Search

The goal of this study is to identify a signature of bioenergetic and functional markers in the muscles of individuals with Parkinson’s disease (PD). Quantitative physiological properties of in vivo hand muscle (FDI, first dorsal interosseus) and leg muscle (TA, Tibialis Anterior) of older individuals with PD were compared to historical age/gender-matched controls (N = 30). Magnetic resonance spectroscopy and imaging (MRS) were used to assess in vivo mitochondrial and cell energetic dysfunction, including maximum mitochondrial ATP production (ATPmax), NAD concentrations linked to energy/stress pathways, and muscle size. Muscle function was measured via a single muscle fatigue test. TA ATPmax and NAD levels were significantly lower in the PD cohort compared to controls (ATPmax: 0.66 mM/s ± 0.03 vs. 0.76 ± 0.02; NAD: 0.75 mM ± 0.05 vs. 0.91 ± 0.04). Muscle endurance and specific force were also lower in both hand and leg muscles in the PD subjects. Exploratory analyses of mitochondrial markers and individual symptoms suggested that higher ATPmax was associated with a greater sense of motivation and engagement and less REM sleep behavior disorder (RBD). ATPmax was not associated with clinical severity or individual symptom(s), years since diagnosis, or quality of life. Results from this pilot study contribute to a growing body of evidence that PD is not a brain disease, but a systemic metabolic syndrome with disrupted cellular energetics and function in peripheral tissues. The significant impairment of both mitochondrial ATP production and resting metabolite levels in the TA muscles of the PD patients suggests that skeletal muscle mitochondrial function may be an important tool for mechanistic understanding and clinical application in PD patients. This study looked at individuals with mid-stage PD; future research should evaluate whether the observed metabolic perturbations in muscle dysfunction occur in the early stages of the disease and whether they have value as theragnostic biomarkers.

Background: Disruption of metabolic and bioenergetic homeostasis related to mitochondrial dysfunction is a key driver of aging biology. Therefore, targeting mitochondrial function would be a rational approach to slowing aging. Elamipretide (Elam, a.k.a. SS-31) is a peptide known to target mitochondria and suppress mammalian signs of aging. The present study was designed to examine the phenotypic effects of long-term Elam treatment on aging in C57BL/6 mice starting at 18 months of age. Methods: Mice were fed regular chow (RC diet) or a diet high in fat and sugar (HF diet) and treated with 3 mg/ kg of Elam or saline subcutaneously 5 days per week for 10 months. Physiological performance assessments were conducted at 28 months of age. Results: Elam improved the physical performance of males but not females, while in females Elam improved cognitive performance and enhanced the maintenance of body weight and fat mass. It also improved diastolic function in both males and females, but to a greater extent in males. The HF diet over 10 months had a negative effect on health span, as it increased body fat and decreased muscle strength and heart function, especially in females. Conclusions: Elam enhanced healthy aging and cardiac function in both male and female mice, although the specific effects on function differed between sexes. In females, the treatment led to better cognitive performance and maintenance of body composition, while in males, performance on a rotating rod was preserved. These overall observations have translational implications for considering additional studies using Elam in therapeutic or preventive approaches for aging and age-related diseases. Keywords: Aging, mitochondria, elamipretide, C57BL/6 mice, high fat and sugar diet

This randomized clinical trial evaluates the improvement in the 6-minute walk distance test and in leg and hand muscle endurance after use of urolithin A in patients aged 65 to 90 years., Key Points Question What is the effect of supplementation with urolithin A, a natural gut microbiome–derived food metabolite, on skeletal muscle performance and mitochondrial health in older adults? Findings In this randomized clinical trial of 66 older adults, those who received supplementation with 1000 mg of urolithin A had a significant improvement in muscle endurance (number of muscle contractions until fatigue) for both hand and leg skeletal muscles compared with those who used placebo. Plasma levels of several acylcarnitines, ceramides (biomarkers of mitochondrial health), and C-reactive protein were decreased after urolithin A supplementation. Meaning These findings indicate that urolithin A was safe and well tolerated as well as beneficial for muscle endurance and mitochondrial health in older adults; it may also be a promising approach to counteracting age-associated muscle decline., Importance Aging is associated with a decline in mitochondrial function and reduced exercise capacity. Urolithin A is a natural gut microbiome–derived food metabolite that has been shown to stimulate mitophagy and improve muscle function in older animals and to induce mitochondrial gene expression in older humans. Objective To investigate whether oral administration of urolithin A improved the 6-minute walk distance, muscle endurance in hand and leg muscles, and biomarkers associated with mitochondrial and cellular health. Design, Setting, and Participants This double-blind, placebo-controlled randomized clinical trial in adults aged 65 to 90 years was conducted at a medical center and a cancer research center in Seattle, Washington, from March 1, 2018, to July 30, 2020. Muscle fatigue tests and plasma analysis of biomarkers were assessed at baseline, 2 months, and 4 months. Six-minute walk distance and maximal ATP production were assessed using magnetic resonance spectroscopy at baseline and at the end of study at 4 months. The analysis used an intention-to-treat approach. Interventions Participants were randomized to receive daily oral supplementation with either 1000 mg urolithin A or placebo for 4 months. Main Outcomes and Measures The primary end point was change from baseline in the 6-minute walk distance and change from baseline to 4 months in maximal ATP production in the hand skeletal muscle. The secondary end points were change in muscle endurance of 2 skeletal muscles (tibialis anterior [TA] in the leg and first dorsal interosseus [FDI] in the hand). Cellular health biomarkers were investigated via plasma metabolomics. Adverse events were recorded and compared between the 2 groups during the intervention period. Results A total of 66 participants were randomized to either the urolithin A (n = 33) or the placebo (n = 33) intervention group. These participants had a mean (SD) age of 71.7 (4.94) years, were predominantly women (50 [75.8%]), and were all White individuals. Urolithin A, compared with placebo, significantly improved muscle endurance (ie, increase in the number of muscle contractions until fatigue from baseline) in the FDI and TA at 2 months (urolithin A: FDI, 95.3 [115.5] and TA, 41.4 [65.5]; placebo: FDI, 11.6 [147.4] and TA, 5.7 [127.1]). Plasma levels of several acylcarnitines, ceramides, and C-reactive protein were decreased by urolithin A, compared with placebo, at 4 months (baseline vs 4 mo: urolithin A, 2.14 [2.15] vs 2.07 [1.46]; placebo, 2.17 [2.52] vs 2.65 [1.86]). The mean (SD) increase from baseline in the 6-minute walk distance was 60.8 (67.2) m in the urolithin A group and 42.5 (73.3) m in the placebo group. The mean (SD) change from baseline to 4 months in maximal ATP production in the FDI was 0.07 (0.23) mM/s in the urolithin A group and 0.06 (0.20) mM/s in the placebo group; for the TA, it was −0.03 (0.10) mM/s in the urolithin A group and 0.03 (0.10) mM/s in the placebo group. These results showed no significant improvement with urolithin A supplementation compared with placebo. No statistical differences in adverse events were observed between the 2 groups. Conclusions and Relevance This randomized clinical trial found that urolithin A supplementation was safe and well tolerated in the assessed population. Although the improvements in the 6-minute walk distance and maximal ATP production in the hand muscle were not significant in the urolithin A group vs the placebo group, long-term urolithin A supplementation was beneficial for muscle endurance and plasma biomarkers, suggesting that urolithin A may counteract age-associated muscle decline; however, future work is needed to confirm this finding. Trial Registration ClinicalTrials.gov Identifier: NCT03283462

More than half a century of skeletal muscle research is continuing at Padua University (Italy) under the auspices of the Interdepartmental Research Centre of Myology (CIR-Myo), the European Journal of Translational Myology (EJTM) and recently also with the support of the A&CM-C Foundation for Translational Myology, Padova, Italy. The Volume 30(1), 2020 of the EJTM opens with the collection of abstracts for the conference “2020 Padua Muscle Days: Mobility Medicine 30 years of Translational Research”. This is an international conference that will be held between March 18-21, 2020 in Euganei Hills and Padova in Italy. The abstracts are excellent examples of translational research and of the multidimensional approaches that are needed to classify and manage (in both the acute and chronic phases) diseases of Mobility that span from neurologic, metabolic and traumatic syndromes to the biological process of aging. One of the typical aim of Physical Medicine and Rehabilitation is indeed to reduce pain and increase mobility enough to enable impaired persons to walk freely, garden, and drive again. The excellent contents of this Collection of Abstracts reflect the high scientific caliber of researchers and clinicians who are eager to present their results at the PaduaMuscleDays. A series of EJTM Communications will also add to this preliminary evidence., Multiple biological pathways contribute to aging. Some prominent pathways and causes are genome instability, immunosenescence, inflammation, the NAD+ salvage pathways, proteostasis, and mitochondrial dysfunction. Altered gene regulation influenced by epigenetics, circadian rhythms, diet and physical activity levels also influences the rate of aging. Our Institute on Aging and collaborators are involved with multiple clinical trials with the goal to target these pathways and to improve physical and cognitive function or slow the rate of decline typically seen during aging. The ultimate goal is to develop interventions that can maintain health and independence of older adults. Trials ongoing and/or completed have utilized compounds shown to extend health-span and/or life span in invertebrates (c. elegans) or vertebrates (mice/rats). The compounds used range from nutritional (nicotinamide riboside, epicatechin, resveratrol, omega 3, fermented papaya) to pharmacological (metformin, aspirin, rapamycin, telmisartin/losartan, testosterone), lifestyle-physiological interventions (calorie restriction, exercise, fasting regimes). These biological compounds and lifestyle interventions target single or multiple pathways to potentially improve mitochondrial function, mitobiogenesis, autophagy, angiogenesis, nitric oxide production, and levels of NAD+ and/or reduce pathways or biomarkers of inflammation or senescence. In most of these trials we collect skeletal muscle, fat, white blood cells and plasma to determine if the compound alters the proposed biological pathway. We will briefly discuss some clinical trials and highlight findings on a recently completed clinical trial using epicatechins a flavonoid, which improved endothelial and mitochondrial function as well as walking speed. We will also highlight the challenges and limitations in conducting clinical trials, such as dose, duration of study, targeting single vs. multiple pathways, combining an intervention with exercise and/or intermittent fasting.1-5, Age-related cognitive decline has become a major public health concern. Even in the absence of frank neurodegenerative disorders, both cognitive and brain function decline as a function of advancing age. At present, there are few effective intervention options for remediating age-related cognitive and neural decline. Fermented Papaya Product has previously shown promise in human and animal models as a potential nutraceutical compound for impacting mitochondrial function, inflammation, and other age-related processes that contribute to cognitive and neural decline.1-3 The current study presents data from a pilot crossover clinical trials in 28 healthy older adults undergoing 8 weeks of treatment on either 9mg/day FPP vs. 9mg/day placebo (table sugar) with a 6-week washout period. Participants underwent multimodal magnetic resonance imaging and spectroscopy before and after each arm, as well as comprehensive neurocognitive assessment. Results demonstrated promising effect sizes on default mode resting state connectivity (Cohen’s d = .44), measures of verbal fluency (d = 1.16) and attention (d = .61), as well as two out of three novel markers of mitochondrial function from 31P magnetic resonance spectroscopy and neuroinflammation from free-water quantification in diffusion weighted imaging (d = .38). While results failed to survive significance after multiple comparison correction, the pattern of effect size improvement in FPP vs. placebo demonstrate significant promise for further investigation in larger Phase II randomized clinical trials. Potential implications for both healthy older adults and adults with neurodegenerative disease will be discussed., Prolonged oxidative stress may play a key role in tumor development. Antioxidants molecules are contained in many foods and seem to have potential role in future anti-tumor strategies. Among the natural antioxidants, the beneficial effect of Fermented Papaya (FPP®) is known. The aim of this study was to investigate the effects of orally administered FPP® in either prevention or treatment of a murine model of melanoma. The tumor growth was analyzed together with the blood levels of both oxidants (ROS) and anti-oxidants (SOD-1 and GSH). The results showed that FPP® controlled tumor growth, reducing the tumor mass of about 3 to 7 times vs untreated mice. The most significant effect was obtained with sublingual administration of FPP® close to the inoculation of melanoma. At the time of the sacrifice, none of mice treated with FPP® had metastases, the subcutaneous tumors were significantly smaller, and amelanotic compared to untreated mice. Moreover, the FPP® anti-tumor effect was consistent with the decrease of total ROS levels and the increase in the blood levels of GSH and SOD-1. This study shows that a potent anti-oxidant treatment through FPP® may contribute to both preventing and inhibiting tumors growth. The results of the above study suggested that FPP® while showing a clear anti-tumor effect it occurred through the in vivo induction of a potent anti-oxidant reaction. In a new set of experiments, we wanted to verify whether FPP had a clear and scientifically solid in vivo anti-aging effect together with the induction of the anti-oxidant reaction. To this purpose we used a mouse model suitable for aging studies (C576J) treating daily each mouse from 4 weeks of life to 10 months with the same dose of IMMUNEAGE dissolved into the daily water as compared to mice receiving only tap water. At the end of the treatment period (10 months) we measured some biological parameters related to aging cell processes: i) the total anti-oxydant capacity in the plasma of mice treated or untreated with FPP®; ii) the telomerase activity in the plasma of mice treated or untreated with FPP®; iii) the telomeres length in the bone marrow and ovaries of mice treated or untreated with FPP®. The results showed that the blood of treated mice, at the end of the treatment period (10 months) had 2-3 folds more anti-oxidant power and telomerase activities than the untreated mice. In the same mice at the sacrifice, we collected both the bone marrow (from the tibias) and the ovaries. We measured the telomere lengths in both cellular preparations. The results showed that daily FPP® assumption induced 3 folds increase in telomeres length in bone marrow and ovary of treated mice as compared to the untreated mice. This suggests that FPP® induce a clear improvement of the aging biomarkers and that the treated mice were for those variables younger than the untreated ones., Increased intake of a diet high in phosphorus (P) or phosphate (Pi) is associated with an increased risk of mortality in patients with chronic kidney disease or cardiovascular disease. Muscle fibers with ectopic calcification are observed in young patients with severe muscle degeneration. Ectopic calcification was also observed in the skeletal and cardiac muscles of mdx mice. In this study, we determined the effects of dietary phosphorus intake on ectopic calcification in skeletal muscle, muscle performance (specific maximal forces and daily running activity), and pathological features during exercise-induced stress in mdx mice. Increased level of dietary P or Pi intake also increased ectopic calcification and decreased muscle performance in mdx mice. On the other hand, decreased level of dietary P or Pi intake decreased ectopic calcification and muscle degeneration and improved muscle performance. When we added calcium absorber in high phosphorus diet, the ectopic calcification decreased significantly. In C2C12 muscle cell cultures, the increased concentration of Pi in culture medium inhibited myogenesis in a dose-dependent manner. Therefore, we propose that phosphorus (P) or phosphate (Pi) is one of the modifiers in regulating muscle degradation/inflammation in vivo and in vitro. In addition, we also found that the long-lasting Vitamin C, ascorbic acid-2 phosphate added into culture medium supported myogenesis even under high Pi- or low temperature-culture conditions. These results suggest that vitamin C may have therapeutic effect on muscle degeneration caused by increased level of Pi or lowered culture temperature., The neuromuscular junctions (NMJ) are specialized synapses between motor neuron and muscle fibers. They are targets for a variety of neuromuscular diseases. Maintenance and remodeling of NMJ are critical to treat these diseases. Different therapeutic strategies for neuromuscular diseases have been developed ranging from gene therapy to antisense-mediated exon skipping and nonsense mutation suppression by small molecules. We are attempting to search for therapeutic potential molecules from natural sources such as several microorganisms and chemical libraries. Among others, natural bioactive molecules, glucocorticoids and vitamin D have been proposed as potential treatments to delay progression in neuromuscular diseases and aging. However, the direct mechanisms for skeletal muscle have not been fully characterized. Recently, using cultured C2C12 myotubes, we showed that glucocorticoids and vitamin D enhance agrin-induced acetylcholine receptors (AChR) clustering compared to agrin alone. However, sex hormones such as estradiol and testosterone are less effective. To elucidate the physiological role of these compounds in agrin-induced AChR clustering, we investigated and will discuss their effects on expression of key molecules involved in AChR cluster formation and maintenance. This work was supported in part by Japan Society for the Promotion of Science, JSPS Grants-in-Aid for Scientific Research, Grant number:17K10089., Japan is facing a “super-aging” society. Seniors over 65 is 28.4% of the population and the life expectancy is the highest in the world, however, the nation’s annual medical cost is now over 42.6 trillion yen. If this trend continues, the Japanese government will not be able to sustain the social services. Therefore, Osato Research Institute has been conducting researches, aimed at reducing medical costs through Preventive Medicine. Our three core activities for achieving this goal are Education, Researches on Fermented Foods, and Improving QOL of senior generation. Firstly, we think the best preventive medicine is education. For example, AIDS is a typical disease that can be prevented by education. We have been working on young people’s education for AIDS prevention with Prof. Montagnier, president of World Foundation AIDS Research and Prevention at UNESCO. Secondly, we have been focusing our attention on “Fermented Food” since traditional fermented foods are considered to be a key to the world's highest longevity of Japanese people. Among our numerous studies, I will present 5 researches on Fermented Papaya Preparation,1-5 which may be a desired candidate for preventive medicine. Finally, we would like to introduce our activities of “Project ORI wine”. We established vineyards to make a place for social activities especially for local retired people. It would give them fun and challenge to live better and keep them productive and healthy. In this way, we hope we could have happy world not only for young but also for senior people., Mitochondria play a crucial role in health and disease, imposing a growing demand for evaluation of mitochondrial function in inherited and preventable degenerative diseases. These are associated with lack of physical exercise and obesity, causing decline of mitochondrial fitness and early aging. High-resolution respirometry (HRR) is based on the state-of-the-art instrument (Oroboros O2k, Innsbruck, Austria) for mitochondrial and cell respiration.1 The O2k combines real-time respirometry with simultaneous measurement of H2O2 production, mt-membrane potential, ADP-ATP exchange, pH, Ca2+, or nitric oxide. HRR can be applied with a wide spectrum of sample preparations (e.g. isolated mitochondria, tissue homogenate, permeabilized fibers, permeabilized cells, and living cells). Optimized substrate-uncoupler-inhibitor titration (SUIT) protocols offer a powerful tool for comprehensive mitochondrial physiology analysis by HRR.2 Peripheral blood mononuclear cells, platelets and permeabilized fibers are relevant models for diagnostic analysis of oxidative phosphorylation (OXPHOS) in clinical applications (e.g. aging, sarcopenia, obesity). Science and particularly mitochondrial respiratory research face a reproducibility crisis.3 Consequently, Oroboros is strongly committed towards reproducibility by promoting: (1) O2k quality control (i.e. calibrations, instrumental background, DatLab software); (2) proficiency tests with reference samples for inter-laboratory harmonization4,5; and (3) training by scientific experts. Supported by the COST Action MitoEAGLE network, we generate reference values for an Open-Access mitochondrial respirometry database, which requires harmonization of nomenclature.6 With a large O2k-Network, the Oroboros Ecosystem operates in the frame of collaborative Open-Innovation (NextGen-O2k with Q-redox sensor, NAD(P)H autofluorescence and PhotoBiology module) to promote quality and reproducibility in mitochondrial physiology. Support. Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE. Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770., Skeletal muscle plays a significant role in the regulation of whole-body metabolism, and mitochondria are essential organelles for ATP production, thus the studies focused on skeletal muscle mitochondrial activity are necessary to understand the molecular mechanisms controlling the skeletal muscle homeostasis.1 One of the key regulators of mitochondrial metabolism is the second messenger Ca2+. Upon physiological stimuli, skeletal muscle mitochondria rapidly and efficiently accumulate Ca2+ into their matrix via an electrogenic pathway, which relies on the driving force of a steep electrochemical gradient. A large [Ca2+]mt peak occurs dynamically in parallel to agonist-induced [Ca2+]cyt increases, thanks to the activity of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ accumulation.2 Mitochondrial Ca2+ stimulates aerobic metabolism, tightly regulating three dehydrogenases of the TCA cycle, thus the alteration of mitochondrial Ca2+ homeostasis leads to a dysregulation of metabolism.3 In this talk, we will show an overview of techniques to monitor mitochondrial activity in skeletal muscle, starting with the ex vivo measure of Ca2+ signaling, in terms of cytosolic Ca2+ concentration and mitochondrial Ca2+ uptake, in single isolated fibers thanks to the use of genetically encoded Ca2+ probes.4 Then, to investigate skeletal muscle mitochondrial metabolism we take advantage of genetically encoded indicators for ATP and NADH (Perceval and Peredox probes), and O2 consumption rate measurements.5 Finally, mitophagy, the removal of damaged mitochondria, is a necessary cellular process to maintain healthy muscle homeostasis. Mt-Keima probe is an easy tool to monitor this phenomenon in muscle fibers. Together, all these techniques allow studying many processes that regulate mitochondrial metabolism, a key aspect in the pathophysiology of skeletal muscle., Mitochondria are implicated in atrophy of aging skeletal muscle, yet the link between specific changes in mitochondrial function and aging muscle atrophy has not been established. Amongst many changes occurring with aging, there is a sensitization to mitochondrial permeability transition (MPT) in aged skeletal muscle.1 Building on this observation, we took advantage of heterogeneity in atrophy with aging between hindlimb muscles of the rat, in combination with a muscle cell culture system, to evaluate the hypothesis that MPT is a mechanism of aging muscle atrophy. Our results showed that, exclusive to muscles that atrophy with aging, mitochondria are sensitized to MPT and there is a marked increase in nuclei positive for the mitochondrial-derived protein, apoptosis inducing factor (AIF). Within a given muscle, aged muscle fibers harboring AIF-positive nuclei are severely atrophied relative to normal muscle fibers. In a muscle cell culture system, induction of MPT using doxorubicin is coincident with AIF translocation to myotube nuclei and causes atrophy that can be blocked using inhibitors of MPT (cyclosporin A, bonkrekic acid), inhibiting mitochondrial reactive oxygen species (ROS; using MitoTempo), or knocking down caspase 3 (using shRNA). Together, these results show that: (1) MPT is linked to aging muscle atrophy from the whole muscle to single fiber level; and (2) MPT causes muscle cell atrophy in cell culture in a manner that depends upon both mitochondrial ROS and caspase 3. Our findings thus support the hypothesis that MPT is a mechanism of aging muscle atrophy, FBXL4-related encephalomyopathic mitochondrial DNA depletion syndrome is an autosomal recessive severe, multi-systemic mitochondrial disease with 48 known pathogenic variants. To better understand FBXL4 function and therapies, we characterized novel C. elegans and D. rerio models of FBXL4 disease. This study particularly focuses on analyzing the neuromuscular function of these genetic models using manual and automated assays. We investigated fbxl-1 worms’ neuromuscular function analyzing pharyngeal pump rate, body bends rate and overall motor activity using manual and automated assays. A novel automated video-tracking approach was used to evaluate overall worms’ activity in liquid media. In the latter case, we created a protocol based on the use of the Zebrabox (Viewpoint), automated software created and used for analyzing zebrafish larvae activity and only in this study, applied to worms. Worms’ neuromuscular function was impaired with significantly increased and uncoordinated pharyngeal pumping rate in young adult and decreased rate during aging. Motility was reduced by ~70% compared to N2 control worms. Automated Video-tracking analysis was also used to analyze swim activity in fbxl4sa12470 zebrafish larvae carrying a missense mutation in the FBXL4 allele. Data shows decreased (~50 %) swimming activity in basic condition and after applying stimuli of light on/off cycles. Overall, these data demonstrate that FBXL4 deficiency can be effectively modeled in simple translational animals; new protocols were created for using automated software applications offering robust translational platforms in which therapies can now be modeled to reverse organ-level and behavioral dysfunction directly relevant to the human disease., The neuromuscular junction (NMJ) plays an essential role in enabling the cross-talk between the motoneuron (MN) and skeletal muscle. With progressive age, oxidative damage due to mitochondrial dysfunction, seems to cause a ‘dying-back’ phenomenon, where damage at the axon terminal, causes degeneration of the entire cell.1 The ensuing death of MNs leads to muscle fibre denervation and loss of the entire motor units, a main cause of sarcopenia.2 NMJ degeneration may also arise from changes at the muscle level. In ageing muscle, several phenomena could also cause NMJ damage: mitochondrial dysfunction, increased ROS production, decreased PGC-1alpha expression, known to trigger NMJ changes with ageing.3 Inactivity also promotes muscle fibre denervation as increased neural cell adhesion molecule (N-CAM), marker of denervation/reinnervation, has been found after short periods of bed rest (10-14 days) in young and older men.4,5 In this study, we tested the hypothesis that regular physical activity, such as recreational dancing, would protect against NMJ degeneration while improving functional performance of older individuals. Thirty-one participants aged 60>years (70.4 ± 5.1 years) were recruited for this study after Ethical approval and participants’ informed consent. Among these, 15 (9 female, 6 male) were sedentary (S), while 16 (10 female, 6 male) practiced regular recreational dancing (D) at least 1.5 h/week for a minimum of 1 year. NMJ degeneration was assessed from serum c-terminal agrin fragment (CAF) levels using a commercially available Elisa kit assay. Gait performance was tested with the timed 10m walk test (10WT), dynamic balance (WD) was tested with a stabilometric tablet measuring ankle oscillations, while timed get-up-and go (TUG) was tested as the time taken to get up from a chair, walk 3m and sit back. Muscle size and architecture of the knee extensors were assessed using ultrasonography. Significance was set at p, Stimulation of myogenic cells with vasopressin (AVP), oxytocin (OT), their analogs and antagonists revealed a previously unknown role of the neurohypophyseal hormones (NH) in modulating myogenic differentiation and trophism1 through the activation of complex intracellular signaling, the expression of the MRFs and the stimulation of the hypertrophy pathways2,3. Other studies demonstrated the expression of functional OT receptors (OTR) in human and murine satellite cells (reviewed in2). Extending these studies to in vivo models, we could show a highly stimulated expression of the V1a AVP receptor (V1aR) in post-injury muscle regeneration and assess that the overexpression of V1aR suffices, without exogenous hormone administration, to sustain muscle regeneration2. Furthermore, the administration of AVP rescued the inhibitory effect of TNF in a mouse model of cancer cachexia (reviewed in2). Works conducted in bovine and ovine livestock showed that animals treated with anabolizing steroids displayed dramatically increased OT expression in skeletal muscle, leading to a significant increase of plasma OT level and that the hypertrophying effect of anabolic steroids is mediated by increased OTR signaling (reviewed in 4). Furthermore, in a mouse model of aging, it was shown that reduced circulating OT levels accompany sarcopenia, and that satellite cells obtained from aged mice express reduced levels of OTR5. Efficient regeneration could be restored in aged mice by administering exogenous OT. Taken together, these studies not only confirm that NH target skeletal muscle increasing differentiation and trophism, but also point to muscle as a physiologic source of OT., Non-coding RNAs are emerging as important players in the regulation of several aspects of cellular biology. The expression and therefore the activity of non-coding RNAs is more tissue and cell specific than that of coding RNAs. Therefore, for a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. We compiled a comprehensive catalog of ncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them. We demonstrated that many ncRNAs can be involved in the biological processes de-regulated during muscle atrophy. Focusing our attention on a specific long non-coding RNA (Pvt1), activated early during muscle atrophy, we revealed that it impacts on mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of long non-coding RNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity., One of the consequences of aging is the reduction of the growth hormone/insulin-like growth factor I (GH/IGF-I) axis. For skeletal muscle, this leads to a loss of anabolic and regenerative capacity, which, in part, underlies the onset of sarcopenia. Maintenance of activity in aging can help offset functional deficits, and these benefits are both systemic and specific to skeletal muscle. It is an open question as to the contribution of muscle IGF-I activity to these beneficial adaptations. To address this, we imposed an endurance exercise regimen on a recently developed mouse model with muscle specific inducible deletion of IGF-I (MID mouse), and strain matched controls (CON), comparing their performance to sedentary mice of the same genotypes. After 4 weeks of daily endurance training at 15 m/min for 60 minutes, both MID and CON mice displayed similar and significant improvements in a run-to-exhaustion test. Body composition also displayed a reduction in proportional fat with training. However, in the subsequent 4 weeks of daily endurance training at 18 m/min for 60 minutes, only the CON mice continued to improve in the run-to-exhaustion test, even though there were no differences in the body composition changes in the exercised groups. Over the course of 8 weeks, the sedentary mice progressively gained fat, and by the end of the study, the sedentary MID mice ran for less time in the run-to-exhaustion test compared to sedentary CON mice. Taken together, this suggests that benefits to moderate intensity training can occur in the absence of muscle IGF-I. However, the capacity of muscle to adapt to higher intensity regimens may be limited when actions of local IGF-I signaling are impaired., Skeletal muscle atrophy induced by immobilization is one of the greatest issues in athletes as well in general population. The mechanisms underlying skeletal muscle atrophy, are reasonable known, however, there is no evidence of works that was able to avoid it. A strategy that could preclude the large recovery needed after immobilization would be of great importance. Our group made a research protocol that aims to study the effect of Branched-Chain Amino Acids (BCAA’s) administration to avoid skeletal muscle atrophy. In order to achieve our goals, we use an animal model divided in several groups that includes control, cast-immobilization, exercise animals and animals which have taken BCAA’s. We observed the structure of skeletal muscle, fibre type changes and the satellite cells turnover. To evaluate satellite cells, we used antibodies against Pax-7, Myf-5 and c-met, identification by fluorescence which allows to count the immunoreactive cells and study its evolution and differentiation. As expected, cast immobilization caused atrophy and muscle damage. The satellite cells were activated to regenerate the skeletal muscle, as seen by the increase in Pax-7 and Myf-5 expression with a decrease of c-met, suggesting mobilization of those cells. Visible cell niches in the muscle reinforced these data. The supplement had a partial protective effect when it was taken during immobilization, but it seems to hamper the muscle regeneration, when exercise is performed after immobilization. Thus, our results suggested that BCAA prevent skeletal muscle atrophy by acting over its satellite cells. Partially Supported: FCT, Portugal (Strategic Project UID/NEU/04539/2013), FEDER-COMPETE., According to Bartholomaei Eustachii, Aristotle was the first to describe the complicate interlacement of canaliculi which are in the petrosal bone, identifyng the “coclea”. Aristotle, to explain his idea from an anatomical point of view, compared the inner ear to the strovmboi, i.e. spiral shell (cochlea). Regarding the term labirinto, probably Falloppio was the first to use the term labyrinthus – of egyptian and not greek origin - to describe the inner ear: quum haec cavitas tot habeat meatus et cuniculos, merito labyrinthus dicetur, in quem prospicit fenestra ovalis, clausa a stapede, ecc.». The Anatomic school of Padova gave during the XVI century an enormous contribution to the knowledge of the ear: • Andreas Vesalius 1514-1564 • Andrea Falloppio 1523-1562 • Jeronimus Fabricius ab Acquapendente 1537-1619 • Bartolomeus Eustachi 1514-1574 • Matteo Colombo 1515-1559 • Giovanni Filippo Ingrassia 1510-1580 • Iulius Casserius 1552-1616 One of the major contributions of Vesalius was his suggestion that the organ of hearing should be removed from the skull for investigation. We remember here 3 important contributions: Bartolomeus Eustachi in his work entitled “Epistola de auditus organis” (Eustachi, 1564); Girolamo Fabrici ab Acquapendente De Visione Voce Auditu (1600) and Iulius Casserius De Vocis Auditusque Organis Historia 1601. In 1740, Antonio Valsalva published his anatomical observations on the human auditory system in which he pointed out the importance of the ossicular chain and the oval window for hearing and also observed that the innervation target for the auditory nerve was not the osseous spiral lamina, but was instead the membranous portions of the cochlea and that these areas of sensory epithelium represented, in the opinion of Valsalva, the true receptors of sound. Later, Antonio Scarpa, pupil of Giovanni Battista Morgagni and Marco Antonio Caldani, reported a liquid present within the cochlea's inner membranous compartment, that is, scala media. The Count Alessandro Volta is generally qualified as the first to stimulate the ear with the electricity. Volta, carried out on himself in the late 1790s the first experiment on electrical stimulation of the auditory nerve. Because of the unpleasant sensation experienced by the scientist, any other experiment was carried out over the next half century to study this effect. From the first half of the 18th century onwards, tremendous curiosity about electrical phenomena spread throughout Europe. Machines producing electrostatic electricity were produced, and lectures on electricity attracted members of academia as well as the ruling elite. A field known at the time as “medical electricity” emerged following the electrical researches and the discovery of the effects of electricity on the human body. In 1950, Lundberg performed one of the first recorded attempts to stimulate the auditory nerve with a sinusoidal current during a neurosurgical operation. His patient could only hear noise. In 1957 by Djourno and Eyries provided the first detailed description of the effects of directly stimulating the auditory nerve in deafness. They placed a wire on the auditory nerves that were exposed during an operation for cholesteatoma. When the current was applied to the wire, the patient described generally high-frequency sounds that resembled a “roulette wheel”. The signal generator provided up to 1,000 Hz and the patient gradually developed limited recognition of common words and improved lip-reading capabilities. At this point the clinical application of a cochlear implant started. A cochlear implant is a surgically implanted electronic device that provides a sense of sound to a person who is profoundly deaf or hard of hearing; at now approximately 500,000 people worldwide had received cochlear implants. Padova Audio/Otological group is strongly engaged in the diagnosis and treatment of deafness particularly in childhood and in basic research (Padua Bioacustic lab). Recently an international center was established in Venice: I APPROVE, the International Auditory Processing PROject in Venice, Maximal oxygen consumption (V̇O2max) denotes the reproducible upper limit of oxygen (energy) flux through the respiratory system into skeletal muscle mitochondria that can be reached during intense exercise with a large muscle mass. A high V̇O2max is a key requisite for success in all endurance sports such as cycling, cross-country skiing or running over longer distances. However, V̇O2max has also been strongly and negatively associated with cardiovascular diseases and all-cause mortality. V̇O2max can vary by more than twofold between untrained, sedentary subjects with a heritability value greater than 50%.1 Trainability for an individual's V̇ O2max also varies massively between subjects. Trainability is independent of sedentary V̇O2max with a similarly high heritability as sedentary V̇O2max.2 The high heritability of sedentary V̇O2max and trainability and its importance for athletic performance as well as health has prompted a massive search for its genetic underpinning. Candidate-gene studies, gene-expression studies and genome-wide-association studies (GWAS) have failed to identify a genetic signature of the high V̇ O2max phenotype.3 This may be due to the fact that there are vast multigenetic regulatory networks in skeletal muscle and in other organs that are responsible both for the set-point and the malleability of V̇O2max. Multigenetic phenotypes such as V̇O2max appear to be emergent properties of multiple underlying transcriptomic networks modified by epistasis, the epigenome and the epitranscriptome. It is unclear currently whether an artificial intelligence approach on sufficiently large datasets can make reliable predictions on multigenetic phenotypes such as V̇O2max., In the last decades sparse evidence of the therapeutic potentialities of surface electrical stimulation for the treatment of facial palsy has been published.1,2,3 Our study represents one of the few systematic evaluations of this approach within a homogeneous population suffering from complete unilateral facial paralysis. For this study the stimulation was delivered on the region closest to the upper mouth corner and denervation recovery was monitored for the entire period. Five patients were recruited, undergoing surface electrostimulation for a maximum of 1y, twice a day for 15min (5min pause every 5min stimulation). The parameters set during the first visit were confirmed/adapted every month thereafter. At each visit the patients underwent 3D photos, electromyography, ultrasound,4 magnetic resonance controls,5 and Sunnybrook evaluation, answered to the FaCE and FDI questionnaire. For all the patients effective parameters leading to an utterly Zygomaticus m.-specific stimulation could be found below the discomfort threshold. The required stimulation pulse width decreased with time without requiring a significant amplitude increase to remain effective and specific. Sunnybrook results significantly (p=0.04) improved after 6m stimulation. FaCE (total score) showed a significant (p=0.04) improvement already after 2m, while the social component of the FDI significantly (p=0.04) improved after 7m. Short- (hours) and mid-term (weeks) improvements of muscle tone could also be shown by 3D imaging. Our results showed that surface electrostimulation can be used to deliver a specific response of the Zygomaticus m. without adverse events and without non-specific activation of other facial muscles. Patients were extremely compliant with the treatment protocol and received a significant improvement in their quality of life., Age-related cognitive decline has become a major public health concern. There is currently a paucity of effective interventions to prevent or treat cognitive decline and prevent dementia in the elderly. Decline in working memory is a central facet of the cognitive aging process. Prior research has attempted to use transcranial direct current stimulation (tDCS) to enhance working memory performance in older adults with and without neurodegenerative disease, often in the context of cognitive training paradigms(1, 2). Alone, these methods have shown a degree of promise in remediating age-related working memory decline. In combination, these interventions may prove more effective by pairing two interventions targeting neuroplasticity. However, the neural mechanisms and efficacy of combined cognitive training and tDCS improvements in working memory is not well understood. This talk will discuss data from 3 studies investigating the benefits neural mechanisms of cognitive training paired with tDCS to remediate age-related cognitive decline.1-3 Study 1 was a mechanistic investigation of the impact of tDCS with CT during BOLD fMRI in 14 healthy older adults. Study 2 was a pilot randomized clinical trial of 28 older adults randomized to active tDCS or sham tDCS with cognitive training. Study 3 is an ongoing Phase III RCT investigating the benefits of tDCS with cognitive training in 360 older adults. Neural mechanisms of working memory improvement, efficacy of near transfer from tDCS vs. sham with cognitive training in working memory measures, and future directions of investigation will be discussed., We present preliminary results from the ongoing study entitled “Icelandic AVH TMS” which aim is to study the effectiveness of repetitive transcranial magnetic stimulation (rTMS) treatment for patients with schizophrenia and with persistent auditory verbal hallucinations (AVH) using high-density EEG system (256 channels). The main objectives of this work were to describe P50 and P300 cortical topography pre and post treatment, and to define a robust methodology of signal quantification using high density EEG.1-4 Our results show differences in sensory gating and a stronger response to rare audio stimulus 1 week post treatments. Moreover we show the value of assessing brain electrical activity from high-density EEG (256 channels) analyzing the results in different regions of interest., Reduction in muscle mass and muscle strength together with alterations in the metabolic and cellular machinery of the skeletal muscle are common in type 1 diabetes (T1D)1 Collectively these negative changes have been termed diabetic myopathy.2 Moreover, whole-body fatigue is a frequent complaint in individuals living with T1D but it is unclear if this would depend on muscle alterations per se or on a concomitant impairment in motor units (MU) recruitment capacity. MU recruitment can be now investigated non-invasively using high-density surface electromyography (HD-EMGs) using multi-channels electrodes.3 Sixteen participants, 8 T1D (4M; 33.7 ± 5.2 yrs.; HbA1c 8.1 ± 3.1 %) and 8 healthy (4M; 31.5 ± 5.6 yrs.) volunteered for the study. In one 2-hour visit to the lab participants were tested for maximal isometric strength (MVIC) of knee extensors (KE), sustained KE isometric contractions at 10, 20, 40 and 60 (%MVIC), plus a sustained contraction at 40% MVIC until task failure. During these measures we recorded HD-EMGs from vastus lateralis (VL) muscle with a grid of 64 electrodes. At baseline no differences, between the 2 groups, were observed in MVIC, rate of torque development, and HD-EMGs parameters. In addition, and contrary to our expectations, no differences were observed concerning the fatiguing test either in terms of time to fatigue or of changes in muscle fiber conduction velocity (a marker of MU recruitment). In conclusion, no major differences were detected between the two groups, which could justify the presence of diabetic myopathy (whether neurogenic or myogenic) in our T1D participants., Skeletal muscle is the driver of whole-body aerobic capacity measured by spiroergometry as VO2max/M [mL O2∙min-1∙kg-1]. Obesity is defined as accumulation of excess fat tissue mass, MFE=MF-MF° [kg/x]. MF° is the fat mass [kg] per individual [x] in the healthy reference population at a given height and total body mass M° without overweight.1 Body fat excess, BFE=MFE/M°, is directly related to total body mass excess, BME=ME/M°, where ME=M-M°. In model 1, BFE does not reduce VO2max [mL O2∙min-1∙x-1], but BFE lowers VO2max/M by increasing M. Experimentally, however, VO2max/M declines with BME much steeper than predicted by model 1. The more pronounced loss of ergometric fitness is due to the decline of mitochondrial respiratory capacity per muscle mass, mM,2,3 as a function of BME. Yet this model 2 predicts an even lower VO2max/M at overweight. Finally, model 3 includes the well-known ‘weight-lifting’ effect of obesity on increasing muscle mass with low mitochondrial density, providing a quantitatively complete link between low mitochondrial and whole body aerobic fitness in obesity before onset of sarcopenia. The decline of muscular mitochondrial fitness in overweight states is a biomarker of the systemic mitObesity syndrome: Compromised mitochondrial fitness across metabolically active organs provides the mechanistic link between obesity and comorbidities such as diabetes, cardiovascular and neurodegenerative diseases and various types of cancer bound to redox imbalance, inflammation, oxidative stress and insulin resistance. Today mitObesity is the world-wide leading cause of deaths and early aging, which can be prevented by an active lifestyle and improvement of the quality of life by exercise and caloric balance. Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770. Contribution to COST Action CA15203 MitoEAGLE funded by the Horizon 2020 Framework Programme of the European Union., By combining the digital holography with the tomography, Nanolive technology is an example of emerging intelligent nanoscopy techniques1. The 3D Cell Explorer microscope guarantees a high spatial-temporal resolution imaging of transparent unlabeled specimens, with the advantage over fluorescence techniques of not requiring sample labeling, thus reduce potential damages to living cells2. This intuitive and affordable technology enables scientists to investigate macro cellular dynamics like cell health, proliferation, movement and function as well as micro organelle dynamics and interactions in a e.g. mitochondrial network label-free characterization.3, Intracellular aggregates of RNA binding proteins and perturbed RNA metabolism are typical features of many human neurological and neuromuscular diseases, including oculopharyngeal muscular dystrophy (OPMD), a rare genetic repeat expansion disease (1). In OPMD, a short abnormal polyalanine expansion in PABPN1 (poly(A)-binding protein nuclear 1) leads to the accumulation of intranuclear tubulofilament aggregates in muscles of patients and ultimately to muscle dysfunction. Symptoms usually appear in the fifth decade of life and we have shown a good genotype phenotype correlation (2). The disease is characterized by weakness of the eyelid and pharyngeal muscles, leading to eyelid drooping and dysphagia. A strong collaboration with clinicians gave us access to a large collection of muscle biopsies from OPMD patients. We characterized PABPN1 nuclear aggregates to evaluate if age and genotype influence their features (submitted). In addition, using mammalian models of OPMD, we assessed the efficacy of the anti-aggregates molecule guanabenz (GA). We showed that aging and genotype of OPMD patients influence the amount, the size, the percentage and the composition of nuclear aggregates. Treatment of cellular and mouse models of OPMD with GA allowed a reduction in the percentage and the size of nuclear aggregates as well as an improvement of the mice muscle phenotype. GA acts through the unfolded protein response to endoplasmic reticulum stress (ER) showing for the first time that ER stress is activated in OPMD. (3) This study suggests that the use of pharmacological molecules modulating ER stress is a promising strategy to treat OPMD., Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive lipid storage myopathy, often associated with ETFDH gene mutations.1 This gene encodes electron transfer flavoprotein dehydrogenase, a mitochondrial protein involved in the electron-transfer system.2 In this study we investigated the correlation between ETFDH mutations, expression profile of serum muscle‐specific miRNAs (myomiRs)3 and response to treatment in 2 Italian MADD patients. Patient 1 (53 years) presented late onset of MADD, at the age of 38 years. She carried two different ETFDH missense mutations.4 In patient 2 (54 years) the symptoms appeared at 2 years of age and molecular analysis of ETFDH showed a missense and a splice site variation.5 Muscle biopsies of both patients displayed type I fibers vacuolated and muscle atrophy. Moreover, MRI of patient 2 revealed slight alteration of posterior thigh muscles. Bioinformatic analysis demonstrated that the missense mutations can determine conformational modification of protein structure, decreasing enzyme stability and activity. Serum myomiRs analysis displayed an up-regulation of miR-1, miR-133a, miR-133b and miR-206 expression in the two patients. Treatment led to an improvement of clinical condition in both patients, in particular of muscle vacuolization. In this preliminary study the results seem to indicate that the presence of two ETFDH missense mutations correlate not only with a minor severity of MADD phenotype but also with a partial dysregulation of circulating myomiRs, mainly miR-1 and miR-206. In these cases, various treatments can improve ETFDH stability, resulting in skeletal muscle recovery., Store-operated Ca2+ entry (SOCE) is a ubiquitous cellular Ca2+ influx mechanism, first described in non-excitable cells, that is triggered by depletion of intracellular Ca2+ stores (endoplasmic reticulum, ER). A major breakthrough in the field was the identification of the two essential molecular players in SOCE: STIM1, the Ca2+ sensor in the ER, and Orai1, a Ca2+ permeable channel in the plasma membrane.1,2 SOCE is also well-documented in skeletal muscle4 where it limits muscle fatigue during repetitive stimulation.3 Also in muscle SOCE is mediated by interactions between STIM1 in the SR and Orai1 channels in the PM.4 However, the precise subcellular location of STIM1-Orai1 SOCE complexes in skeletal muscle is still debated. We discovered that exercise in mice drives formation of new junctions between stacks of sarcoplasmic reticulum (SR) cisternae and transverse-tubules (TTs) containing STIM1 and Orai1, two proteins that mediate store-operated Ca2+ entry (SOCE). We proposed that these previously unidentified SR-TT junctions function as Ca2+ Entry Units (CEUs), providing a preferential pathway for rapid reuptake of Ca2+ into the SR during repetitive muscle activity.5 Using electron microscopy and two different functional assays we also studied muscles from mice subjected to an incremental treadmill running and sacrificed within 1hr or after 6-24hrs.6 Data collected indicates that: a) while the number of SR-stacks increased up to at least 6hrs to return to control values only after 24hrs of recovery, the extension of TTs (significantly increased at 1 hr), returned to control values already after 6hrs; b) fatigue resistance of EDL muscles during high-frequency stimulation and Mn2+ quench of Fura-2 fluorescence in FDB fibers were both increased after 1hr, but were not different from control after 6 and 24hrs of recovery. Our work represents a pioneer study that identified exercise-driven dynamic formation of new intracellular structures, a mechanism potentially quite important for the delay of muscle fatigue. As altered SOCE activity contributes to muscle dysfunction in ageing and various myopathies, our findings may also have implications for the understanding of mechanisms involved in muscular dysfunction., The HERG potassium channel is detected as a heteromultimer of 2 alternative splice variants (1A and 1B) in heart and has been shown to be partially responsible for repolarization of the cardiac action potential.1 Both alternative splice variants have been reported in certain cancer cells, but their role in these cells is not clear.2 The HERG1A variant has been detected at low abundance in normal skeletal muscle, but is up-regulated in atrophying skeletal muscle, where it has been shown to increase protein degradation by modulation of both intracellular calcium levels and ubiquitin proteasome proteolysis (UPP).3,4,5 The pathways by which this modulation occurs is not clear. Therefore, we virally transduced C2C12 myotubes with either an adenovirus encoding HERG or an appropriate control virus (n=6). After 48 hours, we extracted total RNA from these cells and reverse transcribed them into cDNA, selecting for coding sequences (i.e., mRNA) by using poly(T) oligomers; the cDNA libraries were sequenced on Illumina’s NovaSeq platform. Sequence quality was assessed using FastQC (v 0.11.7; https://www.bioinformatics.babraham.ac.uk/projects/fastqc) for all samples and quality trimming was done using FASTX-Toolkit (v 0.0.14; http://hannonlab.cshl.edu. fastx_toolkit/) to remove bases with Phred33 score of less than 30. Resulting reads of at least 50 bases were mapped against the reference genome using STAR.6 STAR derived mapping results and annotation file for reference genome were used as input for HTSeq7 (v 0.7.0) to obtain read counts. Counts from all replicates were merged together to produce a read count matrix for all samples and this count matrix was used for downstream differential gene expression analysis (DGEA). DGEA between treatment and control was carried out using ‘R’ (v 3.5.1; http://www.r-project.org/). The results show that HERG does result in numerous changes in gene expression. Limiting results to those with a p, MRI is a powerful non-invasive imaging technique that exploits different contrast mechanisms to provide an unprecedented view into both structure and function. We have used velocity encoded phase contrast technique to map muscle motion in-vivo1 and applied to study age and disuse atrophy related changes. A limitation of this dynamic technique is that it requires of the order of 70 consistent contractions cycles in order to image muscle motion. Thus, it is not easy to extend the MR dynamic studies to senior subjects and to higher % Maximum Voluntary Contractions (%MVC). We have implemented a fairly recent innovation called ‘compressed sensing (CS)’ to dynamic MRI for mapping muscle kinematics during isometric contraction. The implementation of this ‘fast’ technique allowed us to study muscle kinematics in 3D and at three %MVCs in the calf muscle. Eight young and eight senior subjects were imaged using the CS-VEPC sequence and the 3x3 strain and strain rate tensors were calculated in the principle axis and in the muscle fiber basis at 35, 45 and 60 %MVC at much reduced number of contractions cycles. Region of interest measurements are reported for the medial gastrocnemius and in the soleus: principle strains and strain rates were significantly different for 30 and 60% MVC (MG and soleus) and shear strain and shear strain rates were significantly different with age. Surprisingly, the strain and strain rates in the fiber basis were much smaller than in the principal basis and no significant differences were found between %MVCs or in age. The implications of these findings with respect to muscle force loss with age will be discussed., Soft tissue contrast afforded by MRI makes it an ideal candidate for imaging the musculoskeletal system. We will discuss our results using a combination of advanced MR pulse sequences to explore age related changes in tissue composition and tissue microstructure. Tissue composition includes estimation of fat (adipose) fraction, connective tissue/macromolecular fraction and water fraction in different muscles and compartments1. A combination of MRI sequences, IDEAL, UTEs and Magnetization transfer2 contrast sequences were applied to cohorts of ten young and ten senior subjects to determine the intramuscular and intermuscular tissue composition in calf muscle. Initial results show that fat and connective tissue fraction increased while macromolecular fraction (MTsat) decreased with age. Tissue microstructure was extracted from diffusion tensor imaging3,4 using a STEAM-DTI sequence and Random Permeable Barrier Modeling (RPBM)5. DTI studies were performed after IRB approval on a GE 3T scanner on seven young and six senior subjects. Time dependent diffusion was measured at ten values of the mixing time, TM (20 ms to 600 ms). The RPBM fits were made to the time dependence of the average of the secondary and tertiary diffusion eigenvalues λ2 and λ3 (Dᅩ ); the values of Dᅩ were the average over the medial gastrocnemius (MG) muscle segmented from all three slices. Muscle fiber diameters from the fit showed a small increase with age which is surprising as fiber atrophies with age. Fiber membrane permeability increased with age potentially indicating compromised sarcolemma integrity. Initial results of validation with biopsy analysis will also be presented., Chronic inactivity is a main cause of muscle wasting and weakness.1 However, muscle atrophy may not be solely due to a reduction in mechanical loading since muscle denervation and neuromuscular junction damage seem also triggered by inactivity.2,3,4 The identification of biomarkers of muscle atrophy and of neuromuscular degeneration is therefore needed for an early detection of the alterations of the neuromuscular system induced by inactivity. Hence, the aim of the present study was to investigate the onset of muscle atrophy and of neuromuscular alterations during a short-term inactivity period. Ten healthy male volunteers (aged 23±5 years) were recruited for this horizontal 10-day bed rest (BR) study, after Ethical approval and participants’ informed consent. Blood samples were collected every two days for the assessment of NMJ damage, based on serum levels of c-terminal agrin fragment (CAF) measured with a commercially available Elisa kit. Muscle fibre denervation was determined from the expression of neural cell adhesive molecule (N-CAM) in myofibres obtained from vastus lateralis (VL) muscle biopsies collected at baseline, and at 5 and 10 days of BR. Myofiber atrophy was determined from mean fibre cross-sectional area (CSA) measurements on histological sections. Whole muscle atrophy was assessed from changes in muscle architecture (pennation angle and muscle thickness) measured every two days of BR using ultrasonography. Significance was set at p, Over the last 25 years imaging techniques have been extensively used to study the in-vivo muscle and tendon morphology at tissue scale. Whereas Dual-energy X-ray absorptiometry (DXA), computed tomography (CT) and MRI have been widely adopted in health, ageing and clinical populations to track changes in body composition and muscle mass, ultrasound (US) can provide further information about the structure, length, thickness and area/volume of muscles and tendons. Despite these characteristics could also be obtained by MRI, US has the advantage that it can provide the same quantity of information, but during passive joint rotations, isometric or dynamic contractions. Recent advancements in musculoskeletal imaging have also led to the use of US also for the investigation of strain in soft tissues in response to compression (Shear Wave Elastography - SWE). However, some limitations of conventional B-mode US lay on: a) the small field of view used (especially during SWE or the study of in-vivo dynamic changes in muscle and tendon structures) and b) the impossibility of US to provide any in-vivo insights into adaptations at the sarcomeric level. Recent developments in the extended field-of-view US (EFOV) and 3D US techniques may offer further advantages in providing information at a whole-tissue level, providing software-integrated panoramic images and 3D volume reconstructions, allowing to gain insights into regional changes along the muscle-tendon unit. Furthermore, in-vivo microendoscopy has been recently implemented in human skeletal muscle, laying the foundation of new investigations into the remodeling of skeletal muscle “from the micro to the macro”. The purpose of this talk is to present and discuss these advances in imaging techniques for the study of human skeletal muscle in-vivo., Real time Ultrasound (US) imaging is being increasingly utilized by Physical and Rehabilitation Medicine (PRM) specialists to assess abdominal muscle in healthy subjects and in patients with low back pain (LBP).1 The current study set out to evaluate the variability and the intra- and inter-rater reliability of US measurements not only of the thickness of the abdominal muscles but also of the fasciae.2 Three specialists with different levels of training in US measurement techniques followed a standard protocol based on four reference anatomic landmarks to perform US examinations of the abdominal muscles and fasciae of a healthy volunteer in resting and dynamic condition. Each of the specialists measured 17 anatomical structures six times during two sessions (three per session). Their intra-rater reliability was assessed by evaluating the range of relative error and the coefficient of variation (CV). The inter-rater reliability was evaluated using the Kruskal-Wallis test at probability levels of 0.05 and 0.01. There were no significant differences between the measurements that the three raters registered (inter-rater reliability) with the exception of those referring to the anterior fascia of the external oblique muscle (p-value < 0.01), the fascia between the external and internal oblique muscles (p-value< 0.05) and the fascia between the internal oblique and the transversus abdominis muscles (p-value < 0.05).3 Knowledge about the fascial anatomy of the abdominal wall is essential to carrying out accurate US examinations. These findings confirm that US imaging is a reliable, non-invasive, cost-effective instrument for evaluating the abdominal muscles/fasciae.3, Spinal cord injury produces muscle wasting, which is especially severe after complete and permanent damage of lower motor neurons as occurs in complete Cauda Equina Syndrome.1 Even in this worst-case scenario, we have shown that permanently denervated Quadriceps muscle can be rescued by surface electrical stimulation, and a purpose-designed home-based rehabilitation regime, i.e., Home based Functional Electrical Stimulation (hbFES) for human muscles that are completely denervated and degenerating.2,3 Here the aim is to show that the effects are extended to the antagonist muscles and skin of the thighs. Before and after two years of electrical stimulation, the mass and structure of Quadriceps and Hamstrings muscles were quantitated by force measurements.1 Muscle gross cross-sections were evaluated using color computed tomography.4 Biopsies of skeletal muscles,1-3 and of stimulated skin,5 were analyzed by quantitative histology and immuno-histochemistry. The treatment produced: 1) an increase in the cross-sectional area of stimulated muscles; 2) an increase in muscle fiber mean diameter; 3) improvements in ultrastructural organization; and 4) increased force output during electrical stimulation.1-4 The recovery of Quadriceps muscle force was sufficient to allow 25% of the compliant subjects to perform stand-up and step-in place trainings.1-3 Improvements were extended to antagonist hamstrings,5 and skin.6 In conclusion, the cushioning effect provided by recovered tissues is a major clinical benefit. The hope is that new trials may start soon in Europe and beyond to provide stronger evidence of the positive effects of h-bFES for permanently denervated muscles. This will help to extend the results world-wide, and to all persons who are in need of the help they deserve., Patient oriented research in rehabilitation revolves around maintaining, improving or restoring muscle function in conjunction with pain treatment. Based on our findings in treating patients with long-term denervated, degenerated muscle (DDM) after spinal cord injury with electrical stimulation, (Kern et al. 2010)1 our activities in the past were aimed at transferring the knowledge to rehabilitation of elderly people (Kern et al. 2014).2 The current work comprises investigating age-related sarcopenia, a multifactorial disorder which underlying mechanisms are still not fully known. It could be shown, that alterations of mitochondrial Ca2+ homeostasis regulated by mitochondrial calcium uniporter (MCU) affect muscle function. Therefore, improved muscle function and structure are linked to increased protein levels of MCU (Zampieri et al. 2016).3 Future translational research will investigate cellular and molecular mechanisms of aging and physical activity, especially the effect of electrical stimulation training and physical therapy on these mechanisms, because only little research on this topic exists and therefore the evidence for efficacy of these therapy approaches is still missing. Focus of the future clinical research program is the in- and outpatient therapy of orthopedic patients, the remobilization and the rehabilitation of patients after knee and hip replacement surgery. For this reason, standardized assessments, analyzes and documentation will be developed and the results are collected across locations in a data base for in- and outpatient rehabilitation. Overall aim of the program is the evaluation of existing and new approaches in musculoskeletal rehabilitation also concerning the economic aspects with constant or improved quality of treatment. In the next years the following topics will be in focus: i) Underlying cellular and molecular mechanisms of Physical Medicine and Rehabilitation procedures; ii) In- and outpatient rehabilitation after knee and hip replacement surgery; iii) Proof of efficacy and cost-effectivity., Physical inactivity is a global pandemic that not only causes morbidity and mortality, but also represents a major economic burden worldwide. As a longer-term goal, we must strive to integrate physical activity into our everyday lives.1 Increased physical activity, among other things, has an influence on chronic pain of the locomotor system, minimizes the risk of cardiovascular disease, increases self-esteem and cognitive abilities, maintains mobility and autonomy, and thus has direct effects on healthy life years and direct and indirect health expenditure. The Centre of Active Ageing (CAA) project, funded by the EU cross border cooperation program INTERREG, is intended to be in line with the Austrian health targets (R-GZ) for Austria,2 the National Health Program of Slovakia,3 the recommendations of the Lancet Physical Activity Series Executive Committee, and of the WHO.5 Encouraging policymakers to take physical activity seriously, to motivate people, and to create the opportunities to do so on a regular basis. Over the entire project period of three years, more than 1000 people aged 60 years