Sarcopenia - Molecular mechanisms and open questions.

Sarcopenia represents a muscle-wasting syndrome characterized by progressive and generalized degenerative loss of skeletal muscle mass, quality, and strength occurring during normal aging. Sarcopenia patients are mainly suffering from the loss in muscle strength and are faced with mobility disorders reducing their quality of life and are, therefore, at higher risk for morbidity (falls, bone fracture, metabolic diseases) and mortality. Several molecular mechanisms have been described as causes for sarcopenia that refer to very different levels of muscle physiology. These mechanisms cover e. g. function of hormones (e. g. IGF-1 and Insulin), muscle fiber composition and neuromuscular drive, myo-satellite cell potential to differentiate and proliferate, inflammatory pathways as well as intracellular mechanisms in the processes of proteostasis and mitochondrial function. In this review, we describe sarcopenia as a muscle-wasting syndrome distinct from other atrophic diseases and summarize the current view on molecular causes of sarcopenia development as well as open questions provoking further research efforts for establishing efficient lifestyle and therapeutic interventions.

Measuring redox effects on the activities of intracellular proteases such as the 20S Proteasome and the Immuno-Proteasome with fluorogenic peptides.

Proteolytic enzymes are often strongly affected by redox reactions, free radicals, oxidation, or oxidative stress. The 20S Proteasome and the Immuno-Proteasome are examples of major intracellular proteases whose concentration, transcription, translation, and proteolytic activity are all subject to redox regulation. Proteasomes are essential in maintaining overall protein homeostasis (or proteostasis), and their dysregulation results in detrimental phenotypes associated with various pathologies, including several common age-related diseases. Many studies have used Western blots to assess redox changes in Proteasome protein levels or RT-PCR to study RNA transcript levels, but actual measurements of proteolytic activity are far less common. Since each intact protein substrate exhibits a different proteolytic profile when incubated with proteasome or Immuno-Proteasome [± activators such as 19S or 11S (also called PA28)] and these proteolytic profiles are drastically altered if the protein substrate is denatured, for example by oxidation, heat, acetylation, or methylation. In an attempt to standardize proteasomal activity measurements small fluorogenic protein/peptide substrates were developed to test the three proteolytically active sites of the Proteasome and Immuno-Proteasome: trypsin-like, chymotrypsin-like, and caspase-like activities. Despite extensive use of fluorogenic peptide substrates to measure proteasome activity, there is an absence of a standardized set of best practices. In this study we analyze different parameters, such as sample concentration, AMC conjugated substrate concentration, duration of assay, and frequency of measurements, and examine how they impact the determination of Proteasome and Immuno-Proteasome activities using fluorogenic peptide substrates.

Limitations to adaptive homeostasis in an hyperoxia-induced model of accelerated ageing.

The Nrf2 signal transduction pathway plays a major role in adaptive responses to oxidative stress and in maintaining adaptive homeostasis, yet Nrf2 signaling undergoes a significant age-dependent decline that is still poorly understood. We used mouse embryonic fibroblasts (MEFs) cultured under hyperoxic conditions of 40% O2, as a model of accelerated ageing. Hyperoxia increased baseline levels of Nrf2 and multiple transcriptional targets (20S Proteasome, Immunoproteasome, Lon protease, NQO1, and HO-1), but resulted in loss of cellular ability to adapt to signaling levels (1.0 µM) of H2O2. In contrast, MEFs cultured at physiologically relevant conditions of 5% O2 exhibited a transient induction of Nrf2 Phase II target genes and stress-protective enzymes (the Lon protease and OXR1) following H2O2 treatment. Importantly, all of these effects have been seen in older cells and organisms. Levels of Two major Nrf2 inhibitors, Bach1 and c-Myc, were strongly elevated by hyperoxia and appeared to exert a ceiling on Nrf2 signaling. Bach1 and c-Myc also increase during ageing and may thus be the mechanism by which adaptive homeostasis is compromised with age.

Sex-specific stress tolerance, proteolysis, and lifespan in the invertebrate Tigriopus californicus.

Because stress tolerance and longevity are mechanistically and phenotypically linked, the sex with higher acute stress tolerance might be expected to also live longer. On the other hand, the association between stress tolerance and lifespan may be complicated by tradeoffs between acute tolerance and long-term survival. Here we use the copepod Tigriopus californicus to test for sex differences in stress resistance, proteolytic activity and longevity. Unlike many model organisms, this species does not have sex chromosomes. However, substantial sex differences were still observed. Females were found to have superior tolerance to a range of acute stressors (high temperature, high salinity, low salinity, copper and bisphenol A (BPA)) across a variety of treatments including different populations, pure vs. hybrid crosses, and different shading environments. Upregulation of proteolytic capacity - one molecular mechanism for responding to acute stress - was also found to be sexually dimorphic. In the combined stress treatment of chronic copper exposure followed by acute heat exposure, proteolytic capacity was suppressed for males. Females, however, maintained a robust proteolytic stress response. While females consistently showed greater tolerance to short-term stress, lifespan was largely equivalent between the two sexes under both benign conditions and mild thermal stress. Our findings indicate that short-term stress tolerance does not predict long-term survival under relatively mild conditions.

