finger-like villi. All of the crypt epithelial cells produce the thyroid-stimulating hormone (TSH) and the cytokine interleukin-7 (IL-7), while villus epithelial cells do not. In the crypts, TSH- and IL-7-expressing cells are layered into three compartments. This layering is not present in knockout mice that do not have intestinal T-cells, suggesting that T-cells are targets for activation via TSH and IL-7, through their surface receptors for both (TSHR and IL-7R). In healthy mice, TSH and IL-7 production are generally limited to the crypts, as noted above, but long contiguous blocks of dividing cells, expressing TSH/IL-7, are present on the villi in intestines of mice infected with enteric pathogens. These reactive cells may be a reserve stem cell population or may have dedifferentiated for T-cell signaling and barrier defense. Dividing cells in the intestinal crypts are recognized targets of agents that cause neoplastic transformation (e.g. ionizing irradiation). We suggest here that dividing, back-differentiated villus cells on the villi may also generate tumors, especially when activating conditions persist, maintaining large numbers of dividing cells on the villi, e.g. in inflammatory bowel syndromes. Targeted delivery of TSH or IL-7 agonists or antagonists may have therapeutic effects for these conditions, via their effects on intestinal T-cells, and may prevent tumor initiation in the intestinal epithelium.

To test the hypothesis that caloric and fat intake in a pre-load meal have no subsequent effects upon blood glucose and insulin concentrations, perceived hunger, subsequent food intake and appetite control in lean and obese men.Lean and obese men reported to the laboratory in the morning in a fasted state where they were subject to an eating test based on the pre-load-test meal paradigm, using a double-blind protocol. The breakfast pre-load was either a reduced caloric low-fat (LF) meal or an overfeeding high-fat (HF) meal. LF was 20% of each individual's average daily energy requirement (ADER) and comprised 60% carbohydrate, 27% protein, and 13% fat, whilst HF was adjusted to yield 55% of the ADER, and comprised 45% carbohydrate, 22% protein and 43% fat. The pre-loads on both trials were administered as one single mean, and were given in a random order. After 5(1/2) h, an ad libitum test-lunch was given to determine how much energy was consumed. Between the two meals, blood samples were collected and subjective hunger ratings were assessed hourly. These variables were measured at 30-min intervals for 75 min after the ad libitum meal.Twelve healthy men, six of whom were lean (BMI 22. 50+/-1.08 kg.m2) and six of whom were obese (BMI 39.05+/-11.63 kg. m2) were recruited.When given 55% of their ADER in a HF pre-load meal, the obese group consumed more energy (5426+/-1126 kJ; F1,20=11.45, P0.01), than the lean group did (3473+/-1114 kJ), accounting for 45% of the ADER in that meal setting. However, no differences between lean and obese intake were noted at the test meal following a LF pre-load. The lean group exhibited a significant inverse correlation (r=0.628, P0.05) between serum insulin concentration before eating the test meal and the amount of energy consumed at the test meal, while such a relationship was absent in the obese group.The obese males were unable to compensate for the caloric overloading when fed a HF (55% ADER) pre-load at a subsequent test meal, whereas a calorically reduced pre-load (20% ADER) produced similar intakes to the lean control group. The inverse relationship noted in the lean group between insulin levels before the test meal and the energy intake at that test meal suggests that insulin may play a role in the regulation of appetite - satiety mechanism in lean males. The absence of such a relationship in the obese may suggest the site for possible appetite dysfunction contributing to obesity. These results further suggest that when obese individuals consume a high-fat meal they are prone to passive overconsumption, whereas lean study participants appear to be more resistant to such a phenomenon.