and over will benefit from the offers of the CAAs in the program area. Within the scope of the project, they should carry out a standardized training (10-12 weeks) followed by home training for 12 months. In 2019 the first groups started their “SENIOR aktiv” activity program in Austria. It combines education sessions of training theory and nutrition before and after a 10-week physical activity program for two times a week with alternating strength and gymnastics sessions. The first results show improvements in almost all measured parameters and the subjective feedback of the subjects is consistently positive, When resistance training for the improvement of the neuromuscular function is considered, older adults should base training on submaximal loads (85-80% of their one repetition maximum (1RM); tested or estimated from nRM) and advance to speed-power training regimens (50-75% of 1RM executed explosively). Based on the recent research, the latter is an important supplement facilitating physical ability and functional capacity of older individuals. Recent studies have shown that resistance training stressing performance of repetitions with maximal velocity results in higher performance gains as compared to strength training alone.1 It therefore seems reasonable to include speed-power training into physical conditioning of older adults. As a result, we aim to address this topic in the framework of the current Interreg project Slovakia-Austria, by exposing 60+ subjects to 10-week flywheel speed-power training. This training modality offers innovative solutions for individualised training progression (load, tempo, postural stability), which we will share during with the audience during our conference presentation. Additionally, our plan is to get a better insight into the potential of this kind of resistance training for changing force-velocity-power profile in elderly. This would complement our previously used testing approaches we used in past interventional studies in elderly.2 Namely, improving velocity dominance in the force-velocity profile seems to have significant functional relevance (balance control, gait initiation, change of direction; and prevention of falls as a result).3 Preliminary results on initial methodological considerations and protocols (training and testing) development will be presented, Proper muscle function is controlled by intracellular Ca2+ concentration, which in turn depends on: a) release of Ca2+ from intracellular stores during excitation contraction (EC) coupling, which activates muscle contraction; b) entry of Ca2+ from the extracellular space via store-operated Ca2+ entry (SOCE), a mechanism important to limit muscle fatigue; c) uptake of Ca2+ into the mitochondrial matrix, which stimulates aerobic ATP production; and finally d) sequestration/removal by sarcoplasmic reticulum (SR) and plasma membrane (PM) pumps, which relaxes muscle fibers. Abnormalities in Ca2+ handling underlies many physio-pathological conditions. For instance, reduced SR Ca2+ release has been linked to fatigue and dysfunction in ageing, while excess of SR Ca2+ leak may even underlie life-threatening conditions such as malignant hyperthermia susceptibility (MHS). In the last 15 years we have collected compelling evidence that the proper architecture and function of all those membrane systems involved in Ca2+ handling and aerobic ATP production depends on muscle activity: i) denervation causes disarray of units deputed to EC coupling (calcium release units, CRUs) and mitochondrial apparatuses, while functional electrical stimulation (FES) promotes some rescue of this structural disarray;1 ii) sedentary ageing in mice and humans causes misplacement of mitochondria and partial disarray of CRUs, while long-term training does prevent effectively those changes;2-4 iii) acute exercise promotes functional assembly of SOCE-sites (calcium entry units, CRUs), while post-exercise recovery determines their disassemble;5 iv) sedentary ageing causes accumulation of dysfunctional proteins in tubular aggregates (TAs), while exercise prevents TAs formation (manuscript in preparation). Ca2+ handling is crucial for muscle function and is controlled by diverse membrane systems and intracellular organelles. Our experience collected in different, but complementary, projects: i) indicates the structure and function of intracellular organelles is preserved or rescued by exercise or training; and ii) underlines the importance of physical exercise during ageing to preserve muscle function., The dominant role in muscle regeneration is played by the muscle stem cells known as satellite cells, which reside between the basal lamina and sarcolemma of myofibers. Along with satellite cells, other precursor cells, whose activity is strictly dependent by environmental signals, participate to muscle regeneration.1,2 A tightly regulated interplay between stem cells and other resident cell types, as well as the intimate connection with structural components of the tissue niche, can be responsible for the maintenance of the stem cell pool under steady-state conditions and to guide stem cells activation and differentiation when regenerative signals are provided.1 Because niche factors and components normally control and sustain a physiological stem cell activity and maintenance, the loss of homeostatic input from the niche, as observed under pathological conditions, can deregulate stem cell physiology, critically affecting the ability of muscle tissue to efficiently regenerate and to regain the functional integrity after damage.1-3). Thus, the stem cell niche is not only an anatomical compartment but a complex, integrated network of both cellular and acellular components that provide signals influencing stem cells and muscle homeostasis. The basis of muscle regeneration and the impact of cytokines and growth factors on the physiopathology of skeletal muscle will be discussed.1,4-6, Neuromuscular diseases, ageing, and cancer cachexia are chronic conditions characterized by skeletal muscle wasting (1, 2) resulting from altered muscle metabolism, and catabolic processes. Following muscle pathophysiological changes, proteins are secreted or passively released into systemic circulation. The identification of muscle-derived serum biomarkers can be used to develop a kind of “liquid biopsy” for diagnostic/prognostic approaches and to identify altered muscle physiology. With this aim, we performed combined morphological and biochemical analyses of skeletal muscle biopsies and blood samples obtained from cancer patients, both collected the day of surgery. Samples from non-oncologic patients were also collected as controls. Morphometrical analyses showed a significant reduction of myofiber size in oncologic patients in comparison to controls, and the analyses of myofiber diameter distribution showed a higher prevalence of severely atrophic fibers in oncologic patients, most of them having angulated and flat shaped morphology, typical features of denervation (3). The percentage of in situ Neural cell adhesion molecule (N-CAM) expressing myofibers and serum levels of N-CAM (sN-CAM) and Agrin were also tested, as markers of neuromuscular junction plasticity and remodelling (4, 5). Consistently with morphological observations, in cancer patients with respect to controls, the percentage of N-CAM positive myofibers was significantly higher and circulating levels of sN-CAM and Agrin were also significantly increased. These results correlate morphological changes of skeletal muscle trophism and innervation with serum alterations in NMJ-derived proteins. Moreover, they show that sN-CAM and Agrin are promising and reliable serological biomarkers for neuromuscular junction plasticity and remodeling in conditions characterized by skeletal muscle wasting, Functional electrical stimulation (FES) is used to induce skeletal muscle contractions in persons who are unable or reluctant to move usually by surface neuromuscular electrical stimulation (NMES), also known as transcutaneous electrical nerve stimulation (TENS) in out-patients or hospital. If properly instructed, persons may continue at home with FES-induced training with a daily frequency and for the long duration of time needed to achieve clinically-relevant outcomes. Standing on the results of the EU Project RISE,1 and on those in aging persons,2,3 we are confident that FES may contribute to a better life for an expanding population of aged persons and of early aged patients suffering with mobility impairments due to neuromuscular and metabolic disorders as well as systemic diseases. We will describe in detail results of FES for denervated degenerated muscles (DDM) delivered by large anatomically shaped surface electrodes at the level of the stimulated skin. Indeed h-bFES for DDM is able to recover skin from SCI-induced atrophy and flattening.4,5 Further, we will discuss the value of surface morphometry of neck skin, and of non-invasive blood analyses of circulating myokines (using sweat and mouth fluids6,7 as biological markers of aging progression., Male hypogonadism is a clinical syndrome manifesting with low testosterone (TT) including symptoms like decline in lean mass, muscle strength, increased adiposity, visceral obesity and incidence of insulin resistance and metabolic syndrome. 1 There are only few studies dealing with effect of physical activity on hypogonadal males.2 The aim of the study was to examine the effect of 12-week strength training program on body composition, selected biochemical parameters and physical performance in hypogonadal men. The study compared the effect of resistance training (RT) on hypogonadal patients without hormonal therapy (HP, n=6, 48.41±6.38 yrs, TT= 7.9±1.75 nmol/L) and control group of eugonadal males (EM, n=8, 49.31±5.84 yrs, TT= 15.81±3.99 nmol/L). The subjects performed RT twice a week, the training program consisted of 6 exercises at an intensity from 60-80% of 1RM. Body composition was measured by DXA, muscle strength was measured by predicted dynamic leg press 1RM from multiple repetition maximum, handgrip strength using hand dynamometer. Fasting morning venous blood samples were collected. The parameters analyzed from serum were glucose, total cholesterol, LDL cholesterol, HDL cholesterol, SHBG, insulin, total testosterone, and cortisol. Subjects from both the HP and EM groups significantly decreased relative fat mass by 6.2 % and 4.91% (p>0.05 and p, Central Core Disease (CCD) is a congenital myopathy characterized by presence of amorphous central areas (or cores) lacking glycolytic/oxidative enzymes and mitochondria in skeletal muscle fibers.1 Most CCD families are linked to mutations in ryanodine receptor type-1 (RYR1), the gene encoding for the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle.2 As no treatments are available for CCD, currently management of patients is essentially based on a physiotherapic approaches. Functional electrical stimulation (FES) is a technique used to deliver low energy electrical impulses to artificially stimulate selected skeletal muscle groups.3-5 Here we tested the efficacy of FES in counteracting muscle loss and improve function in the lower extremities of a 55-year-old female patient which was diagnosed with CCD at the age of 44. Genetic screening of the RyR1 gene identified a missense mutation (c.7354C>T) in exon 46 resulting in an amino acid substitution (p.R2452W) and a duplication (c.12853_12864dup12) in exon 91. The patient was treated with FES for 26 months and subjected before, during, and after training to a series of functional and structural assessments: measurement of maximum isometric force of leg extensor muscles, magnetic resonance imaging, a complete set of functional tests to assess mobility in activities of daily living, and analysis of muscle biopsies by histology electron microscopy. All results point to an improvement of muscle structure and function induced by FES suggesting that this approach could be considered as an additional supportive measure to maintain/improve muscle function and reduce muscle loss in CCD patients., Over the last decade, transcutaneous auricular nerve stimulation (tANS) become established method of VNS and has been verified in various disorders including neurological and psychological trauma, addiction of drugs, inflammation and tinnitus. Several groups examined also effects of tANS on central and peripheral nervous system, effects on behaviour in neuropsychiatric populations and effects on subjets with disorder of cognitive and social functioning. tANS of particular areas at the cymba conchae (CC) require electrodes with high spatial selectivity, low impedance and safe reversible charge delivery to specific populations of receptors through the electrode-tissue interface. Cortisol is a naturally occurring steroid hormone that has many important functions in the body and is released into the bloodstream at times of stress. Cortisol is blamed for anxiety, high blood pressure and stroke. It helps to control blood sugar levels, regulate metabolism, help reduce inflammation and assist with memory formulation. All treatments to correct eventual cortisol imbalance involve medication. The present study was aimed at demonstrating that selective tANS can be potentially used as a method for the induction of Cortisol hormone secretion. tANS was accomplished in a 62-year-old subject with angina pectoris, coronary artery disease, and moderate insomnia. Sites at the CC were selectively stimulated using a silicone plug with four platinum cathodes. For the tANS, current regulated stimulating pulses with an intensity of ic=20 mA, a pulse width of 200 μs, and a frequency of f=25, were used. Results show that in six out of eight samples of the mouth fluids, the cortisol level was significantly lower after tANS that before tANS., The environment is critical when, in order of intrinsic morphological or geographical characteristics, or extrinsic features (caused by human presence or human behavior), it induces strenuous changes (independent of emergency accidents) in the physiological conditions of people who require emergency aid.1 Moreover, in an extrinsic critical environment the possibility to have a not homogenous population to be rescued is greater, consequently we can act only on the variable physical capacities and competence of emergency team, to have more success. This is the purpose of this work that has a background of more than 1200 physical evaluations over 10 years, on Italian Alpine Soldiers.2 Every subject had three evaluations of fitness profile during the year (time 0, after three months of programmed training and after six months). Evaluation and training methods are studied in order to be applied to a large number of subjects. The fitness profile includes: anthropometric assessment, aerobic fitness, muscle endurance, heart rate, diastolic and systolic pressure, respiratory rate, hydration, postural assessment by assessing the spine with spinal mouse system.3 The obtained results have shown that the physical preparation carried out, has improved and made homogeneous the physical capabilities of both the team and individual subjects that make it up. In addition to making the members of the emergency team more conscious, the increase in the level of coordinated physical preparation has objectively improved the safety (for team members) and effectiveness of intervention in the various crisis situations., In daily life situations, if we are exposed to unusual, challenging situations, our body reacts in a special way, called acute stress. This mechanism helps to cope with these situations efficiently. Contrary, exposing the body to stress over a longer time period, called chronic stress, like in working environments, can cause various serious health problems.1 The assessment of electrodermal activity (EDA, skin conductance) might be an easy approach to evaluate individual stress conditions over a longer period of time in an objective way. With the help of cvxEDA,2 the monitored skin conductance,3 can be split into the slow skin conductance level (SCL) and the fast skin conductance response (SCR). 20 volunteers were exposed to a relaxing-challenging-relaxing situation to mimic different stress conditions. The SCR rate proofed to be a reliable measure to evaluate sympathetic arousal. During the relaxation phases, the SCR rate was around 6 min-1and during mathematical challenges, and it raised to 9 min-1(p, The nonlinear trimodal regression analysis (NTRA) method,1,2 based on radiodensitometric computed tomography (CT) distributions,3,4 is here used for building predictive models of cardiovascular health parameters, through tree-based Machine Learning (ML) algorithms. This study reports the use of NTRA parameters for classifying elderly subjects from the AGES-Reykjavik database;5 with coronary heart disease (CHD), cardiovascular disease (CVD), and chronic heart failure (CHF). ML models employing the random forests algorithm yielded the highest classification performance for all analyses, and overall classification scores for all three conditions were excellent: CHD (AUCROC: 0.936); CVD (AUCROC: 0.914); CHF (AUCROC: 0.994). The present work introduces a substantial step forward in the construction of non-invasive, standardized tools for associating adipose, loose connective, and lean tissue changes with cardiovascular health outcomes in elderly individuals., Postural Control is the complex feedback system that allows humans to keep balance and maintain the naturally unstable upright stance. This is the result of an articulate interplay between sensorimotor systems (vision, proprioception, somatosensory, etc.) and the Central Nervous System (CNS)1,2. Lesions to the CNS, pathologies like unilateral vestibular loss (UVL) and disorders like Motion Sickness disrupt the postural feedback system, severely impairing balance. In these adverse postural circumstance, prompt adaptive and habituating processes are activated in the CNS to ensure upright posture and gait. By integrating the traditional dynamic posturography with HD-EEG analysis on a cohort of thirtythree healthy subjects3, we investigate cortical involvement in the phases of adaptation and habituation to a postural control challenge, where the balance disruption is induced through a randomized sequence of vibratory stimuli applied to the gastrocnemius muscles of the participant’s calves Keywords: Balance, cerebral cortex, HD-EEG, kinematics, postural control, power spectral density., During skeletal muscle contraction Ca2+ release units (CRUs), the sites of excitation-contraction coupling (EC) provide Ca2+,1 while mitochondria, the organelles deputed to cellular respiration, produce ATP.2 In fast-twitch fibers from adult mice CRUs and mitochondria are structurally linked by small strands or tethers,3 and interact functionally, as the uptake of Ca2+ into the mitochondrial matrix stimulates the respiratory chain.4 Aging causes separation of mitochondria from CRUs and a cross-talk impairment between the two organelles.5 However, whether this age-related uncoupling is the result of aging per-se or the consequence of reduced muscle activity remains still unclear. Here we tested if muscle activity maintains the correct association of mitochondria to CRUs in a) extensor digitorum longus (EDL) muscles from 2 year old mice, either sedentary or trained for 1 year in wheel cages; and b) EDL muscles from denervated adult mice and rats. We analyzed muscle samples using a combination of structural, biochemical, and functional experimental procedures. The results collected in structural studies indicate that: a) ageing and denervation result in partial uncoupling between CRUs and mitochondria; b) exercise and re-innervation either maintains (in old mice) or restores (in transiently denervated rats) the association between the two organelles. Functional studies support the hypothesis that CRU-mitochondria cross-talk is important for mitochondrial Ca2+ uptake, optimal force generation, and muscle performance. Taken together, our results show that muscle activity maintain/improve proper Association between CRUs and mitochondria, providing a potential tool to counteract muscle function decline in elderly and sedentary persons., Wartenberg introduced the pendulum test in the 1950s as a method to assess spasticity in the clinical setting.1 It has proven to be sensitive to the presence and severity of spasticity.2,3 The pendulum test is based on letting the lower leg swing freely under the influence of gravity while recording joint kinematics. In the presented work, the reflex period, extracted from the pendulum test, will be proposed as a new parameter to measure spasticity. The reflex period is defined as the time period from 50% of the maximum velocity of the leg to the first EMG signal indicating muscle contraction. Data from two separate studies, one on spinal cord injury patients (Halla Kristín Guðfinnsdóttir)4 and the other on stroke patients (Belinda Chenery)5 were analyzed. In both studies a pendulum test with goniometers on the knee joint was performed and simultaneously recording electromyography (EMG) of the quadricpes m. Both studies consisted of 3 trials and data from 4 subjects of each study were analyzed. The EMG data was processed with Matlab R2014b (The MathWorks, Inc.) using the open-source toolbox EEGLab. A 4th order Butterworth high-pass filter with cutoff frequency at 10 Hz and a low-pass filter at 500 Hz was applied along with notch filters at 50 Hz, 100 Hz, 150 Hz and so forth. The data was smoothed with a Gaussian filter prior to the analysis. The mean reflex period of SCI patients was 370 ms with a standard deviation of 72 ms. The mean reflex period of stroke patients was 215 ms with a standard deviation of 54 ms. This suggests a difference between the reflex period of stroke and SCI patients which might be explained by the location of their lesion., The demonstrated expression of endocannabinoids receptors in myofascial tissue suggested the role of fascia as source and modulator of pain.1 It is known that the fibroblasts can modulate the production of the various components of the extracellular matrix according to different stimuli: physical, mechanical, hormonal and pharmacological.2 In this work we isolated the fascial fibroblasts from small samples of human fascia lata of the thigh collected from 3 volunteers patients during orthopedic surgery, 2 males and 1 female. We demonstrated for the first time that the agonist of cannabinoid receptor 2, HU-308, permits the in vitro production of hyaluronan-rich vesicles in only 3-4 hours after the treatment, quickly released in the extracellular environment. The cells treated with the synthetic cannabinoid showed a large number of vesicles near the Golgi apparatus of the cells, and also in the cytoplasmic extensions. We demonstrated by Alcian Blue and Toluidin Blue stainings, immunocytochemistry and Transmission Electron Microscopy that the content of these vesicles was rich in hyaluronan. The hyaluronan is a critical element for the extracellular matrix composition and remodeling,3 and in the deep fascia, it affects the movement of hyaluronan-containing fluid layers within and underlying the deep fascia, facilitating the smooth gliding between these structures during movement.4 So its production induced by the cannabinoids can be able to increase the ability of the collagen bundles inside the fasciae to glide one respect to the other, and consequently to improve the tissue adaptability. Furthermore, the stimulation of the endocannabinoid system could be able to provide an anti-fibrotic activity by suppression of pro-inflammatory cytokines and a relief