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Background: Degeneration of the motoneuron and neuromuscular junction (NMJ) and loss of motor units (MUs) contribute to age-related muscle wasting and weakness associated with sarcopenia. However, these features have not been comprehensively investigated in humans. This study aimed to compare neuromuscular system integrity and function at different stages of sarcopenia, with a particular focus on NMJ stability and MU properties., Methods: We recruited 42 young individuals (Y) (aged 25.98 ± 4.6 years; 57% females) and 88 older individuals (aged 75.9 ± 4.7 years; 55% females). The older group underwent a sarcopenia screening according to the revised guidelines of the European Working Group on Sarcopenia in Older People 2. In all groups, knee extensor muscle force was evaluated by isometric dynamometry, muscle morphology by ultrasound and MU potential properties by intramuscular electromyography (iEMG). MU number estimate (iMUNE) and blood samples were obtained. Muscle biopsies were collected in a subgroup of 16 Y and 52 older participants., Results: Thirty-nine older individuals were non-sarcopenic (NS), 31 pre-sarcopenic (PS) and 18 sarcopenic (S). A gradual decrease in quadriceps force, cross-sectional area and appendicular lean mass was observed across the different stages of sarcopenia (for all P < 0.0001). Handgrip force and the Short Physical Performance Battery score also showed a diminishing trend. iEMG analyses revealed elevated near fibre segment jitter in NS, PS and S compared with Y (Y vs. NS and S: P < 0.0001; Y vs. PS: P = 0.0169), suggestive of age-related impaired NMJ transmission. Increased C-terminal agrin fragment (P < 0.0001) and altered caveolin 3 protein expression were consistent with age-related NMJ instability in all the older groups. The iMUNE was lower in all older groups (P < 0.0001), confirming age-related loss of MUs. An age-related increase in MU potential complexity was also observed. These observations were accompanied by increased muscle denervation and axonal damage, evinced by the increase in neural cell adhesion molecule-positive fibres (Y vs. NS: P < 0.0001; Y vs. S: P = 0.02) and the increase in serum concentration of neurofilament light chain (P < 0.0001), respectively. Notably, most of these MU and NMJ parameters did not differ when comparing older individuals with or without sarcopenia., Conclusions: Alterations in MU properties, axonal damage, an altered innervation profile and NMJ instability are prominent features of the ageing of the neuromuscular system. These neuromuscular alterations are accompanied by muscle wasting and weakness; however, they appear to precede clinically diagnosed sarcopenia, as they are already detectable in older NS individuals., (© 2024 The Author(s). Journal of Cachexia, Sarcopenia and Muscle published by Wiley Periodicals LLC.)