The rabbit could be a superior animal model to use in bone physiology studies, for the rabbit does attain true skeletal maturity. However, there are neither normative bone mineral density (BMD) data on the rabbit nor are there any validation studies on the use of dual-energy X-ray absorptiometry (DXA) to measure spinal BMD in the rabbit. Therefore, our aim was twofold: first, to investigate whether DXA could be used precisely and accurately to determine the bone mineral content (BMC). bone area (BA). and BMD of the rabbit lumbar spine: Second. to evaluate the new generation fan-beam DXA (Hologic QDR-4500) with small animal software by comparing two DXA methodologies QDR-1000 and QDR-4500 with each other, as well as against volumetric bone density (VBMD) derived from Archimedes principle. As expected. there was a magnification error in the QDR-4500 (BMC, BA. and BMD increased by 52%. 38%. and 10%, respectively, when the vertebrae were positioned flat against the scanning table). With the magnification error kept constant (vertebrae positioned 10 cm above the scanning table to match the height in vivo). there were no differences among the mean BMC. BA. and BMD of the rabbit vertebrae (Ll-L7) in vivo and in vitro using the QDR-4500 (p0.05). BMC, BA, and BMD differed between QDR-1000 and QDR-4500 in vitro because of a magnification error when the vertebrae were flat on the table (p0.0001). and, consequently. the machines did not correlate with one another (p0.05). However, the BMC, BA, and BMD of the two DXAs did significantly correlate with each other in vivo and in vitro when the magnification error was compensated for (r = 0.44 and 0.52. i2 = 0.45 and 0.63. and 12 = 0.41 and 0.60. respectively. p0.05-0.008). The BMC and BMD (in vivo and in vitro) of the rabbit vertebrae measured by QDR-4500 was significantly correlated with VMBD, ash weight, and mineral content (,2 = 0.67-0.90,j0.01-0.0001). Therefore, the QDR-4500 can be used to yield precise and accurate measurements of the rabbit spine.

Meals of varying energy content and episodes of sleep influence body temperature. We compared the effect of an evening meal, varying from high-energy (11.91 +/- 0.86 MJ) to average (5.74 +/- 0.88 MJ) and a 10-h fast (no evening meal), on nocturnal body temperature and sleep. Seven healthy men (20-24 years, mean body mass index of 23.4 +/- 2.6 kg/m2) reported to the laboratory for an evening meal at 2000 h having consumed similar amounts of food before 1300 h. After completing the meal, subjective hunger ratings were assessed, and a venous blood sample taken. The subjects spent 4 nonconsecutive nights (an adaptation night, followed by either of the two meal conditions or the fast in random order) in the sleep laboratory when polysomnographic recordings were made from 2300 to 0700 h. Meal energy content and serum concentrations of insulin, triglyceride, and low-density lipoproteins (LDL) varied significantly. Lower rectal temperatures were measured during the fast than following the meals. Over the 8-h recording period, thermal response indices (TRI) varied with higher body temperatures following the higher energy meal. Similar rectal temperatures were attained by the end of the sleep periods. There were no significant differences in any of the subjective or objective sleep measures. The physiological responses associated with the transient dietary changes of an evening meal or a 10-h fast altered nocturnal body temperature but did not significantly affect sleep of good sleepers when sleep was initiated 2 to 3 h after finishing the meal.

Eight healthy males of age 22.9+/-4.2 years (mean+/-SD) and body weight 73.26+/-11.50 kg, with BMI of 23.11+/-2.84 kg/m(2)underwent two different eating meal frequency patterns on 2 separate days. On both days they were fed approximately 33.3% of their average daily energy requirements as a breakfast pre-load meal: served either as a single meal (SINGLE) or divided into five equal portions (served hourly) (MULTI). Five and a half hours after the initial meal, an ad libitum meal was served. Venous blood was tested to determine plasma glucose and serum insulin concentrations every hour until the ad libitum lunch, and at 15-, 45- and 75-min after lunch. Visual analogue scales (VAS) were completed every hour until the ad libitum lunch, and at 15-, 45- and 75-min after lunch as a measure to determine hunger, appetite and satiety indices. Although both groups were fed isocaloric and identical macronutrient "breakfast pre-loads" (3450+/-466 kJ), the SINGLE group consumed 26.6% more (p

We studied roost microclimates, thermal preferences and temperature-related variation in body temperatures and flight abilities of M. condylurus from three roosts in man-made structures in South Af...

Urine and plasma concentrations and haematocrits were measured in free-ranging Angolan free-tailed bats (Mops condylurus) inhabiting thermally-challenging roosts in the Komatipoort region of South Africa. Samples were collected in both autumn and summer, from bats caught emerging from roosts before feeding (pre-feed- ing), and those returning after foraging (post-prandial). Post-prandial bats exhibited higher body fluid concentrations, but lower haematocrits, than individuals caught prior to feeding, reflecting raised excretory mineral and nitrogenous loads and replenishment of body water pools during nocturnal foraging. Pre-feeding concentrations of both urine (2637 ± 506 mOsm/kg; n = 16) and plasma (331.5 ± 25.9mOsm/kg; n = 24) were significantly higher in summer than autumn (urine: 2157 ± 454mOsm/kg; n = 8; plasma: 294.5 ± 35.2 mOsm/kg; n = 18) reflecting the greater dehydration stresses within hotter roost microclimates, and a moderate kidney concentrating ability in this species. Haematocrits of pre-feeding animals were not, however, influenced by season and in both instances exceeded 53%, indicative of the higher oxygen carrying capacity needed for sustained flight in volant insectivores and also the defense of the rheologica! properties of blood. The ability of Mops condylurus to withstand a thermally-challenging roost milieu reflects, in part, its tolerance to dehydration, rather than the maintenance of water balance through exceptional renal concentrating ability.