of the myofascial pain.5 These results can help to understand how the cells of fascia can answer to the endocannabinoid system regulating and remodeling the extracellular matrix formation. This is a first step in the comprehension of how the therapeutic applications of cannabinoids for the treatment of pain can have also a peripheral effect, altering the biosynthesis of extracellular matrix in fasciae and consequently remodeling the tissue and its properties., Muscles generate active forces in each half-sarcomere, the smallest contractile Unit, through the coordinated action of myosin motors, assembled in the thick filament, with actin monomers, assembled in the thin filament. Filaments are then arranged in parallel, forming a main axis in the half-sarcomere along which active forces are directed. However, in the musculoskeletal system these active forces must follow non-linear paths from tendon to tendon. This task is achieved thanks to the ability to transmit active forces between half-sarcomeres, along fibre direction (longitudinal axis) through intra-sarcomeric proteins, and between different fibres, in perpendicular directions, through extra-sarcomeric proteins, generally referred as extra-cellular matrix (ECM).1 This ability is affected by age.2 In this work, we analyzed the passive tension generated in elongated fibres alone and when they are arranged in small bundles young and aged healthy humans.3,4 The mechanical properties of the extracellular passive components in a bundle of fibres were deduced by the subtraction of the passive tension observed in single fibres from the passive tension observed in the bundle itself. ECM-related components of passive force are non-negligible in both cases and of the same order of magnitude of intra-sarcomeric components. However, in young humans the increase in passive tension observed in bundles respect to fibres is smaller than in aged humans. Based on our data, we propose to quantitatively characterize the constitutive parameters of a Hill-type three-elements model incorporated in a finite element (FE) mesh representing a fiber bundle. The characterization can be used in future FE models of whole human muscles., Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder, associated with mutations of patatin like phospholipase domain containing 2 (PNPLA2) gene.1 PNPLA2 encodes adipose triglyceride lipase, that plays a key role in triacylglycerol breakdown1. NLSDM patients mostly present progressive skeletal myopathy, with both proximal and distal involvement.2 Cardiomyopathy, hepatomegaly and diabetes are often observed. NLSDM clinical severity appears to be highly variable and it is difficult to establish the effects of different PNPLA2 mutations on disease phenotype. Recently, alteration in the expression profile of specific microRNAs involved in skeletal muscle development (myomiRs) and lipid metabolism have been observed in neuromuscular disorders.3,4. In this study, we perform the evaluation by qRT-PCR of some circulating microRNAs levels in serum samples obtained from three NLSDM siblings (two brothers and one sister), carrying two PNPLA2 missense mutations.5 Progressive skeletal myopathy was detected in the two brothers and muscle imaging showed fibro-fatty replacement. The sister presented severe hepatosteatosis and diabetes. The analysis of serum microRNAs revealed that NLSDM patients exhibited an increased amount of myomiRs. In particular, a significant correlation between muscle mass and the level of miR-206 and miR-133a was found. Moreover, an elevation of some microRNAs, mainly expressed in liver, was observed, when there was a hepatic involvement. These results show that the changes of serum muscle and lipid metabolism-specific microRNAs might represent biomarkers of skeletal and hepatic involvement. Moreover, the dysregulation of these small molecules might provide a tool to monitor the progression of NLSDM., Spinal muscular atrophy (SMA) is a common autosomal recessive disorder caused by mutations in the gene for the survival motor neuron 1 (SMN 1), and it leads to progressive muscle weakness. A major goal of disease-modifying therapies is to increase the expression of the SMN protein. Although a number of therapies are under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing the efficacy of therapeutic interventions, particularly those targeting the upregulation of the SMN protein levels. Sensitive methods for quantifying SMN protein in peripheral blood are needed. Accordingly, we developed an imaging flow cytometry (IFC) method for evaluation of SMN protein using peripheral blood and fibroblasts. First, we demonstrated that IFC successfully identified different expression patterns and subcellular localization patterns of SMN protein in SMA patient-derived fibroblasts1. Second, we tested the sensitivity and utility of IFC in identifying the differences in the expression of SMN protein between SMA patients and normal subjects using cultured Epstein–Barr virus-transformed B cells2. Subsequently, we developed an IFC method for evaluating the functional SMN protein using < 1.5 mL of peripheral blood. IFC is advantageous for the analysis of peripheral blood because of its capacity to analyze heterogeneous cell populations3. IFC analysis can be implemented in future studies to optimize its application as a tool for assessment of the effectiveness of spinal muscular atrophy treatment., Prolonged oxidative stress may play a key role in tumor development. Antioxidants molecules are contained in many foods and seem to have a potential role in future anti-tumor strategies. Among the natural antioxidants the beneficial effect of Fermented Papaya (FPP®) is known. The aim of this study was to investigate the effects of orally administered FPP® in either prevention or treatment of a murine model of melanoma. The tumor growth was analyzed together with the blood levels of both oxidants (ROS) and anti-oxidants (SOD-1 and GSH). The results showed that FPP® controlled tumor growth, reducing the tumor mass of about 3 to 7 times vs untreated mice. The most significant effect was obtained with sublingual administration of FPP® close to the inoculation of melanoma. At the time of the sacrifice none of mice treated with FPP® had metastases and the subcutaneous tumors were significantly smaller and amelanotic, compared to untreated mice. Moreover, the FPP® anti-tumor effect was consistent with the decrease of total ROS levels and the increase in the blood levels of GSH and SOD-1. This study shows that a potent anti-oxidant treatment through FPP® may contribute to both preventing and inhibiting tumors growth. The results of the above study suggested that FPP® while showing a clear anti-tumor effect it occurred though the in vivo induction of a potent anti-oxidant reaction. In a new set of experiments we wanted to verify whether FPP had a clear and scientifically solid in vivo anti-aging effect together with the induction of the anti-oxidant reaction. To this purpose we used a mouse model suitable for aging studies (C576J) treating daily each mouse from 4 weeks of life to 10 months with the same dose of IMMUNEAGE dissolved into the daily water as compared to mice receiving only tap water. At the end of the treatment period (10 months) we measured some biological parameters related to the aging processes of the cells: i) the total anti-oxydant capacity in the plasma of mice treated or untreated with FPP®; ii) the telomerase activity in the plasma of mice treated or untreated with FPP®; iii) the telomeres length in the bone marrow and ovaries of mice treated or untreated with FPP® .The results showed that the blood of treated mice, at the end of the treatment period (10 months) had 2-3 folds more anti-oxidant power and telomerase activities than the untreated mice. In the same mice we obtained both the bone marrow (from the tibias) and the ovaries at the scarify, and from the cellular preparations we measured the telomere lengths in both. The results showed that daily FPP® assumption induced 3 folds increase in telomeres length in bone marrow and ovary of treated mice as compared to the untreated mice. This suggests that FPP® induces a clear improvement of the aging scientific parameters and that the treated mice were younger than the untreated controls., The muscle spindle (MS) plays an important role in proprioception and coordination,1 which is surrounded by a strong capsule of connective tissue.2 A gelatinous fluid rich in glycosaminoglycans fills the space of capsule, probably mainly hyaluronan (HA).3 Age-related proprioceptive and coordinative deficits are unclear, although some hypothesises have been proposed to explain those mechanisms both on the peripheral and CNS changes. Age-related changes of MS were compared in unmature (1month, M); young adults (4M); and old (27 M) C57BL/6J male mice. Hematoxylin Eosin (HE), Sirius-red,4 Van Gieson staining were used to evaluate age-related MS morphology changes The collagen type I antibody, biotin labeled HA binding protein immunostainings,5 were used to monitor age-related changes in collagen type I and HA of MS. The Purple-Jelley HA assay was used to measure age-related changes HA contents. MS is surrounded by the capsule in continuity with the perimysium and epimysium and HA filled the capsule. The capsule of the MS, perimysium, epimysium underwent thicking with aging, primarily consisting of collagen type I. Van Gieson showed that the presence of elastin in the capsule. The amount of HA in Triceps Surae is 27.74g/g in 4M. The presence of the collagen fiber, elastin and HA in the capsule of MS and its continuous with fascia suggested that they may effect the MS function. If their proportion changed, they will influence the function of MS, which properly explain part of the peripheral Neuro-physiological mechanisms of Age-related proprioceptive and coordinative deficits., Muscle structure,1 and performance2 differ between female and males when compared across the same age groups. Masters athletes compete in age groups of five-year divisions, ranging from 35 to more than 100 years of age. Here, our aim is to investigate the gender differences in the rate of age-related decay using the complete series of female and male normalized Masters’ World records. The World record series are lists of up to 16 data points that can be interpolated with polynomial trend-lines with a very high R2, after normalization to allow comparisons of the values among the different sports events.3 Here, gender comparisons were performed for 19 Track and Field specialties using weighted regression analyses. As expected, the aging decline began at 35 years for both women and men. Despite higher values of the male athletes in all the 19 Track and Field Masters world records,4 the rates of aging performance decay were very similar, if not identical.5 This lack of difference is a unique exception to the general rule of gender differences in sports activities, suggesting that neuro-hormonal mechanisms poorly influence the rate of aging muscle power decay. We discuss implications and limitations of our hypothesis that, at least in humans, the rates of age-induced decline are related to fundamental cellular mechanisms, perhaps those that control energy metabolism., The exponential increase of scientific publications in the mitochondrial field shows the growing interest in mitochondria. However, the lack of methodological consistency in many published projects on mitochondrial respiratory function complicates a quantitative inter/intra-laboratory comparison of datasets. This deficiency manifests the need to improve the quality in science.1 In this context, the MitoEAGLE COST Action is a powerful framework committed to evaluate and enhance the reproducibility in mitochondrial physiology as a basis to establish a novel mitochondrial database related to Evolution, Age, Gender, Lifestyle and Environment. Permeabilized muscle fibers are widely used to evaluate mitochondrial function in health and disease.2 Therefore, our main goals are to: 1) compare protocols used in different research laboratories, 2) analyze factors which contribute to experimental variability, 3) define optimal experimental conditions in muscle studies, 4) elaborate guidelines for evaluating mitochondrial function in muscle tissue, 5) establish reference values on mitochondrial respiration, particularly as a test of the skills in preparing high-quality permeabilized muscle fibers, and 6) generate a database. To achieve our aims, two unique studies are currently in progress: 1) 17 international research groups performing independently experiments on respiration in permeabilized fibers of mouse soleus muscle, following the same experimental procedure;3 2) a blinded international study measuring simultaneously in the same laboratory respiration of permeabilized human skeletal fibers by high-resolution respirometry and assessing the effect of different experimental conditions.4 Our results contribute to face the reproducibility crisis and provide the basis for establishing the first database on mitochondrial respiratory parameters in muscle tissues. Support. MitoEAGLE Task Group WG2. Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE., In the search for systemic changes of biomarkers1 in physical activity, within aging and rehabilitation studies, a major goal is to identify methods less invasive than blood sampling. Thus, the clinical use of mouth fluids is increasing.2 The consensus is that the presence of blood in mouth fluid compromises its diagnostic value. However, we have been looking at its’ contamination as a major opportunity for non-invasive serological analyses of systemic biomarkers. A major preliminary result has been obtained by evaluating the presence of serum in mouth fluids of healthy seniors and the eventual change after a modest trauma, i.e., tooth brushing. Seven healthy persons, aged older than 65, provided the fluids for the analyses by drooling saliva into a test tube. After low speed centrifugation, small aliquots of supernatants were frozen in liquid nitrogen and stored at -80°C until use. Aliquots were thawed and used to quantify by the total protein content using the Lowry method. Serum albumin, fibrinogen and lysozyme were determined by colorimetric ELISA (enzyme-linked immunosorbent assay), and hemoglobin content was quantified by use of spectrophotometry. After a preliminary test using colorimetric ELISA, saliva dilutions were adjusted to better determine the content of analytes. The control reference was a pool of sera from age-matched healthy seniors, to judge the quantity of serum in the fluid obtained from mouths of the elderly. Saliva collected from the seven healthy elderly persons before and after tooth-and-gum brushing presented measurable amount of the analytes, including fibrinogen, which is a minor component of the pooled sera. Tooth brushing did not induce a statistically significant difference in analytes’ contents, suggesting that a measurable blood contamination is a frequent event in elderly persons. In conclusion, fibrinogen is a promising reference to quantify serological biomarkers through a non-invasive procedure. Its determination will increase acceptability and frequency of analyses during follow-up in aging and rehabilitation., Older and oldest persons, i.e., early and late octogenarians, spend small amounts of time performing daily physical activity, which can aggravate their limited independence. This may force them to stay in bed, and/or to incur more frequent hospitalizations. All progressive muscle contractile impairments, including those related to advancing age, need permanent management.1,2 Inspired by the proven capability to recover skeletal muscle by home-based functional electrical stimulation (hbFES) and guided by common sense, we suggest to these elderly persons to perform a 10 to 20 min daily routine of 12 easy and safe physical exercises, that will train almost all the main skeletal muscles of the body. Elderly persons can do many of these exercises in bed (full-body in-bed gym), so that if hospitalized they may continue light training. The routine is an extension of the well-established cardiovascular and ventilatory rehabilitation trainings, usually performed under a guidance and supervision of a physiotherapist.5 If properly instructed in an outpatient clinic or during hospitalization, elderly persons may continue these exercises at home, and eventually integrate into their permanent lifestyle.3,4 Monitored arterial blood pressure before and after the daily routine described demonstrates that peripheral resistance decreases in a few minutes by functional hyperemia of the trained body muscles. The exercise intensity ought to be slightly challenging, up to the manifestation of visible sweating. In the long term, this will add systemic resistance to fatiguing arising from strength training against the own weight of an arm, leg and other body parts.2 Continued regularly, home-based full-body in-bed gym can help to maintain independence of the most frail elderly persons (old and oldest), reducing risks of accidental falls, and thus of serious clinical consequences., Scientific literature suggests that fitness characteristics in patients with Multiple Sclerosis (MS) may be compromised. Because nanotechnological devices have shown improvements in different healthcare application,1,2 the purpose of this study was to investigate the use of a nanotechnological device integrated with a postural exercise program (PEP) on cervical range of motion and handgrip strength in patients affected by MS. Seventeen participants with MS were recruited and randomly assigned to the Experimental Group (EG) and the Control Group (CG) including 9 and 8 subjects respectively. All participants carried out a cervical range of motion evaluation using an inertial motion sensor (Moover®;Sensor Medica®) and a handgrip test using an isometric mechanical dynamometer (KernMap model 80K1;Kern®) before and after a PEP (twenty-session/1 hour each, twice/week). A nanotechnological device (Taopatch®;Tao Technologies srls) was applied to the skin of the participants of the EG at the level of the C7 vertebra throughout the period of the PEP. No differences (p>0.05) were found in the EG on the cervical range of motion, while the CG showed a significantly increase on left rotation (p=0.01). Regards the handgrip test, a significantly improvement was found on the non-dominant hand in the CG (p=0.001) and on the non-dominant hand (p=0.04) and on the dominant hand (p=0.01) in the EG. Our preliminary results suggest that the use of a nanotechnological device could influence muscle strength. The results of this pilot study suggest that we should continue testing larger sample of subjects, diversifying also type and stage of the disease. The authors thank the patients and the Italian Association for Multiple Sclerosis (AISM) of Trapani, Italy for collaboration., Athletes have to pay particular attention on the eating patterns and to dietary intake in the hours before exercise, because pre-exercise nutritional strategies can influence not only exercise performance but also other biological responses to exercise and training including bone remodeling. In fact, inadequate nutrition leads to bone injuries. Twenty-eight preadolescent female gymnasts, playing artistic gymnastics at a pre-competitive level, were examined to investigate the effects of two different pre-exercise meals on athletic performance and bone resorption post exercise. Exercise trial were preceded ninety minutes by an isocaloric meal with carbohydrates or proteins. Urine was sampled at four different time points: pre-meal, ninety minutes post-meal/pre-exercise, ninety and hundred and fifty minutes post-exercise. In urine was analyzed the biomarker of bone resorption CTX by using an established enzyme-linked immunosorbent assay technique. Energy Self-Perception Questionnaire (ESPQ) was used to evaluate energy status of the athletes after the performance. Pre-workout supplementation with a carbohydrate-rich meal reduced significantly post-exercise bone resorption, compared with the protein-rich meal as evidenced by the reduction in CTX levels sixty minutes after the end of physical activity. Moreover, the consumption of both pre-workout meal improves performance indifferently. In conclusion the study shows that a carbohydrate rich pre-exercise meal is able to reduce bone resorption after exercise in pre pubertal age gymnasts and improve the athletic performance., The Main objective of the study was to evaluate the effects of treatment with Cannabis-based drugs combined or not with a proprioceptive training, on patients affected by Multiple Sclerosis (MS), a chronic autoimmune demyelinating disease which can have different symptoms; we estimated the possible synergic interaction between therapy and exercise particularly to reduce the collateral adverse effects of the drugs, over all the spasticity, one of the effect of the disease. We enrolled in our study 10 patients randomly assigned in two groups; both was treated with a cannabinoids drug (group 2) but only one group performed a physical activity for 12 weeks two times per week in session of 60 minutes each (group 1). Blood assessment (serum levels of BDNF, proBDNF, interleukine10 and interleukine17) was performed and the results highlighted a changes. As to concern BDNF levels were 111 vs 33 group 1 compare with 82 vs 22 in group 1; whilst proBDNF levels were 190 vs 153 group 1 compare with 121 vs 138 in group 1. About IL’s, we highlighted this results: IL10 (anti-inflammatory) 61,84 vs 75,52 in group 1 compare with 50,64 vs 54,65 in group 2; IL 17 (pro-inflammatory) 31,12 vs 16,87 in group 1 compare with 24,53 vs 9,68 in group 2. The interpretation of the results is still in progress but the changes has occurred; further investigations are needed improving the sample size and the time of the intervention.

Background\ud Building both strength and endurance has been a challenge in exercise training in the elderly, but dietary supplements hold promise as agents for improving muscle adaptation. Here, we test a formulation of natural products (AX: astaxanthin, 12 mg and tocotrienol, 10 mg and zinc, 6 mg) with both anti‐inflammatory and antioxidant properties in combination with exercise. We conducted a randomized, double‐blind, placebo‐controlled study of elderly subjects (65–82 years) on a daily oral dose with interval walking exercise on an incline treadmill.\ud \ud Methods\ud Forty‐two subjects were fed AX or placebo for 4 months and trained 3 months (3×/week for 40–60 min) with increasing intervals of incline walking. Strength was measured as maximal voluntary force (MVC) in ankle dorsiflexion exercise, and tibialis anterior muscle size (cross‐sectional area, CSA) was determined from magnetic resonance imaging.\ud \ud Results\ud Greater endurance (exercise time in incline walking, >50%) and distance in 6 min walk (>8%) accompanied training in both treatments. Increases in MVC by 14.4% (±6.2%, mean ± SEM, P < 0.02, paired t‐test), CSA by 2.7% (±1.0%, P < 0.01), and specific force by 11.6% (MVC/CSA, ±6.0%, P = 0.05) were found with AX treatment, but no change was evident in these properties with placebo treatment (MVC, 2.9% ± 5.6%; CSA, 0.6% ± 1.2%; MVC/CSA, 2.4 ± 5.7%; P > 0.6 for all).\ud \ud Conclusions\ud The AX formulation improved muscle strength and CSA in healthy elderly in addition to the elevation in endurance and walking distance found with exercise training alone. Thus, the AX formulation in combination with a functional training programme uniquely improved muscle strength, endurance, and mobility in the elderly.