Purpose: to investigate the early consequences of type 1 diabetes (T1D) on the neural strategies of muscle force production., Methods: motor unit (MU) activity was recorded from the vastus lateralis muscle with High-Density surface Electromyography during isometric knee extension at 20 and 40% of maximum voluntary contraction (MVC) in 8 T1D (4 males, 4 females, 30.5 ± 3.6 years) and 8 matched control (4 males, 4 females, 27.3 ± 5.9 years) participants. Muscle biopsies were also collected from vastus lateralis for fiber type analysis, including myosin heavy chain (MyHC) isoform content via protein and mRNA expression., Results: MVC was comparable between groups as well as MU conduction velocity, action potentials' amplitude and proportions of MyHC protein isoforms. Nonetheless, MU discharge rate, relative derecruitment thresholds and mRNA expression of MyHC isoform I were lower in T1D., Conclusions: young people with uncomplicated T1D present a different neural control of muscle force production. Furthermore, differences are detectable non-invasively in absence of any functional manifestation (i.e., force production and fiber type distribution). These novel findings suggest that T1D has early consequences on the neuromuscular system and highlights the necessity of a better characterization of neural control in this population., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ageing is a biological process caused by the malfunctioning of multiple cellular mechanisms, ascribable to nine hallmarks: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. These ageing pillars have three common traits: (i) they appear during normal ageing; (ii) their experimental intensification accelerates ageing; and (iii) their experimental reduction delays ageing. The evidence that the elderly are more prone to develop pathologies such as cancer, diabetes and degenerative diseases, together with data showing that the elderly population is steadily increasing, has stimulated an important effort to find specific countermeasures to physiological ageing. Unfortunately, the investigation of ageing processes and the search for countermeasures in humans is very difficult. Therefore, researchers must rely on a wide range of experimental models that span from unicellular to more complex organisms. Unfortunately, experimental models are not devoid of pitfalls, flaws or obstacles that can have an impact in ageing research. In the present review we describe the most exploited experimental models in the field, such as in vitro, animal and human models, highlighting the characteristics that justify their application in the laboratory routine, and translation to human research., (©The Author(s) 2023. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Background: Ageing is accompanied by a progressive loss of skeletal muscle mass and strength, potentially determining the insurgence of sarcopenia. Evidence suggests that motoneuron and neuromuscular junction (NMJ) degeneration contribute to sarcopenia pathogenesis. Seeking for strategies able to slow down sarcopenia insurgence and progression, we investigated whether a 2-year mixed-model training involving aerobic, strength and balance exercises would be effective for improving or preserving motoneuronal health and NMJ stability, together with muscle mass, strength and functionality in an old, sarcopenic population., Methods: Forty-five sarcopenic elderly (34 females; 11 males) with low dual-energy X-ray absorptiometry (DXA) lean mass and Short Physical Performance Battery (SPPB) score <9 were randomly assigned to either a control group [Healthy Aging Lifestyle Education (HALE), n = 21] or an intervention group [MultiComponent Intervention (MCI), n = 24]. MCI trained three times per week for 2 years with a mix of aerobic, strength and balance exercises matched with nutritional advice. Before and after the intervention, ultrasound scans of the vastus lateralis (VL), SPPB and a blood sample were obtained. VL architecture [pennation angle (PA) and fascicle length (Lf)] and cross-sectional area (CSA) were measured. As biomarkers of neuronal health and NMJ stability status, neurofilament light chain (NfL) and C-terminal agrin fragment (CAF) concentrations were measured in serum. Differences in ultrasound parameters, NfL and CAF concentration and physical performance between baseline and follow-up were tested with mixed ANOVA or Wilcoxon test. The relationship between changes in physical performance and NfL or CAF concentration was assessed through correlation analyses., Results: At follow-up, MCI showed preserved VL architecture (PA, Lf) despite a reduced CSA (-8.4%, P < 0.001), accompanied by maintained CAF concentration and ameliorated overall SPPB performance (P = 0.007). Conversely, HALE showed 12.7% decrease in muscle CSA (P < 0.001), together with 5.1% and 5.5% reduction in PA and Lf (P < 0.001 and P = 0.001, respectively), and a 6.2% increase in CAF (P = 0.009) but improved SPPB balance score (P = 0.007). NfL concentration did not change in either group. In the population, negative correlations between changes in CAF concentration and SPPB total score were found (P = 0.047), whereas no correlation between NfL and SPPB variations was observed., Conclusions: The present findings suggest that our 2-year mixed aerobic, strength and balance training seemed effective for preventing the age and sarcopenia-related increases in CAF concentration, preserving NMJ stability as well as muscle structure (PA and Lf) and improving physical performance in sarcopenic older individuals., (© 2023 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Human skeletal muscle atrophy and a disproportionate force loss occur within a few days of unloading in space and on Earth, but the underlying mechanisms are not fully understood. Disruption of neuromuscular junction homeostasis has been proposed as one of the possible causes. Here, we investigated the potential mechanisms involved in this neuromuscular disruption induced by a 10-day unilateral lower limb suspension (ULLS) in humans. Specifically, we investigated hemichannels' upregulation, neuromuscular junction and axonal damage, neurotrophins' receptor downregulation and inflammatory transcriptional signatures. Biomarkers were evaluated at local and systemic levels. At the sarcolemmal level, changes were found to be associated with an increased expression of connexin 43 and pannexin-1. Upregulation of the inflammatory transcripts revealed by deep transcriptomics was found after 10 days of ULLS. The destabilisation of the neuromuscular junction was not accompanied by changes in the secretion of the brain-derived neurotrophic factor and neurotrophin-4, while their receptor, BDNF/NT growth factors receptor (TrkB), decreased. Furthermore, at 5 days of ULLS, there was already a significant upregulation of the serum neurofilament light chain concentration, an established clinical biomarker of axonal injury. At 10 days of ULLS, other biomarkers of early denervation processes appeared. Hence, short periods of muscle unloading induce sarcolemmal hemichannels upregulation, inflammatory transcripts upregulation, neuromuscular junction instability and axonal damage.