A double-blind, placebo-controlled, multicenter study, was performed to evaluate the efficacy and safety of ziprasidone in 139 patients with an acute exacerbation of schizophrenia or schizoaffective disorder. Patients were randomized to receive ziprasidone 40 mg/day, 120 mg/day or placebo for 28 days. Ziprasidone 120 mg/day was significantly more effective than placebo in improving the BPRS total, CGI-S. BPRS depression cluster and BPRS anergia cluster scores (all P0.05). Similarly, the percentages of patients classified as responders on the BPRS (or = 30% reduction) and the CGI improvement (scoreor = 2) were significantly greater with ziprasidone 120 mg/day compared with placebo (P0.05). The number of patients who experienced an adverse event was similar in all three treatment groups, and discontinuation due to adverse events was rare (five of 91 ziprasidone-treated patients). The most frequently reported adverse events, that were more common in either ziprasidone group than in the placebo group, were dyspepsia, constipation, nausea and abdominal pain. There was a notably low incidence extrapyramidal side-effects (including akathisia) and postural hypotension and no pattern of laboratory abnormalities or apparent weight gain. Ziprasidone-treated patients were not clinically different from placebo-treated patients on the Simpson-Angus Rating scale, Barnes Akathisia scale and AIMS assessments. These results indicate that ziprasidone 120 mg/day is effective in the treatment of the positive, negative and affective symptoms of schizophrenia and schizoaffective disorder with a very low side-effect burden.

This study investigated the relative satiating hierarchy of the four energy-providing macronutrients (fat, carbohydrate (CHO), protein, and alcohol) in lean women. On four separate occasions, the composition of an iso-energetic lunch preload was manipulated in 12 lean (BMI25 kg/m2) women. The four treatments comprised a 1-MJ baseline meal and drink (40% fat, 48% CHO, 12% protein) to which was covertly added: 1) + 1MJ protein; 2) + 1MJ alcohol; 3) + 1MJ CHO; and 4) + 1MJ fat. Prior to and at 30-min intervals, subjects completed 100-mm visual analogue scales rating subjective hunger and satiety. Ninety min following the preload, an ad lib. lunch meal was given (40% fat, 48% CHO, and 12% protein) and energy intake (EI) measured. Energy intake at the lunch meal was 2195 (880, SD) kJ, 2772 (1191, SD) kJ, 2502 (681, SD), kJ and 2558 (1050, SD) kJ for the protein, alcohol, CHO, and fat preloads, respectively. There was no significant difference between the pleasantness of the preloads (p0.05). Macronutrient composition had a significant effect on short-term hunger (F = 3.19; p0.05), subjects being less hungry after the protein preload. Subjects also had a lower energy intake after the protein preload (F = 3.11; p0.05). We conclude that only protein has a differential short-term satiating effect when incorporated iso-energetically and at a similar energy density into the diet.

The effect of 18 months of lactation on indexes of calcium and bone metabolism was studied in 60 Gambian women accustomed to a very low calcium intake. Half the women consumed a calcium supplement from 10 days postpartum for 52 weeks (supplement, 714 mg Ca/day; total Ca intake, 992 ± 114 mg/day), and half consumed placebo (total Ca intake, 288 ± 128 mg/day). Fasting blood and 24-h urine samples were collected at 1.5, 13, 52, and 78 weeks of lactation and analyzed for calciotropic hormones (intact PTH, 1,25-dihydroxyvitamin D, and calcitonin), bone turnover markers (osteocalcin, bone alkaline phosphatase, and urinary deoxypyridinoline), and plasma minerals (calcium and phosphate). The first months of lactation were associated with increased bone turnover and plasma phosphate, and decreased PTH and 1,25-dihydroxyvitamin D. These effects diminished by 52 weeks, although breast milk volumes remained high. The Gambians had higher PTH, 1,25-dihydroxyvitamin D, and bone formation than British women with a greater customary calcium intake. None of the biochemical indexes was affected by calcium supplementation, with the possible exception of bone alkaline phosphatase (−29% at 52 weeks; P = 0.015). These data demonstrate that lactation-associated changes in calcium and bone metabolism are physiological and are independent of dietary calcium supply in women with very low calcium intakes.