The San Antonio Nathan Shock Center Conferences have attracted international speakers and participants since 1995. This annual conference, held in Bandera, Texas, addresses a different topic in the biology of aging each year. The venue's intimate setting, relatively remote location and common areas are ideal for a small conference (80–100 participants) where informal intellectual interchange supplements that of the formal sessions. The 2012 meeting, part of an annual series sponsored by the Nathan Shock Center of Excellence in the Biology of Aging and the Barshop Institute for Longevity and Aging Studies at the University of Texas Health Science Center San Antonio, addressed the concept that healthy aging and assessment of physiological performance are important parameters, in addition to longevity, to measure quality of life with increasing age. The purpose of the 2012 conference was to provide a forum for the presentation and discussion of various assays of measuring physiological performance and function and determining what assays of function could be used to asses healthspan of a mouse. Longevity is a precise endpoint (binary, the individual is either alive or it is dead), but the true goal of aging research is to increase the health of the elderly, not their longevity. That is, the goal is to enhance and extend healthspan, defined as the portion of our lives spent free of serious illnesses and disabilities. The assumption is that the only way an organism can increase its lifespan is by increasing its healthspan. This is a plausible assumption, but it still needs to be proven each time a manipulation is assessed for its potential for translation into humans. While the invertebrate models are particularly useful in genetic studies, they are generally not very good models for mammalian health, physiology, disease susceptibility, etc. Mice age with a constellation of diseases and functional losses that in some aspects resemble those observed in humans. Therefore, the conference focused on healthspan measures in mice. To this end, speakers were recruited who are working on assays (both simple and complex) to evaluate the functional status of various organ and physiological systems that are important in the health/physiological performance in mice and/or humans. In addition, attention was given to clarification of the molecular mechanisms underlying physiological decline, and its causal relationship to metabolic changes, muscle wasting, neurodegenerative diseases, cardiovascular disease, cancer, and inflammation and immunity, as well as targets for prophylactic intervention. Thus, the conference gave investigators a panel of assays that would allow them to determine the effect of genetic or pharmacological/nutritional manipulation on healthspan. Abstracts from posters presented at the meeting are presented in this special abstract issue to provide an overview of the breadth and depth of the program., Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, a drug that extends lifespan and delays aging, prevented the development of AD-like disease in mice modeling AD. Here we show that rapamycin administrated after the onset of AD-like deficits reversed brain vascular breakdown through endothelial nitric oxide (NO) synthase activation and NO-dependent vasodilation, decreased cerebral amyloid angiopathy and brain microhemorrhages, and improved memory in AD mice. These data suggest a mechanism by which chronic rapamycin ameliorates established AD-like deficits through the preservation of brain vascular integrity and function. Rapamycin, an FDA-approved drug already used in the clinic, may have promise as a therapy for AD and possibly for vascular dementias., A widely accepted cause of the functional losses that accompany aging is decreased brain metabolism (i.e., glucose oxidative capacity in mitochondria). It is generally believed that preserving bioenergetics is critical for optimizing lifespan and healthspan. Interventions have been introduced to preserve metabolism in aging process. Caloric restriction (CR) perhaps is the most well-studied one for various model organisms of extended longevity. In addition, in the neuronal system of rats (F344BNF1), CR also enhances cognitive function. However, the underlying physiology in the brain remains unclear. In the study, we used carbon-13 magnetic resonance spectroscopy (C-13 MRS) to investigate CR effect on brain metabolism in aged rats (24 months of age). CR-treated and control F344BNF1 rats (N = 6 for each group) were purchased from NIA. C-13 labeled glucose was continuously infused through the femoral vein of the rat for two hours and MRS was acquired simultaneously. The results show that CR rats had significantly increased oxidative metabolism rate (Voxi) in neurons (p < 0.01) and neurotransmission rate (glutamate-glutamine recycling rate; Vcyc) (p < 0.01) compared to the controls. The aged CR rats’ Voxi (4.5 µmol/g/min) and Vcyc (2.2 µmol/g/min) were comparable to those of young control rats reported in literature. However, CR and control rats did not have significant difference of glucose uptake and lactate production in the brain. The results suggest that alternative fuel subtract (e.g., ketone bodies) may be used to meet the brain energy demand. Our data provide a possible explanation of CR-induced increased lifespan and healthspan in rats., Chronic administration of rapamycin by transgenic (Tg) PDAPP mice allows them to perform better in hippocampal-associated learning and memory tasks compared with controls. We found, using conventional brain slice methods, that rapamycin had no significant effect on excitatory synaptic transmission, neuronal excitability, or the induction of long-term potentiation (LTP) in the CA1 region of the hippocampus. Surprisingly, we observed no significant effect on LTP in the control Tg group compare with wild type (Wt). We were concerned that some factor, such as stress due to transportation, might have enhanced the likelihood for LTP. To test for this possibility, we examined the relationship between stimulus strength and the magnitude of LTP induction. It is well known that LTP is a function of stimulus strength before induction due to the properties of NMDA receptors; with greater depolarization there is more calcium influx and, in turn, larger LTP. However, we found no correlation for either of our non-rapa control groups (Wt or Tg). In contrast, there was a correlation when animals were administered rapamycin, and the correlation was greater for Wt over Tg animals. Our working hypothesis is that stress, possibly due to transport, depressed inhibitory circuits lowering the threshold for LTP induction. Monte Carlo simulations comparing the amount of LTP produced by variations in the ratio of excitation to inhibition (E/I) support this hypothesis. Chronic rapamycin may protect the hippocampal network from dis-inhibition, maintaining E/I to sustain normal cognitive function., Chronic treatment with the mTOR inhibitor rapamycin (“Rapa”) extends lifespan in mice. Whether Rapa slows specific aging processes to increase “health span” is unknown. During aging, visual performance declines, and retinal neurons decrease in number. Here, we find and quantify a specific age-related decline in vision in mice. We also show that Rapa does not prevent this decline in visual function or affect neuron number. Instead, Rapa was detrimental to vision. Vision was tested using optokinetic tracking (“OKT”) to measure spatial frequency threshold at maximum contrast (“SPFT”) of the head-tracking behavior to horizontally drifting sinusoidal gratings. Male B6 mice were tested at 5, 21, 29, and 33 months of age (“mos”). Two other lines of mice were fed chow ad libitum containing micro-encapsulated rapamycin from 3 until 18 mos. In another group of B6 mice treated with rapamycin from 4 to 25 mos, retinas were immunostained with markers to count neuron subtypes. During normal aging, OKT SPFT significantly declined by 31%. Rapa did not protect against this age-related OKT decline in either treated strain but significantly decreased OKT performance for male, but not female, mice at 18 mos. Rapa male mice had decreased IPL thickness in the retinal periphery, but numbers of dopaminergic and cholinergic amacrine neurons and retinal ganglion cells were unchanged. Thus, Rapa does not prevent age-related declines in OKT visual function or in retinal neuron number. It instead causes an OKT SPFT deficit in male mice. These findings suggest Rapa does not increase vision health span during aging., Decline in sensory acuity is a general hallmark of aging, which in humans decreases quality of life. We report here creation and successful utilization of a novel sensory acuity assay in mice. Three features of the assay merit attention. First, as mice are primarily nocturnal in nature, olfaction is an important sensory modality for them. Second, our assay instead of using artificial olfactory cues employs major urinary proteins, which are important in both intrasexual and intersexual communication of mice in nature. Third, the assay can be performed in the mouse's home cage, thus avoiding artifacts from distracting, novel environments. Procedurally, the assay uses serial dilutions of urine and preference for the urinary odor relative to a water control to measure olfactory acuity. Age-related changes in olfactory acuity have not previously been reported in mice. We created this assay, which compares time spent sniffing a sample relative to time spent at a distilled water control. It has been used numerous times and proves to be sensitive, repeatable and encompass particularly informative urinary dilution ranges. Specifically, previous testing revealed that of any age, mice usually cannot distinguish urine from water at a dilution of 1:10,000 (Rendón, unpublished data). The range of experimental dilutions between 1:10,000 to 1:5,000 has been narrowed down through successive modifications. Sampling in this range, we have detected a clearly defined age-related decline in mouse olfactory acuity. Therefore, this assay will serve useful in assessing changes in health span of mice and can be combined with therapeutic agents to assist in evaluation of their effect on health span., The accumulation of oxidative damage is a proposed mechanism regulating the aging process and the development of disease. Proteins are sensitive to such oxidative stress, which can cause them to accumulate, altering conformational structure, and thus the function, of cellular proteins. Methionine sulfoxide reductase A (MsrA) plays an important role in the antioxidant defense, but is unique in that it repairs protein oxidative damage. MsrA reduces methionine sulfoxide residues to non-oxidized methionine, thus participating in the antioxidant defense system of cells specifically by protecting proteins from oxidative stress. We have found that mice that lack MsrA (MsrA−/−) and mice that over express MsrA (MsrATg) are phenotypically similar to wildtype (WT) mice under normal conditions, but that MsrA levels can regulate susceptibility to oxidative stress. Because these mice are grossly normal, this suggests that excess methionine oxidation may not occur under these physiological conditions. In vivo, increasing adiposity has been associated with increases in oxidative stress, altered redox signaling and increased oxidative damage to cellular macromolecules in several disease models, including obesity-induced metabolic diseases. When placed on a high fat (HF) diet, MsrA−/− mice become more insulin resistant than WT mice whereas MsrATg mice are protected from development of insulin resistance. The increase in insulin resistance in MsrA−/− mice fed HF diets correlated with reduced insulin-stimulated signaling in the insulin signaling pathway. We found that HF fed MsrA−/− mice had reduced phosphorylation of both insulin receptor and Akt with administration of insulin under high fat fed conditions. Also, increased insulin sensitivity seen in the HF fed MsrATg mice correlated with an increase in insulin-stimulated signaling in the insulin signaling pathway. These results suggest that oxidative damage, specifically to proteins, may play an important role in obesity-induced insulin resistance. To address how protein oxidation may cause insulin resistance, we have utilized in vitro studies in primary myoblasts to test the effect of MsrA on oxidative stress-induced insulin resistance. By utilizing these models, this study will test the hypothesis that MsrA can regulate the development of insulin resistance by repairing oxidative damage in proteins involved in the insulin signaling pathway in vitro. Insulin resistance can be induced in vitro by H2O2. In this study, skeletal muscle precursor cells isolated from MsrA−/−, MsrATG, and WT mice, and then differentiated into myotubes, were tested for resistance to oxidative stress. Insulin signaling protein phosphorylation correlates with in vivo signaling observations, determined by western blot after insulin stimulation. Our hypothesis is that the level of protein oxidation can be correlated with the degree of insulin resistance in this system. Protein oxidation can be globally measured in the cell using a carbonyl assay. Once labeled, individual proteins can also be measured for total carbonyl content via immune precipitation. Because oxidation of proteins can lead to a decline in their function, these studies will focus on both function of the insulin signaling proteins isolated from these models as well as oxidation status of these proteins., Loss of skeletal muscle function is severely debilitating and sarcopenia profoundly affects the quality of life in the aged population. Impaired mitochondrial energetics in skeletal muscle is associated with loss of function and increased mitochondrial oxidative stress. To explore age-related mitochondrial energy deficits we use chronic (transgenic) and acute (pharmacological) targeting of mitochondrial oxidative stress. Previous work demonstrated that mitochondrial targeted catalase (mCAT) delays the onset of age-related pathology and extends lifespan in mice. However, little is known about how the relationship of mitochondrial energetics and cellular redox status changes with age. We demonstrate that there is a decline in mitochondrial quality in aged permeabilized skeletal muscle, particularly in the fast-twitch extensor digitorum longus, that was prevented in mice expressing mCAT. We also demonstrate that acute treatment (~1hr) of aged mice with the mitochondria-targeted small peptide SS-31 results in immediate improvement of skeletal muscle energy metabolism and performance. These results provide further evidence that decreased mitochondrial function with age may be due to an altered oxidative status of mitochondria and we propose that there are two facets of mitochondrial deterioration with age: a structural component that is attenuated with long-term expression of MCAT, and a regulatory component dependent upon the oxidative status of the cell that is rapidly reversible with acute treatment of SS-31. These results suggest that the oxidative state of skeletal muscle is a practical therapeutic target, and raises questions about how oxidative status and mitochondrial content affect the adaptive and pathological response of mitochondrial metabolism to age., Recently, our laboratory made the surprising observation that overexpressing Cu/ZnSOD [Tg(SOD1-SD)+/0] in Sprague-Dawley (SD) rats resulted in a significant increase in lifespan and a reduction in age-related pathologies. The purpose of this study was to determine why overexpressing Cu/ZnSOD increases lifespan in SD rats. The Tg(SOD1-SD)+/0 rats showed lower levels of oxidative damage to DNA and lipids in vivo and higher resistance to oxidative stress in vitro. Both Tg(SOD1-SD)+/0 and wild-type rats showed an age-related increase in body fat and Cu/ZnSOD overexpression did not attenuate adiposity. Interestingly, Tg(SOD1-SD)+/0 rats showed a significant increase in insulin sensitivity at a young age and lower plasma glucose levels at an old age. To further investigate the role of Cu/ZnSOD overexpression on aging, we generated transgenic rats with F344 overexpressing Cu/ZnSOD [Tg(SOD1-F344)+/0]. Tg(SOD1-F344)+/0 rats showed similar levels of Cu/ZnSOD overexpression to Tg(SOD1-SD)+/0. The Tg(SOD1-F344)+/0 rats showed lower levels of oxidative damage to lipids in vivo, however, neither Tg(SOD1-F344)+/0 nor wild-type rats showed any age-related changes in body fat, insulin insensitivity, and plasma glucose levels. Furthermore, Tg(SOD1-F344)+/0 rats showed little increase in lifespan compared to wild-type rats. Our results are very exciting because these data indicate that overexpression of Cu/ZnSOD could be more protective against oxidative stress and could attenuate aging and age-related diseases under obese conditions in mammals. (Supported by grants from the VA Merit Review, the American Federation for Aging Research, and the Glenn Foundation), A reduced ability to effectively regulate glucose metabolism is one of the most common markers of declining healthspan in aging mammals. Advancing age is an independent risk factor in the development of glucose intolerance, insulin resistance, and diabetes mellitus. Understanding the mechanisms responsible for this could significantly contribute to developing effective therapeutics or preventatives for those most at risk. Our data support a hypothesis that oxidation of proteins involved in insulin signaling may play a significant role in this process. Using a cell culture model, we show that oxidative stress inhibits the cellular response to insulin. The binding of insulin to insulin receptor normally promotes auto-phosphorylation of the β-subunit which regulates downstream insulin signaling through its kinase activity. Our data show that oxidative stress inhibits insulin signaling partly by causing oxidative damage that inhibits this process. Oxidative stress promotes formation of protein carbonyl adducts within insulin receptor; these adducts lead to diminished auto-phosphorylation function. We then addressed whether insulin receptor oxidation occurs in vivo with metabolic dysfunction. Insulin receptor isolated from high fat-fed C57BL/6 mice also show significantly elevated insulin receptor oxidative damage and reduced auto-phosphorylation function. Our preliminary studies suggest a similar process of oxidative damage is associated with reduced glucose metabolism in aging mice. These data support the idea that accumulating oxidative damage is a common molecular mechanism by which several primary risk factors (i.e., obesity, aging) promote insulin resistance. Targeting therapeutics that reduce/remove/repair oxidative damage might then develop as a valuable treatment option among the geriatric population., Longevity and aging are influenced by common intracellular signals of the insulin and IGF-1 pathway. Enhanced availability of IGF-1 is promoted by cleavage of IGF binding proteins (IGFBPs) by proteases, including the pregnancy-associated plasma protein-A (PAPP-A). PAPP-A (-/-) mice live 30% longer than their normal littermates and have decreased bioactive IGF-1 on normal diets. Our objective was to elucidate the effects of a high-fat (58 % kcal)/ high-sucrose (25.5 % kcal) diet that promotes obesity and increase pro-inflammatory cytokines in normal and PAPP-A(-/-) female littermates. The results indicate that PAPP-A (-/-) mice fed a high energy diet are more glucose tolerant than normal littermates fed a low energy diet (P ≤ 0.05) while insulin tolerance did not change. The high energy diet increased IGF-1 levels in PAPP-A (-/-) mice compared to littermates (-/-) fed a low energy diet (P ≤ 0.002). PAPP-A (-/-) mice fed with a high energy diet had lower levels of pro-inflammatory cytokines (IL-2, IL-6 and TNF-a) compared to normal littermates fed a high energy diet (P < 0.05). In contrast, anti-inflammatory cytokine levels (IL-4 and adiponectin) were higher in PAPP-A (-/-) mice fed a high energy diet compared to normal littermates on high energy diet (P < 0.05). We conclude that PAPP-A (-/-) mice when compared to normal littermates are resistant to the effects of diet-induced metabolic dysfunction. Furthermore, high energy fed PAPP-A (-/-) mice have higher levels of anti-inflammatory cytokines and lower levels of inflammatory cytokines, possibly rescuing them from the detrimental effects of a high energy diet., Obesity is a major risk factor for the development of age-related metabolic diseases. The mammalian target of the rapamycin (mTOR) pathway plays critical roles in eukaryotic cell growth, survival, and translation and hyperactivation of mTOR pathway due to excess nutrients causes insulin resistance, a major risk factor for type 2 diabetes. Rapamycin is a potent inhibitor of mTOR pathway suggesting its beneficial effects on metabolism. Paradoxically, rapamycin treatment causes glucose intolerance in mice. While most of the studies focus on the effect of rapamycin on metabolism in normal mice, no study has addressed the metabolic effects of rapamycin in diabetic mouse models. Here, we are studying the effects of rapamycin in db/db mice, a model of diabetic dyslipidemia. Administration of rapamycin for 9 months, starting at 2 months of age, significantly reduced body weight (43%) in female db/db mice compared to db/db mice fed the control diet (eudragit), due to a reduction in fat mass. This reduction in fat mass is not due to alterations in fat synthesis (PPARξ and SREBP1) or fatty acid transport (CD36 and FATP1) or lipolysis (P-HSL/HSL ratio), rather due to increased levels fatty acid oxidation as indicated by increased levels of carnitine palmitoyltransferase I (CPT1, 5-folds), large-chain acyl-coenzyme A dehydrogenase (LCAD, 2.5-folds) and medium-chain acyl-coenzyme A dehydrogenase (MCAD, 1.5-folds) in rapamycin-fed db/db mice compared to eudragit-fed db/db mice. Consistent with this observation, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) are significantly up-regulated both at the transcriptional and translational levels. In addition, markers of mitochondrial biogenesis CREB-regulated transcription coactivator 3 (CRTC3), nuclear respiratory factor 1 (NRF1) and estrogen-related receptor alpha (ERRα) were significantly elevated in the adipose tissue of rapamycin-fed db/db mice. While rapamycin did not decrease the levels of circulating triglycerides and glucose in db/db mice, levels of circulating free fatty acid was significantly reduced and adiponectin levels were significantly increased by rapamycin treatment, suggesting improved insulin sensitivity. Finally, insulin sensitivity assessed by insulin tolerance test showed significant improvement in insulin sensitivity in rapamycin-fed db/db mice. In summary, our study demonstrates for the first time that rapamycin exhibits anti-obesity effect in db/db mice and improves insulin sensitivity due to the up-regulation of the mitochondrial biogenesis and increased fatty acid oxidation in the white adipose tissue., Cytochrome c oxidase (COX) is an essential transmembrane protein complex in the mitochondrial respiratory electron chain. Mutations in genes responsible for the assembly of COX are associated with Leigh syndrome, cardiomyopathy, spinal muscular atrophy and other fatal metabolic disorders in humans. Paradoxically, mice lacking the COX assembly protein SURF1 show increased longevity associated with upregulation of mitochondrial biogenesis and stress response pathways despite significant reductions 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 similar molecular and physiologic changes. A complete knockout of the sco2 gene in mice is embryonic lethal, however mice harboring a mutated sco2 knock-in (KI) allele that is commonly found in human patients with sco2 mutations is viable, and despite the 30–60% reduction in COX activity, no significant phenotypic abnormalities are readily apparent. Interestingly, these mice have a decrease in lean mass and increase in fat mass. Preliminary evidence suggests that these mice are insulin resistant and glucose intolerant as compared to wild-type mice. The sco2 KI/KI mice also have decreased running endurance on the treadmill suggesting that these mice have muscle weakness. Interestingly, the COX-deficient mice do not have changes in the blood lactate levels suggesting that these mice do not upregulate glycolysis to compensate for decreased rates of respiration. This is counter to studies done in another COX deficient Surf1-/- mice, illuminating the complex nature of mitochondrial dysfunction on physiology. Results from this study will further our understanding of the role of complex IV in physiological outcomes due to mitochondrial dysfunction., Vitamin D insufficiency, sarcopenia of aging, and obesity exert profound impacts on physical performance and overall healthspan. Although human clinical studies have demonstrated significant relationships between vitamin D and physical performance, they contain confounding factors such as age, obesity, diet, and lifestyle that make understanding the specific pathophysiology difficult. Therefore, we are developing a novel mouse model capable of isolating individual and combinatorial impacts of vitamin D insufficiency, aging and obesity on physical performance. We provided 6 month-old male mice with either 1000IU or 125IU vitamin D3/kg chow over 4 months. Longitudinal serum 25-OH vitamin D measurements show levels change rapidly (both depletion and repletion) and consistently to the degree of supplementation, allowing for comparisons between sufficient and insufficient mice. Furthermore, our data indicate body weight and fat percentage are higher in vitamin D insufficient mice after 4 months. Additionally, our data suggest that vitamin D insufficient mice have higher levels of IL-6 and TNF- in their epididymal fat tissue. Rotarod treadmill performance and grip strength were similar regardless of vitamin D status. However, we found that elderly mice (24 months) exhibit functional decline compared to young mice despite both groups being sufficient (25-OHD ≥ 30 ng/ml). These data lay the foundation for our continuing investigation on vitamin D insufficiency, aging, obesity and physical performance and will further our understanding of the underlying mechanisms driving health span decline., Synucleinopathies are age-related neurodegenerative disorders characterized by expression of pathological α-synuclein inclusions. Synucleinopathies include Parkinson's disease (PD), multiple system atrophy and dementia with Lewy bodies (DLB), which affect millions of patients worldwide. Parkinsonian motor symptoms like rigidity and slow movement are common in synucleinopathies. A53T mutation is the first α-synuclein mutation linked to PD, and it is linked to both sporadic and familial PD. Autophagy is reduced in PD brain. Levels of mTOR are increased and ATG7 levels are reduced in DLB brain. Rapamycin, an mTOR inhibitor and autophagy enhancer, is protective in mouse models of neurodegenerative diseases like Alzheimer's disease and PD. Rapamycin reduces a-synuclein accumulation and neurodegenerative phenotype in neuronal cells. Feeding rapamycin diet extends mouse lifespan and the mechanisms are hypothesized to be mediated via delaying age-related diseases including neurodegenerative diseases. 