Physical inactivity (PI) is a major risk factor of chronic diseases. A major aspect of PI is loss of muscle mass and strength. The latter phenomenon significantly impacts daily life and represent a major issue for global health. Understandably, skeletal muscle itself has been the major focus of studies aimed at understanding the mechanisms underlying loss of mass and strength. Relatively lesser attention has been given to the contribution of alterations in somatomotor control, despite the fact that these changes can start very early and can occur at multiple levels, from the cortex down to the neuromuscular junction (NMJ). It is well known that exposure to chronic inactivity or immobilization causes a disproportionate loss of force compared to muscle mass, i.e. a loss of specific or intrinsic whole muscle force. The latter phenomenon may be partially explained by the loss of specific force of individual muscle fibres, but several other players are very likely to contribute to such detrimental phenomenon. Irrespective of the length of the disuse period, the loss of force is, in fact, more than two-fold greater than that of muscle size. It is very likely that somatomotor alterations may contribute to this loss in intrinsic muscle force. Here we review evidence that alterations of one component of somatomotor control, namely the neuromuscular junction, occur in disuse. We also discuss some of the novel players in NMJ stability (e.g., homer, bassoon, pannexin) and the importance of new established and emerging molecular markers of neurodegenerative processes in humans such as agrin, neural-cell adhesion molecule and light-chain neurofilaments., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

A growing number of disorders has been associated with mutations in the components of the vesicular transport machinery. The early secretory pathway consists of Endoplasmic Reticulum, numerous vesicles, and the Golgi Complex (GC), which work together to modify and package proteins to deliver them to their destination. The GC is a hub organelle, crucial for organization of the other secretory pathway components. As a consequence, GC's form and function are key players in the pathogenesis of several disorders. Skeletal muscle (SKM) damage can be caused by defective protein modifications and traffic, as observed in some Limb girdle muscular dystrophies. Interestingly, in turn, muscle damage in Duchenne dystrophic SKM cells also includes the alteration of GC morphology. Based on the correlation between GC's form and function described in non-muscle diseases, we suggest a key role for this hub organelle also in the onset and progression of some SKM disorders. An altered GC could affect the secretory pathway via primary (e.g., mutation of a glycosylation enzyme), or secondary mechanisms (e.g., GC mis-localization in Duchenne muscles), which converge in SKM cell failure. This evidence induces considering the secretory pathway as a potential therapeutic target in the treatment of muscular dystrophies.

The mammalian target of rapamycin (mTOR) is a major regulator of skeletal myocyte viability. The signaling pathways triggered by mTOR vary according to the type of endogenous and exogenous factors (e.g., redox balance, nutrient availability, physical activity) as well as organismal age. Here, we provide an overview of mTOR signaling in skeletal muscle, with a special focus on the role played by mTOR in the development of sarcopenia. Intervention strategies targeting mTOR in sarcopenia (e.g., supplementation of plant extracts, hormones, inorganic ions, calorie restriction, and exercise) have also been discussed.