To adapt or not to adapt: Consequences of declining Adaptive Homeostasis and Proteostasis with age.

Many consequences of ageing can be broadly attributed to the inability to maintain homeostasis. Multiple markers of ageing have been identified, including loss of protein homeostasis, increased inflammation, and declining metabolism. Although much effort has been focused on characterization of the ageing phenotype, much less is understood about the underlying causes of ageing. To address this gap, we outline the age-associated consequences of dysregulation of 'Adaptive Homeostasis' and its proposed contributing role as an accelerator of the ageing phenotype. Adaptive Homeostasis is a phenomenon, shared across cells and tissues of both simple and complex organisms, that enables the transient plastic expansion or contraction of the homeostatic range to modulate stress-protective systems (such as the Proteasome, the Immunoproteasome, and the Lon protease) in response to varying internal and external environments. The age-related rise in the baseline of stress-protective systems and the inability to increase beyond a physiological ceiling is likely a contributor to the reduction and loss of Adaptive Homeostasis. We propose that dysregulation of Adaptive Homeostasis in the final third of lifespan is a significant factor in the ageing process, while successful maintenance of Adaptive Homeostasis below a physiological ceiling results in extended longevity.

The role of oxidative stress in anxiety disorder: cause or consequence?

Anxiety disorders are the most common mental illness in the USA affecting 18% of the population. The cause(s) of anxiety disorders is/are not completely clear, and research in the neurobiology of anxiety at the molecular level is still rather limited. Although mounting clinical and preclinical evidence now indicates that oxidative stress may be a major component of anxiety pathology, whether oxidative stress is the cause or consequence remains elusive. Studies conducted over the past few years suggest that anxiety disorders may be characterised by lowered antioxidant defences and increased oxidative damage to proteins, lipids, and nucleic acids. In particular, oxidative modifications to proteins have actually been proposed as a potential factor in the onset and progression of several psychiatric disorders, including anxiety and depressive disorders. Oxidised proteins are normally degraded by the proteasome proteolytic complex in the cell cytoplasm, nucleus, and endoplasmic reticulum. The Lon protease performs a similar protective function inside mitochondria. Impairment of the proteasome and/or the Lon protease results in the accumulation of toxic oxidised proteins in the brain, which can cause severe neuronal trauma. Recent evidence points to possible proteolytic dysfunction and accumulation of damaged, oxidised proteins as factors that may determine the appearance and severity of psychotic symptoms in mood disorders. Thus, critical interactions between oxidative stress, proteasome, and the Lon protease may provide keys to the molecular mechanisms involved in emotional regulation, and may also be of great help in designing and screening novel anxiolytics and antidepressants.

Long-term laboratory culture causes contrasting shifts in tolerance to two marine pollutants in copepods of the genus Tigriopus.

Organismal chemical tolerance is often used to assess ecological risk and monitor water quality, yet tolerance can differ between field- and lab-raised organisms. In this study, we examined how tolerance to copper (Cu) and tributyltin oxide (TBTO) in two species of marine copepods, Tigriopus japonicus and T. californicus, changed across generations under benign laboratory culture (in the absence of pre-exposure to chemicals). Both copepod species exhibited similar chemical-specific changes in tolerance, with laboratory maintenance resulting in increased Cu tolerance and decreased TBTO tolerance. To assess potential factors underlying these patterns, chemical tolerance was measured in conjunction with candidate environmental variables (temperature, UV radiation, diet type, and starvation). The largest chemical-specific effect was found for starvation, which decreased TBTO tolerance but had no effect on Cu tolerance. Understanding how chemical-specific tolerance can change in the laboratory will be critical in strengthening bioassays and their applications for environmental protection and chemical management.

The Oxygen Paradox, the French Paradox, and age-related diseases.