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 α-synuclein transgenic mice, which express human A53T α-synuclein richly in brain and spinal cord and develop motor deficits. Mouse diet containing microencapsulated rapamycin (14 ppm in diet; 2.25 mg/kg body weight/day) or the microencapsulation material 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 of A53T mice. Rapamycin also increased front stride length in male A53T mice. In conclusion, rapamycin attenuated motor deficits in the A53T mice. Further experiments will determine whether the effects of rapamycin are through reducing human α-synuclein expression in brain regions that control and regulate motor function including motor cortex, spinal cord, midbrain, striatum and cerebellum. In addition, it is reported that rapamycin improves myelination in explant cultures from neuropathic mice. Thus, effect of rapamycin on demyelination in A53T mice will also be determined in the brain regions mentioned above., Parkinson's disease (PD) is the second most prevalent neurodegenerative disease. Degeneration of dopamine neurons within the substantia nigra, leads to a substantial decrease in dopamine release in the substantia nigra and the striatum, as well as impaired motor function. Motor symptoms associated with PD include resting tremor, rigidity, and bradykinesia. Although the cause of this disorder remains unclear, several lines of evidence implicate mitochondrial dysfunction and oxidative stress. Major cytosolic enzymes ALDH1 (aldehyde dehydrogenase 1) and GPX1 (glutathione peroxidase 1), are involved in the metabolism of biogenic aldehydes and the reduction of hydrogen peroxide, respectively. ALDH1 is selectively expressed in the midbrain and found to be co-localized with tyrosine hydroxylase within the substantia nigra and ventral tegmental area. Gene profiling studies have been reported showing a decreased expression of ALDH1 in surviving dopaminergic neurons of PD patients. GPX1 gene expression in the substantia nigra of PD patients is also markedly reduced. Therefore, we hypothesize that deletion of both Aldh1a1 and Gpx1 will lead to the accumulation of reactive oxygen species and highly reactive biogenic aldehydes leading to motor deficits. To test this hypothesis, our lab crossed two mouse lines deficient in Aldh1a1 and Gpx1 genes. Here we report impaired locomotor function in Aldh1a1 x Gpx1 knockout mice. These data are consistent with the idea that elevated levels of reactive oxygen species and/or biogenic aldehydes may lead to motor deficits similar to those found in Parkinson's disease., Traumatic Brain Injury (TBI) is a known risk factor for ALS. The goal of this study is to elucidate the mechanism linking TBI to motor neuron disease, by testing the hypothesis that TBI will accelerate disease progression in animal models of ALS. We used the well-characterized mouse models of familial ALS (G93A SOD1) and sporadic ALS (TDP43, TDP25) to study the effect of TBI on ALS progression. Mice were subjected to a closed head traumatic brain injury and magnetic resonance imaging (MRI) was used 3 days after injury to characterize structural central nervous system pathology and the severity of brain injury. Histological techniques showed neuronal loss (NeuN), astrocyte infiltration (GFAP) and edema (Nissl) following mild TBI in wildtype (WT) and transgenic mice (TG). Our preliminary results indicate that TBI leads to a reduction in grip strength, decreased rotarod performance and muscle denervation via electromyography abnormalities. Also, we have characterized an acceleration of disease related weight loss and overall disease score following TBI in G93A mice. Our results are the first to show that TBI, in an animal model of ALS, results in significantly increased muscle denervation and potentiates disease onset and progression. This work is supported by an individual fellowship grant, 1F31NS080508-01, as well as the Barshop Institute for Longevity and Aging., Mechanical inactivity or disuse causes muscle loss and bone loss in both men and women; however, it is not known whether food restriction (FR) has any effect on mechanical inactivity-associated muscle and bone loss. Disuse-associated musculoskeletal atrophy could be associated with nerve injury. The present study aimed to investigate the effect of 40% FR on sciatic nerve injury associated muscle and bone loss and also to analyze if there is any time dependent effect of FR after sciatic nerve injury. Two-month-old male C57BL/6 mice were randomly allocated into two groups: (1) ad libitum (AL) (2) 40% FR fed lab chow for 8 months. The left hind limb of each mouse was then subjected to sciatic nerve crush to induce mechanical inactivity of the particular leg. After different time points (2 days, 7 days, 14 days, 21 days, 28 days and 42 days) of mechanical inactivity, mice were sacrificed and analyzed for muscle mass (wet weight) and bone mass (dual energy x-ray absorptiometry (DXA)). AL fed mice showed significant loss of gastrocnemius and tibia due to mechanical inactivity whereas, FR mice showed protection of both gastrocnemius and tibia from inactivity associated loss. Interestingly, this gastrocnemius and tibial loss protection was stable up to 42 days of mechanical inactivity, we have tested. This data suggests that FR may be beneficial in case of disuse situation commonly happened during aging. Further studies are necessary to determine the musculoskeletal quality and the molecular mechanisms involved in FR mediated protection of musculoskeletal loss due to disuse., Oxidative stress is implicated in loss of muscle mass with age in the CuZnSOD deficient mice (Sod1-/-). However, the mechanisms of oxidative stress-dependent loss in muscle mass are currently unknown. Since oxidative stress is considered to be an important contributor to muscle atrophy and muscle activity is dependent upon nerve stimulation, this study proposes that oxidative stress damages protein integrity which leads to impaired nerve conduction velocity and myelination. To test our hypothesis, we chose the Sod1-/- mouse model and control c57bl/6 mouse to determine declines in nerve conduction velocity and myelination. Gastrocnemius muscle isolated from the Sod1-/- mice have significant atrophy at 6 and 18 months of age. Sciatic nerve conduction velocity was significantly impaired at both 6 and 18 months of age in the Sod1-/- mice. 6 month old Sod1-/- mice had reduced axon and fiber diameter with what appeared to be changes in myelin morphology which by 18 months of age resulted in reduced myelin thickness and increased g-ratio (axon/fiber diameter). Also, the sciatic nerves from the Sod1-/- mice exhibited significant global increase in protein carbonyls and alteration in exposure of surface hydrophobic domain in proteins. Taken together, these data suggest that loss in nerve conduction velocity and myelin might play a significant functional outcome in gastrocnemius atrophy., Half of all Americans over the age of 50 either already have or will develop osteoporosis and osteoporotic fracture is associated with increased mortality rates. Fracture can be considered a chronic condition as complications from fracture can extend well past healing of the initial fracture, thus preventing fracture is required for prolonging healthspan. Bone mineral density (BMD) is highly correlated to fracture risk and environmental factors, such as diet impact BMD. As diet can be modulated, identification of what types of and how dietary constituents decrease BMD will increase our general knowledge about the etiology of osteoporosis and could illuminate opportunities to intercede to prevent fracture. Preliminary studies have suggested that a high fat diet negatively impacts bone mass, but it remains unknown which type of fat mediates these negative effects. In this study, we specifically examined the consequence of increased cholesterol intake on bone mass and osteoblast maturation. We determined that dietary cholesterol, independent of other types of dietary fat negatively impacts on BMD in C57BL/6J female mice. We then established that dietary cholesterol appears to decrease the marrow osteoblast progenitor pools in the femur. In the vertebrae, a high cholesterol diet was associated with a decrease in trabecular bone thickness and with an increase in osteoclastic activity in the vertebrae. Together, this shows that dietary cholesterol, independent of other types of dietary fat, negatively impacts bone mass. In the femur, cholesterol affects the osteoblast linage where as in the vertebrae its effects are mediated via osteoclastic bone resorption., Maintenance of skeletal muscle regenerative capacity is crucial for preservation of muscle mass and function with age. Skeletal muscle precursor (SMP) cells are myogenic stem cells that play a predominant role in muscle regeneration. These cells are located beneath the basal lamina of the myofiber and respond to tissue injury with activation, differentiation and fusion into an existing myofiber. Previous studies have identified a panel of cell surface markers to isolate pure populations of SMP cells from mice with minimal contamination by flow cytometry. Using this technique, the current study assessed the impact of age on SMP content and function. By analyzing male C57Bl/6 mice aged 18–100 weeks on a standard ad libitum diet, it was found that the SMP population decreases by 20–60% with age, depending on the muscle depot. The greatest decline was found in the triceps bracii, which is composed predominantly of Type IIb fibers (fast glycolytic). Furthermore, the regenerative capacity of isolated cells was impaired in older mice, as measured by proliferation and differentiation of SMP cells into myofibers. This study highlights the negative effect of aging on skeletal muscle stem cells. Future work will explore interventions to prevent the loss of regenerative capacity with age., Osteoporosis is a silent disease characterized by excess bone resorption by osteoclasts compared to bone formation by osteoblasts. Our lab has shown that old male mice deficient in caspase-2 (Casp2-/-), a cysteine protease involved in apoptosis, exhibit severe age-related osteoporosis. Interestingly, these mice also have higher numbers of osteoclasts compared to age-matched wild-type (WT) mice. This could mean that more osteoclasts are being created or less osteoclasts are dying in Casp2-/- animals compared to WT. However, the role of caspase-2 in osteoclasts remains to be elucidated. We hypothesize that caspase-2 plays a dual role in both osteoclast apoptosis and differentiation. With regards to apoptosis, caspase-2 as an upstream component of the apoptotic pathway has been well described in a variety of cell types. Furthermore, cells lacking caspase-2 have been shown to be more resistant to oxidative stressors. Here, we show that osteoclasts derived from Casp2-/- mice are more resistant to 6 hour treatment with various doses of the general stressor H2O2 and the mitochondrial stressor rotenone compared to osteoclasts from WT mice. Osteoclasts are formed through macrophage fusion that is spurred by the osteoblast-derived cytokines CSF-1 and RANKl. We show that macrophages from Casp2-/- animals form increased numbers of osteoclasts compared to WT. In addition, we have seen that caspase-2 levels decrease in macrophages after RANKl stimulation, suggesting that low caspase-2 expression may be important during osteoclast differentiation. Delineating the role of caspase-2 in the osteoclast may provide new information that will aid in the development of novel osteoporosis treatments., Neuromuscular junction (NMJ) degeneration and muscle atrophy occur with age and in various neuromuscular diseases. Previously we have demonstrated that mice deficient in Cu/Zn superoxide dismutase (CuZnSOD or SOD1) exhibit age-dependent NMJ degeneration, muscle weakness and functional motor deficits. The purpose of this study was 1) to determine whether these changes are associated with alterations in NMJ neurotransmission; 2) to determine whether the NMJ phenotype is a cell-autonomous trait of CuZnSOD deficiency in muscles or neurons. Electrophysiological studies of CuZnSOD knockout mice (KO) demonstrate pathological decrement in compound muscle action potential (CMAP) amplitude with repetitive nerve stimulation (RNS), which is indicative of faulty neurotransmission. To test the second aim, we utilized tissue specific knockout and transgenic mice of SOD1. Neuron-specific SOD1 knockout mice (NKO) developed a moderate reduction in muscle mass, while muscle-specific SOD1 knockout mice (MKO) showed no muscle atrophy. Neither NKO nor MKO mice showed alterations in RNS, suggesting the NMJ deficits in KO mice may be a synergistic effect from both cell types. However MKO mice exhibit multiple characteristics of myopathy including denervation potentials, central nuclei and increased muscle damage upon exercise. It suggests that CuZnSOD plays an essential role in maintaining skeletal muscle integrity. Meanwhile, neuronal SOD1 overexpression rescued muscle atrophy and aberrant CMAP parameters in the KO mice. In conclusion, the complete NMJ phenotype in KO mice is likely caused by deficiency of CuZnSOD in both muscle and neurons. Our data indicate that muscle atrophy in KO mice may be secondary to the neuronal defect., Aging is associated with chronic low-grade inflammation, due in part to the pro-inflammatory secretory profile of replicative senescent cells (i.e. senescence associated secretory phenotype [SASP]). Paradoxically, macrophages from aged animals fail to produce the pro-inflammatory cytokines necessary to recruit and activate other immune cells and have poor bacteria killing ability (i.e. age-dependent macrophage dysfunction [ADMD]). A recent publication examining LPS-induced macrophage anergy [Park SH, et al., Nat. Immunol. 2011, 22:12(7): 607–15] triggered us to test the hypothesis that pro-inflammatory cytokines produced by senescent cells may be responsible for ADMD. Bone-marrow derived and J774A.1 macrophages exposed to senescent type II epithelial lung cells (A549 cell line) overnight demonstrated a decreased ability to kill Streptococcus pneumoniae, a gram-positive bacteria and the leading cause of community-acquired pneumonia, versus those exposed to normal cells in vitro. J774A.1 macrophages, but not bone-marrow derived macrophages, exposed to filtered conditioned media from senescent cells also showed an attenuated ability to kill bacteria versus controls. Likewise, they demonstrated an inability to produce de novo Interleukin-6 following stimulation with ethanol-killed pneumococci. Ongoing studies are focused on determining the component produced by senescent cells that is responsible for macrophage dysfunction., The ubiquitin proteasome system (UPS) is responsible for the controlled cleavage of damaged and misfolded proteins and antigen-producing peptides. Commonly reported declines in efficiency of the UPS with age may play a critical role in age-associated dysfunction of protein homeostasis and immune function. The longest-lived rodents, naked mole-rats (Heterocephalus glaber), maintain robust, cancer-free good health for 75% of their 32 year lifespan suggesting that decline in protein homeostasis, observed in other animals, is attenuated or delayed. We compared age-related changes in proteasome activity in whole cell and sub-fractionated lysates from spleen tissues of naked mole-rats and physiologically age-matched mice. Naked mole-rat lysates, as well as cytosolic and nuclear fractions had significantly higher proteasome chymotrypsin-like (ChTL) and trypsin-like (TL) activity than those of age-matched mouse samples. The age-related decline in naked mole-rat ChTL and TL proteasome activity in spleen lysates was negligible; in contrast mice showed a significant age-related decline. By 70% of maximum lifespan proteasome activity of naked mole-rat was unchanged (p > 0.05) whereas mouse declined by 48% (p < 0.02). Similar age-related species differences were observed in all three fractions. Attenuation of age-related UPS decline in naked mole-rats was further supported by sustained maintenance of the 26S proteasome with age, and higher levels of constitutive and immunoproteasome-related proteasome subunits in the naked mole-rat compared to mice. Given the importance of the spleen in immune function, high and sustained UPS in splenic tissue may contribute significantly to prolonged good health in this extraordinary long-lived rodent., Natural aging processes cause gradual degradation or senescence of the immune system at the humoral and cellular levels. A diminished immune capacity due to aging correlates clinically with decreased vaccine efficacy and increased susceptibility to infection and cancer. Due to this loss in immunity the protective capacity of new vaccines should be determined in older individuals. Animal models for vaccine development should embody the immunosenescent effects observed in aging humans. A baboon model was tested by immunizing young (5–6 years of age) and old (17–22 years) animals with the LcrV and F1 candidate vaccine antigens from Yersinia pestis. Contrary to the expected loss of immunity, older baboons demonstrated high antibody titers and exhibited strong T cell proliferation, particularly in response to LcrV. These findings suggest that aging has less effect on the baboon immune system. The cellular and cytokine responses to antigen stimulation were measured to better characterize the effects of aging on T cell fine specificity. T cell proliferation and IFN-γ ELISpots were used to map which of 32 overlapping synthetic F1 peptides stimulated T cells from the immunized baboons. Spectratype analysis of T cell receptor (TCR) expression indicates no age associated loss in T cell activation of the overall repertoire diversity. Currently, F1-specific T cell lines are being generated using herpesvirus papio transformed B cell lines as antigen presenting cells. Future efforts will focus on characterizing the TCR repertoire of an F1-speific response., The anti-tumor action of calorie restriction (CR) and the possible underlying mechanisms on tumor growth were investigated using ethylnitrosourea (ENU)-induced glioma in rat. ENU was given transplacentally at gestational day 15. The brain from 4, 6, and 8-month-old rats fed either ad libitum (AL) or calorie restricted diets (40% restriction of total calories compared to AL rats) were studied. Tumor burden was assessed by comparing the size and number of gliomas present in the brain. Immunohistochemical analysis was used to detect the lipid peroxidation products [4-hydroxy-2-nonenal (HNE), malondialdehyde (MDA), and acrolein] and nitrotyrosine to document oxidative stress, levels of glycated end products, cell proliferation activity (PCNA), and cell death (ssDNA) associated with the development of gliomas. The results showed that the number of gliomas did not change with age in the AL groups; however, the average size of the gliomas was significantly larger in the 8-month-old group compared to that of the younger groups. Immunopositive areas for HNE, MDA, acrolein, nitrotyrosine, and glycated end products increased with the growth of gliomas. The CR group showed both reduced number and size of gliomas, less accumulation of oxidative damage, and less glycated end products compared to the AL group. Furthermore, the CR group showed less PCNA positive and more ssDNA positive cells. Interestingly, we also discovered that the anti-tumor effects of CR were associated with less accumulation of hypoxia inducible factor-1α (HIF1α) levels and a reduction in the mammalian target of rapamycin (mTOR) signaling. Our results are very exciting because they could not only demonstrate the anti-tumor effects of CR on oligodendroglioma, but also indicate the possible underlying mechanisms, i.e., anti-tumor effects of CR could be mediated by the changes in redox-sensitive and/or nutrient sensing signaling pathways. (Supported by grants from the VA Merit Review, the American Federation for Aging Research, the Glenn Foundation, and San Antonio Nathan Shock Center), Our laboratory has conducted the first detailed study on the effect of overexpressing or down-regulating thioredoxin 1 (Trx1: cytosol) or thioredoxin 2 (Trx2: mitochondria) on aging. Interestingly, we found that the Trx2Tg mice showed an extension of median lifespan compared to wild-type mice, although we observed little increase in survival of the Trx1Tg mice. The extension of lifespan of Trx2Tg mice was correlated to less reactive oxygen species (ROS) production from mitochondria and less oxidative stress. These data show that overexpressing Trx in the mitochondria may be more important than in the cytosol on aging because mitochondria are a major source of ROS. When we tested the effects of reduced levels of Trx in cytosol or mitochondria on aging, we surprisingly observed the reversed effects, i.e., an increase in survival of the Trx1KO mice compared to wild-type mice, while the Trx2KO mice showed little effects on lifespan. The extension of lifespan of Trx1 KO mice was associated with less cancer compared to wild-type mice at 22–24 months of age. These results indicate that reduced cancer in the Trx1KO mice could be one of the contributing factors of extended lifespan. Our data are exciting in that we show 1) overexpressing Trx in the mitochondria increases lifespan, but overexpressing Trx in the cytosol has little effect on lifespan, which is similar to the results of mCAT mice; and 2) down-regulating Trx in the cytosol increases lifespan and reduces cancer, but down-regulating Trx in mitochondria has no effect on lifespan or cancer. These paradoxical, but intriguing results could indicate that the Trx2Tg and Trx1KO mice attenuate aging through different mechanisms, e.g., protection of mitochondria against oxidative stress and reduced age-related pathology, e.g., cancer. (Supported by grants from the VA Merit Review, the American Federation for Aging Research, and the Glenn Foundation), Long-lived animal models across multiple phyla have a marked resistance to toxins and other xenobiotics. The longest-lived rodent, the naked mole-rat, has a maximum lifespan of 32 years and is the size of mouse yet lives almost 8 times longer. During their very long lifespan, naked mole-rats show minimal declines in many physiological and molecular age-related characteristics, and most interestingly, an incidence of spontaneously occurring cancer has never been reported. Naked mole-rats are also very resistant to an extensive array of toxins in vitro. We hypothesize that cytoprotective mechanisms in this species are contributing to their protection. We focus on pathways regulated by nuclear factor-erythroid 2-related factor-2 [Nrf2] as the key cytoprotective signaling pathway facilitating this broad resistance to cytotoxins and stressors. This ubiquitously expressed and highly conserved transcription factor has been heavily researched with regards to toxin resistance and cancer, and has been shown to interact with p21 and tumor suppressor p53, implying a role for Nrf2 in cell cycle regulation and cancer progression. Naked mole-rats show an in vitro and in vivo constitutive upregulation of Nrf2-cytoprotective signaling as well as resistance to toxins in both fibroblasts and whole animals. These long-lived rodents also show pronounced resistance to carcinogenesis in vivo and our data reveal that oncogenic and apoptotic activation may be more sensitive in naked mole-rats. By utilizing the naked mole-rat as a model of impeccable healthspan and lengthened lifespan, we may not only identify novel mechanisms that contribute to toxin resistance and cancer prevention, but also longevity., The molecular mechanisms behind aging are complex, and one emerging theory asserts that aging occurs as a result of changes in the epigenetic landscape. Here we test the hypothesis that dietary restriction (DR) mediates its anti-aging effects through epigenetic modifiers and modifications. To test the hypothesis that DR mediates its protective effects through epigenetic modifiers, we used surgical nerve crush to model the denervation that occurs in aging skeletal muscle. We demonstrate that DR, even when initiated after surgery, protects against denervation-induced muscle atrophy as measured by gastrocnemius wet weight. DR inhibited the induction of histone deacetylase 4 (HDAC4), a known mediator of atrophic signaling in skeletal muscle. Using the general HDAC inhibitor sodium butyrate (NaBu), we demonstrate that pharmacologically inhibiting HDACs protects against the muscle loss induced by nerve crush, thus mimicking the effects of dietary restriction. To investigate the effects of aging and DR on histone modifications, we analyzed liver histones from young and old animals fed ad libitum or dietary restricted for acetylation at specific residues. We found an age-related decrease in histone H3K9 acetylation, and importantly this decrease was prevented by dietary restriction. To simulate the increase in histone acetylation seen with dietary restriction, we fed old animals the HDAC inhibitor NaBu which resulted in reduced fat mass and increased glucose tolerance over time, consistent with known effects of dietary restriction. Overall, our data support the epigenetics theory of aging and indicates that dietary restriction uses epigenetic mechanisms to protect against age-related pathologies. (This work was funded by the UTHSC at San Antonio Biology of Aging Training Grant to Steve N. Austad (MEW T32AG021890-10)., Protein homeostasis has been implicated in the aging process in a variety of model organisms. We are utilizing a range of marine bivalve mollusk species, with lifespans ranging from under a decade to over five hundred years, in a comparative study to investigate the hypothesis that 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, and their relative proteome stabilities. Specifically, we are testing their ability to maintain structure and function under various stressors, as well as prevent protein damage and aggregation. Furthermore, the influence of each species’ isolated metabolite fraction is being investigated on each of these proteostasis aspects. Protein damage and unfolding were quantified by incorporation of two fluorescent probes, specific for carbonyls and exposed hydrophobic surfaces. Preservation of function was measured by representative enzyme activity, such as GAPDH, when stressed in-vitro. Stress induced aggregation of both endogenous proteins and exogenous, aggregation prone bait proteins were also. The bait proteins used include amyloid beta, the aggregation prone peptide associated with Alzheimer's disease. The macromolecules facilitating enhanced proteostasis in the longest lived animal species could have dramatic importance to various age-related protein diseases., Rapamycin (Rapa) and dietary restriction (DR) are two manipulations consistently shown to increase the lifespan of mice. To investigate whether Rapa and DR affect similar pathways in mice, we compared the effects of feeding mice ad libitum (AL), Rapa, DR, or a combination of Rapa and DR (Rapa + DR) on the transcriptome and metabolome of the liver. The principle component analysis of the transcriptome shows that Rapa and DR are distinct groups. Of the 2724 genes that significantly change with either Rapa or DR compared to mice fed AL, 79% are unique to DR or Rapa; only 21% of the genes are common to DR and Rapa. A similar observation was made when genes were grouped into pathways by Ingenuity Pathway analysis; 76% of the pathways are uniquely changed by DR or Rapa. The metabolome shows an even greater difference between Rapa and DR; only 6% of the metabolites that change significantly from AL mice are common to Rapa and DR. Interestingly, the number of genes significantly changed in Rapa + DR mice compared to AL mice was twice as large as the number of genes significantly altered by either DR or Rapa. In summary, while both Rapa and DR increase lifespan, their global effect on liver is quite different and a combination of Rapa and DR results in alterations in a large number of genes that are not significantly changed by either manipulation alone., A key component of my research is to develop new generations of techniques to understand how oxidative stress-mediated protein oxidation and perturbation of functional structure contribute to aging and age-related diseases. Over the past nine years, I have been actively involved in developing techniques related to measurement of protein oxidation and conformational changes that occur during aging and in disease conditions (Chaudhuri et al. 2001, 2006; Pierce et al. 2006, 2008; Perez et al. 2009; Salmon et al. 2009; Perez et al. 2010; Bhattacharya et al. 2011; Wei et al. 2012). One of the common and unique aspects of all these technologies is the use of fluorescent molecules as probes to detect changes in protein oxidation and conformation. As fluorescent probes in general give high quantum yield, it helps to identify and quantify the potential target proteins that are present in low level and have subtle changes in conformation in any patho-physiological condition. Development of these techniques is an important part of biological research considering the fact that the oxidative stress plays an important role in aging and various diseases including Alzheimer's, Parkinson's, ALS, cancer, heart disease, arthritis, etc. As a result, many investigators are interested in determining the underlying mechanism of the role of imbalanced protein thiol homeostasis; protein oxidation and alteration of conformation contribute to aging and diseases. Most importantly, researchers want to determine if the imbalanced protein homeostasis can be modulated by experimental manipulations, such as calorie restriction and pharmacological intervention. These new sets of techniques will give investigators the necessary tools to delve into the molecular mechanisms involved in aging and age-related diseases., Caspase-2 has been shown to play a role in aging, neurodegeneration and cancer. The contributions of capase-2 have been attributed to its regulatory role in apoptotic and non-apoptotic processes including cell-cycle, DNA repair, lipid biosynthesis, and regulation of oxidant levels in cells. Recently, our lab demonstrated that caspase-2 modulates autophagy during oxidative stress. Here we report the novel finding that caspase-2 is an endogenous repressor of autophagy. Knockout (KO) or knockdown of caspase-2 resulted in upregulation of autophagy in variety of cell types and tissues. Reinsertion of caspase-2 in caspase-2-knockout mouse embryonic fibroblast (MEF's), suppressed autophagy suggesting its role as a negative regulator of autophagy. Loss of caspase-2-mediated autophagy involved down regulation of mTOR and upregulation of AMPK activation; knocking-down of AMPK1/2 inhibited autophagy. Interestingly, siRNA-mediated knockdown of ATG5 and ATG7 failed to inhibit autophagy induced by the loss of caspase-2 suggesting involvement of the non-canonical pathway of autophagy. Our results also indicate involvement of enhanced intracellular reactive oxygen species levels, down regulation of p38 and upregulation of ERK/MEK activation in autophagy-induction due to loss of caspase-2. In response to a variety of apoptotic stimuli that induce caspase-2-mediated apoptosis, caspase-2-KO cells demonstrated further upregulation of autophagy compared to WT MEFs. Enhanced autophagy improved the survival of caspase-2-deficient cells, which maintained high ATP levels. In conclusion, we document a novel role for caspase-2 as a negative regulator of autophagy, which may provide important insight into the role of caspase-2 in aging, neurodegeneration and cancer. The current findings are the first to provide evidence for regulation of caspase activity by autophagy and thus broaden the molecular basis for the observed polarization between autophagy and apoptosis., C57BL6 mice were studied in youth (4–6 mo), middle-age (18 mo) and old-age (26–32 mo). Albuminuria increased, and, rise in serum cystatin C indicated that renal clearance function fell with aging; there was marked heterogeneity. Kidney hypertrophy and expansion of glomerular and tubulo-interstitial matrix were progressive. Increased mRNA correlated with increase in type III collagen in middle-aged and old mice, suggesting transcriptional regulation. In old-age, increase in mRNA correlated with type I collagen protein; however, in middle age, type I collagen was increased despite unchanged mRNA. Data from ChIP analysis of binding of transcription inhibitors ZEB1/ZEB2 to the type Iα2 promoter, and, polysome assay for mRNA translation did not explain type I collagen increase in middle-age. Thus, decreased degradation could lead to type I collagen increment in middle-age. Matrix changes coincided with TGFβ/SMAD3 activation. SMAD3 binding to collagen type Iα2 promoter was increased. Since microRNAs (miRs) control protein synthesis, we studied TGFβ-regulated miRs. The renal cortical content of miR-21 and miR-200c was increased but that of miR-192, miR-200a or miR-200b was unchanged suggesting selectivity. Increase in miR-21 and miR-200c was associated with reduced expression of their targets, Sprouty-1 and ZEB2, respectively; another miR-21 target, PTEN, was unchanged. Sprouty and ZEB2 inhibit growth factor signaling and expression of miR-21, respectively. Conclusion: Distinct transcriptional and post-transcriptional mechanisms contribute to kidney matrix protein increment in middle and old age. Kidney integrity is essential for maintenance of health span. Understanding mechanisms contributing to renal senescence could identify targets for intervention to improve health span., The development of animal models targeting different components of the TOR signaling pathway has accelerated our understanding of the role of mTOR in animal development, metabolism, diseases, and aging. In addition, the discovery of mTOR inhibitors has further enhanced our ability to define the role of mTOR signaling in various patho-physiological conditions and to develop therapeutic strategies for the treatment of different diseases. Here, we report the development and characterization of a new mouse model, which overexpresses TSC1 (named Tsc1tg mice), part of the mTOR inhibitor complex TSC1/2 (tuberous sclerosis complex 1/2). Overexpression of TSC1 stabilized TSC2 and inhibited mTOR signaling in most tissues including the heart, liver, kidney, skeletal muscle and spleen. The levels of several important cell signaling pathways were found altered in Tsc1tg mice. The body weight of Tsc1tg mice exhibits slight gender difference, with significant increase in male mice at both young and advanced ages while only slight increase in female mice at both ages, when compared to age-matched wild type littermates. Body composition of Tsc1tg mice exhibits an age-associated change; with significantly higher fat mass but lower lean mass at advanced ages. At 4–8 months of age, Tsc1tg mice have normal cardiac function as measured by echocardiography. But, when challenged with isoproterenol, Tsc1tg mice developed less cardiac hypertrophy than age-matched wild type littermates. Importantly, Tsc1tg mice performed significantly better with treadmill test. Finally, the immune response of Tsc1tg mice exhibit subtle changes over wild type control mice. In conclusion, this model will be very useful to study the role of mTOR in such diseases that are associated with a deregulation of mTOR signaling, including cancer, cardiovascular diseases, and metabolic disorders. It will also be an interesting model to study the role of mTOR in mammalian aging, complementary to the rapamycin-feeding approach., Sex differences in life and health span are ubiquitous in humans. Women in the developed world live longer than men even if heart disease, the number one cause of death in men, were completely eliminated. Analogously, female mice respond better to a number of senescence-retarding genetic or pharmacological interventions. Particularly notable in this respect, inhibition of TOR signaling via deletion of S6K1 improves both life- and health span in female mice but has no discernible effect in males. Here we show that aging male and female C57BL/6 mice respond to rapamycin in an age and sex-specific manner. There is a larger and more robust effect on longevity in females compared with males and measures of health span have multiple age and sex-specific effects. Age, sex and age · sex-specific differences in body composition, rotarod performance, gait, measures of activity, sleep and metabolism were observed in animals treated with enteric rapamycin (=e-rapa) relative to controls. There has been very little research on the basic biological mechanisms involved in sex differences in aging, in part because past research suggested that laboratory mice and rats do not show clear consistent trends in sex-specific longevity or health span. Our results suggest that sex differences in some measures of mouse health span may only become apparent late in life and that there are sex-specific responses to senescence-retarding treatments that merit further exploration., Loss of mitochondrial function with age has been implicated as an influencing factor in the aging process. However, studies from model organisms ranging from yeast to mammals have shown that moderate disruption of the electron transport chain can enhance longevity. In the Surf1 knockout mouse, there is a 50–75% decline in cytochrome c oxidase (complex IV of the electron transport chain) activity and a 20% extended median lifespan. Previous studies of fibroblasts from long-lived rodents have shown a correlation between increased resistance to cellular stresses and longevity. Here we investigate whether fibroblasts from Surf1 knockout animals are more resistant to stress than wild type controls. Interestingly, these results are dependent upon the passage of the cells. Early passage (, Metformin, a drug commonly prescribed to treat type-2 diabetes, has been found to extend healthspan, delay cancer incidence and progression and to increase lifespan in laboratory animals. We show here that treatment with metformin (0.1% w/w in diet) starting at one year of age extends healthspan and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with metformin mimicked some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels without a decrease in caloric intake. At a molecular level, metformin increased AMP-activated protein kinase activity and increased antioxidant protection, resulting in reductions in both oxidative damage accumulation and chronic inflammation. Our results indicate that these actions may contribute to the beneficial effects of metformin administration on health span and lifespan. These findings are in agreement with current epidemiological data and raise the possibility of metformin-based interventions to promote healthy aging., We tested the effects of two Class I histone deacetylase inhibitors (HDAcI) on the longevity of normal-lived (Ra) and long-lived (La) strains of Drosophila melanogaster. Only deleterious effects are noted when the first HDAcI tested (sodium butyrate, NaBu) is fed to the La strain at any developmental or adult stage. When fed to the Ra strain, this drug also has negative effects when administered over the entire larval and/or adult life span, or when administered over the adult health span only. Importantly, however, it significantly decreases mortality rates and increases longevity when administered only in the adult transition or senescent spans. A different HDAcI (suberoylanilide hydroxamic acid, SAHA) administered to the same strain also showed significant late-life extending effects, suggesting that this is not an isolated effect of one drug. These results suggest that the stage-specific gene regulatory mechanisms affected by NaBu or SAHA are those intimately involved in inducing gene expression patterns characteristic of a healthy senescence. Epigenetically active molecules, if given at the appropriate stage, allow the fly to shift from a senescent span characterized by a high age-specific mortality rate to one with a lower age-specific mortality rate. These studies may provide an experimental basis with which to shed light on the fraility syndrome affecting some aging organisms., Feeding larvae of a normal-lived strain, but not a long-lived strain, with curcumin induces an extended adult health span with significantly increased median and maximum longevities. This phenotype shows no additive effect on longevity when combined with an adult dietary restriction (DR) diet, suggesting that curcumin and DR operate via the same or overlapping pathways for this trait. This treatment significantly slows the age-specific mortality rate so that it is comparable with that of genetically selected long lived animals. The larval treatment also enhances the adults’ geotactic activity in an additive manner with DR, suggesting that curcumin and DR may use different pathways for different traits. Feeding the drug to adults during only the health span also results in a significantly extended health span with increased median and maximum life span. This extended longevity phenotype is induced only during these stage-specific periods. Feeding the drug to adults over their whole life results in a weakly negative effect on median longevity with no increase in maximum life span. There are no negative effects on reproduction, although larval curcumin feeding increases development time; but it apparently accelerates the normal late-life neuromuscular degeneration seen in the legs. Gene expression data from curcumin-fed larvae shows that the TOR pathway is inhibited in the larvae and the young to midlife adults, although several other genes involved in longevity extension are also affected. These data support the hypothesis that curcumin acts as a stage-specific DR mimetic neutriceutical; and suggest that the search for DR mimetics may be enhanced by the use of stage-specific screening of candidate molecules., Mitochondrial mutations in Caenorhabditis elegans can lead to either a shortening of lifespan or, unexpectedly, lifespan extension. Long-lived mitochondrial mutants (Mit mutants) live twice as long as wild-type animals, have delayed development, and reduced adult sizes. We have used a GC-MS-based metabolic footprinting approach to show that Mit mutants employ a common metabolism, distinct from wild-type animals and from short-lived mitochondrial mutants. The hallmark feature of the Mit metabolism is overproduction of pyruvate and various branched-chain ketoacids. We postulate that these compounds may act as mitokines, signaling molecules emanating from the mitochondria, to result in organismal lifespan extension. We have shown that at least four compounds found in the exometabolome of the Mit mutants can delay development when administered to wild type animals. At least one of these compounds, pyruvate, has also previously been reported to increase lifespan when fed to worms. Lifespan studies on the remaining compounds are underway. We have recently begun additional studies to determine whether the Mit mutants also produce a characteristic profile of ascarosides. Ascarosides are small signaling molecules based on the dideoxysugar ascarylose, and compose the pheromone which signals dauer development in C. elegans. This alternate larval state is resistant to stress and is considered non-aging, since upon leaving the dauer state animals live out their normal lifespan. Interestingly, we found a complete absence of one ascaroside in short-lived mitochondrial mutants. Experiments are underway to determine whether this molecule is capable of recovering lifespan in these mutants., Emerging evidence suggests that both diet composition and genetic make-up have a key role in the beneficial effects of calorie restriction (CR). CR-mediated improvements in health and/or longevity may not be universal, even within species. Furthermore the responsiveness to CR may depend on subtleties of the treatment protocol, diet composition or the “intensity of CR”. In this study we determined the differential response to CR levels of DBA/2J mice. We are testing two main hypotheses: (i) that a milder CR intervention will provide beneficial effects on lifespan and healthspan in DBA/2 mice and (ii) regardless of the lack of effect on longevity, there are healthspan benefits even at the higher CR level. Male and female DBA/2J and C57BL/6 mice on one of three diets: ad libitum (AL), 20% CR, 40% CR starting at 6 months of age. Preliminary data indicates that in female mice there is no difference in median lifespan extension between 20% and 40% CR. In male mice it appears that 20% CR is more beneficial in extending median lifespan. Insulin levels are significantly lower in all DBA mice compared to their C57BL/6 counterparts. CR lowers insulin levels in all groups. We observed a stepwise decrease in insulin resistance with increasing CR, but only in males. In female mice, there was no difference in insulin levels between 20% or 40% CR groups. These results indicate that DBA/2J do respond to CR and supports the idea that there is an “ideal” CR dose for a particular strain., The Health Span Study data are an unprecedented cross-sectional window into the biology of rodent aging, and our newly-developed Health Span Database makes it possible to organize, curate, share, and analyze this information in ways that would have otherwise not been practical. Here we present a range of measurements (e.g. body composition, grip strength, and gait analysis) that significantly change with chronological age of the animal. We go on to identify measurements that are positively and negatively correlated with each other, which can be used to construct a performance score for the corresponding organ systems with a minimum of redundant variables, irrelevant variables, and untested assumptions about the data. This in turn sets the stage for choosing variables from which the chronological age of an animal can be estimated. An animal whose actual age is greater than its estimated age can be interpreted as being healthier for its age an animal whose actual age is lower than its estimated age. We present several such sets of candidate variables. The software portion of the Healthspan database is freely available from the first author under the GNU Public License v2. Keywords: aging, healthspan, functional assessment, animal studies of aging, longevity, bioinformatics, variable selection, physiology of aging., Sleep fragmentation is associated with aging in human populations. As part of a larger study designed to find robust, reproducible assays of health span, we used a sleep phenotyping method developed by Pack et al. (2006) to assess age-related changes in sleep patterns. Using EEG and video monitoring, Pack et al. (2006) developed and validated a simple an operational definition of sleep as a bout of /inactivity lasting ≥40s. Using their method, we developed a sleep fragmentation index by measuring the number of bouts of sleep per hour of sleep (=sleep fragmentation index) during the light and dark phases over a 24-hour period. We then used this technique to measure sleep fragmentation in 4, 20, 28, and 32-month-old male and female C57BL/6 mice to explore sex, age-related changes in sleep and sleep disruption. In combination with other assays, age-related changes in sleep patterns may offer a relatively simple, non-invasive tool for assessing healthspan in aging mice., Reduction of target of rapamycin (TOR) signaling has been shown to extend lifespan in invertebrates as well as in adult mice. In other genetic models of longevity in invertebrates and mice, specific manipulations in the nervous system are sufficient to extend lifespan. To determine whether the reduction of mammalian TOR (mTOR) signaling in mature neurons of adult mice is sufficient to extend lifespan and improve health span we inducibly knocked out The mTOR complex 1 specific protein, Raptor, in adult mouse neurons after brain development was complete (2.5 months). Cre-mediated recombination of genomic DNA was detected in brain, but not in liver, and Raptor protein levels were significantly reduced after induction of Cre expression. To determine whether decreasing Raptor in neurons affected health span, we measured body mass composition, metabolism, motor coordination, muscle strength, and brain metabolite concentrations. While no significant differences in motor coordination, strength or body weight were observed among experimental groups, genetic reduction of Raptor in neurons of adult mice induced significant changes in body composition, with neuronal Raptor knock-out males becoming significantly leaner than non-transgenic controls. Adult neuronal Raptor, conditional knock-outs also showed increased levels of neuronal N-acetylaspartate, a marker of neuronal health and function. Future experiments will determine if decreased mTOR complex I signaling in adult mouse neurons is sufficient to extend lifespan and improve health span. Included in the evaluation of health span will be measures of neurological function as determined by electrophysiological and behavioral experiments., While frailty has long been recognized by physicians in the clinical setting, only recently has effort been made to standardize and quantify definitions of frailty. Fried et al. 2001 and others have used multiple measures in the hopes of developing an easily used index to evaluate age-related risks of morbidity and mortality. Among the most commonly used measures are activity, walking speed, involuntary weight loss and strength (grip strength). In order to assess age-related frailty, as opposed to ill-health more generally, two conditions should be met: firstly, the traits measured should change with age; secondly, the traits should have predictive value for increased risk of morbidity and mortality. Here we assess a combination of several potential measures of frailty in mice, including motor function (e.g. walking speed), activity (e.g. spontaneous activity), strength (e.g. grip strength), body composition and caloric expenditure (e.g. resting metabolic rate) to determine whether age-related morbidity and mortality in C57BL/6 mice can be predicted using a multivariate analysis to produce a relatively non-invasive measure of health similar to the frailty index used with humans., It has been reported that dietary supplementation of male and female genetically heterogeneous (UM-HET3) mice with rapamycin increased median and maximum lifespan suggesting that it slows aging (Harrison et al., 2009; Miller et al., 2011). Therefore, we hypothesized that if rapamycin treatment slows aging it should also prevent or delay age-related deficits that have previously been reported in cognition and motor performance in UM-HET3 mice (Sumien et al., 2006). To test this hypothesis, we have used male and female CB6F1×C3D2F1 (UM-HET3) mice. Three groups of (N = 26 to 50) were tested: young control (4 months old), old control (24 months old) and old mice treated with rapamycin in the diet started from 12 months of age. We administered a battery of behavioral tests. Our results showed that age-related decline in locomotor and rearing activity was attenuated by rapamycin treatment in both the genders. Rapamycin treatment also attenuated the age-related decline in rotarod performance in both the genders. In addition, rapamycin treatment improves the swimming speeds of males in morris water maze test. However, we did not found any effect of rapamycin on age-related decline in grip strength. Interestingly, rapamycin improves the age-related decline in recognition memory in males. To measure anxiety and motivation, we employed the elevated plus maze and tail suspension tests respectively. No change was observed with age and treatment on anxiety and stress levels in males. However, in females rapamycin reduced the basal anxiety levels and depressive-like behavior. Altogether, our findings reveal that the increase in lifespan resulting from rapamycin supplementation is accompanied by improvements in age-sensitive behavioral traits. This study was supported by the National Institute on Aging at the National Institutes of Health (U01-AG022307).