A paradox is a seemingly absurd or impossible concept, proposition, or theory that is often difficult to understand or explain, sometimes apparently self-contradictory, and yet ultimately correct or true. How is it possible, for example, that oxygen "a toxic environmental poison" could be also indispensable for life (Beckman and Ames Physiol Rev 78(2):547-81, 1998; Stadtman and Berlett Chem Res Toxicol 10(5):485-94, 1997)?: the so-called Oxygen Paradox (Davies and Ursini 1995; Davies Biochem Soc Symp 61:1-31, 1995). How can French people apparently disregard the rule that high dietary intakes of cholesterol and saturated fats (e.g., cheese and paté) will result in an early death from cardiovascular diseases (Renaud and de Lorgeril Lancet 339(8808):1523-6, 1992; Catalgol et al. Front Pharmacol 3:141, 2012; Eisenberg et al. Nat Med 22(12):1428-1438, 2016)?: the so-called, French Paradox. Doubtless, the truth is not a duality and epistemological bias probably generates apparently self-contradictory conclusions. Perhaps nowhere in biology are there so many apparently contradictory views, and even experimental results, affecting human physiology and pathology as in the fields of free radicals and oxidative stress, antioxidants, foods and drinks, and dietary recommendations; this is particularly true when issues such as disease-susceptibility or avoidance, "healthspan," "lifespan," and ageing are involved. Consider, for example, the apparently paradoxical observation that treatment with low doses of a substance that is toxic at high concentrations may actually induce transient adaptations that protect against a subsequent exposure to the same (or similar) toxin. This particular paradox is now mechanistically explained as "Adaptive Homeostasis" (Davies Mol Asp Med 49:1-7, 2016; Pomatto et al. 2017a; Lomeli et al. Clin Sci (Lond) 131(21):2573-2599, 2017; Pomatto and Davies 2017); the non-damaging process by which an apparent toxicant can activate biological signal transduction pathways to increase expression of protective genes, by mechanisms that are completely different from those by which the same agent induces toxicity at high concentrations. In this review, we explore the influences and effects of paradoxes such as the Oxygen Paradox and the French Paradox on the etiology, progression, and outcomes of many of the major human age-related diseases, as well as the basic biological phenomenon of ageing itself.

Latitudinal Clines in Temperature and Salinity Tolerance in Tidepool Copepods.

Local adaptation has been understudied in marine systems, but might be expected to be pronounced in the tidepool copepod Tigriopus californicus, which has a broad geographic range and extremely restricted dispersal. Tolerance to temperature and salinity was assessed in 14 populations over a 20° latitudinal range. Adaptive differentiation to temperature and salinity was found at scales as low as 5.6 km. Latitudinal clines were significant, with northern populations being more tolerant of low salinity and less tolerant of high temperature and high salinity. Both temperature and salinity tolerance were more closely associated with long-term thermal maxima than with long-term precipitation data. Hyperthermal and hyposmotic tolerance were inversely correlated, a pattern that could potentially slow adaptation to future conditions. Together, these studies of intraspecific geographic patterns in resistance to multiple stressors are important in predicting how environmental change may effect range shifts and local extinctions.

Variation in tolerance to common marine pollutants among different populations in two species of the marine copepod Tigriopus.

Geographical variation in chemical tolerance within a species can significantly influence results of whole animal bioassays, yet a literature survey showed that the majority of articles using bioassays did not provide detail on the original field collection site of their test specimens confounding the ability for accurate replication and comparison of results. Biological variation as a result of population-specific tolerance, if not addressed, can be misinterpreted as experimental error. Our studies of two marine copepod species, Tigriopus japonicus and Tigriopus californicus, found significant intra- and inter-specific variation in tolerance to copper and tributyltin. Because both species tolerate copper concentrations orders of magnitude higher than those found in coastal waters, difference in copper tolerance may be a by-product of adaptation to other stressors such as high temperature. Controlling for inter-population tolerance variation will greatly strengthen the application of bioassays in chemical toxicity tests.

Acclimation and adaptation to common marine pollutants in the copepod Tigriopus californicus.

Establishing water quality criteria using bioassays is complicated by variation in chemical tolerance between populations. Two major contributors to this variation are acclimation and adaptation, which are both linked to exposure history, but differ in how long their effects are maintained. Our study examines how tolerance changes over multiple generations of exposure to two common marine pollutants, copper (Cu) and tributyltin oxide (TBTO), in a sexually reproducing marine copepod, Tigriopus californicus. Lines of T. californicus were chronically exposed to sub-lethal levels of Cu and TBTO for 12 generations followed by a recovery period of 3 generations in seawater control conditions. At each generation, the average number of offspring produced and survived to 28 d was determined and used as the metric of tolerance. Lines exposed to Cu and TBTO showed an overall increase in tolerance over time. Increased Cu tolerance arose by generation 3 in the chronically exposed lines and was lost after 3 generations in seawater control conditions. Increased TBTO tolerance was detected at generation 7 and was maintained even after 3 generations in seawater control conditions. It was concluded from this study that tolerance to Cu is consistent with acclimation, a quick gain and loss of tolerance. In contrast, TBTO tolerance is consistent with adaptation, in which onset of tolerance was delayed relative to an acclimation response and maintained in the absence of exposure. These findings illustrate that consideration of exposure history is necessary when using bioassays to measure chemical tolerance.