Electrophysiological alterations of the neuromuscular junction (NMJ) and motor unit potential (MUP) with unloading are poorly studied. We aimed to investigate these aspects and the underlying molecular mechanisms with short-term unloading and active recovery (AR). Eleven healthy males underwent a 10-day unilateral lower limb suspension (ULLS) period, followed by 21-day AR based on resistance exercise. Quadriceps femoris (QF) cross-sectional area (CSA) and isometric maximum voluntary contraction (MVC) were evaluated. Intramuscular electromyographic recordings were obtained during 10% and 25% MVC isometric contractions from the vastus lateralis (VL). Biomarkers of NMJ molecular instability (serum c-terminal agrin fragment, CAF), axonal damage (neurofilament light chain) and denervation status were assessed from blood samples and VL biopsies. NMJ and ion channel transcriptomic profiles were investigated by RNA-sequencing. QF CSA and MVC decreased with ULLS. Increased CAF and altered NMJ transcriptome with unloading suggested the emergence of NMJ molecular instability, which was not associated with impaired NMJ transmission stability. Instead, increased MUP complexity and decreased motor unit firing rates were found after ULLS. Downregulation of ion channel gene expression was found together with increased neurofilament light chain concentration and partial denervation. The AR period restored most of these neuromuscular alterations. In conclusion, the human NMJ is destabilized at the molecular level but shows functional resilience to a 10-day unloading period at least at relatively low contraction intensities. However, MUP properties are altered by ULLS, possibly due to alterations in ion channel dynamics and initial axonal damage and denervation. These changes are fully reversed by 21 days of AR. KEY POINTS: We used integrative electrophysiological and molecular approaches to comprehensively investigate changes in neuromuscular integrity and function after a 10-day unilateral lower limb suspension (ULLS), followed by 21 days of active recovery in young healthy men, with a particular focus on neuromuscular junction (NMJ) and motor unit potential (MUP) properties alterations. After 10-day ULLS, we found significant NMJ molecular alterations in the absence of NMJ transmission stability impairment. These findings suggest that the human NMJ is functionally resilient against insults and stresses induced by short-term disuse at least at relatively low contraction intensities, at which low-threshold, slow-type motor units are recruited. Intramuscular electromyography analysis revealed that unloading caused increased MUP complexity and decreased motor unit firing rates, and these alterations could be related to the observed changes in skeletal muscle ion channel pool and initial and partial signs of fibre denervation and axonal damage. The active recovery period restored these neuromuscular changes., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Obesity is a complex condition characterized by abnormal and excessive fat accumulation, resulting in an increased risk for severe health problems. Skeletal muscles play a major role in movement and fat catabolism, but the insulin resistance that comes with obesity makes it difficult to fulfill these tasks. In this study, we analyse two types of training protocols, moderate intensity continuous training (MICT) versus high intensity interval training (HIIT), in a cohort of obese subjects to establish which muscle adaptations favour fat consumption in response to exercise. Mitochondria play a role in fat oxidation. We found protein upregulation of mitochondrial biomarkers, TOMM20 and Cox-4, in HIIT but not in MICT, without detecting any shifts in fibre composition phenotype of the vastus lateralis in both training groups. Interestingly, both MICT and HIIT protocols showed increased protein levels of perilipin PLIN2, which is involved in the delivery and consumption of fats. HIIT also augmented perilipin PLIN5. Perilipins are involved in fat storage in skeletal muscles and their upregulation, along with the analysis of circulatory lipid profiles reported in the present study, suggest important adaptations induced by the two types of training protocols that favour fat consumption and weight loss in obese subjects.

Sarcopenia starts around the age of 40, causes the loss of 8% of muscle mass every 10 years, and is accompanied by functional deficit, chronic low-grade inflammation, and can result in several negative health outcomes. Considering the early and gradual onset of sarcopenia, the time window of the potential interventions could be crucial for the exertion of a beneficial effect. We recently showed that the long-term supplementation with Resveratrol contrasts sarcopenia in naturally ageing C57BL/6 mice. Aiming to understand the effects of a short term treatment, we administered intraperitoneally middle aged male mice with 20 mg/kg body weight Resveratrol daily for 5 weeks. Although we could not observe major differences in the histological properties of SKMs, we detected a significant decrease of Cox-2 in RES-treated muscles, confirming the anti-inflammatory action of Resveratrol, and suggesting that its anti-inflammatory action precedes modifications to SKM fibres.

Genetic background may partly explain differences in muscle responses to internal or external stimuli. Muscle disuse involves various degrees of skeletal muscle atrophy due to inactivity and mechanical unloading. Whether and to which extent genetic background impacts disuse atrophy and retraining in individuals of different ages are currently unclear. Here, we provide a brief overview of relevant literature on the contribution of genetics to muscle disuse, retraining, and aging, and offer a perspective on unanswered questions on the subject that may open new venues for research.

Motor neuron degeneration, denervation, neuromuscular junction (NMJ) fragmentation and loss of motor units (MUs), play a key-role in the development of sarcopenia. The aim of the present study was to investigate the beneficial effects of regular practice of dancing in physically active elders on concentration of C-terminal Agrin fragment (CAF), a marker of NMJ instability, muscle mass, strength, and physical performance in a group of 16 recreationally active older dancers (AOD; 70.1 ± 3.4 yr) compared to 15 age-matched sedentary peers (OS; 70.9 ± 6.2 yr). Circulating concentration of CAF was measured in serum, while morphology of the vastus lateralis and multifidus muscles was assessed by ultrasound imaging. In addition, the participants underwent two functional performance tests, the Timed Up and Go (TUG) and the 10-meter walk test (10-MWT), a lower and upper limb isometric strength test, a static and a dynamic balance test. Although no statistically significant differences were detected for both muscle morphology and isometric strength, higher CAF concentration (20%, p < 0.01) was found in OS. AOD showed a better performance in TUG (22%, p < 0.001), 10-MWT (17%, p < 0.001) and dynamic balance (25%, p < 0.01) than OS. Notably, CAF concentration correlated with dynamic balance performance (r = 0.3711, p < 0.05). Our results provide evidence that the regular practice of dancing in older age, together with non-structured light aerobic physical activities, is associated to lower CAF concentration and improved walking and balance performance. Our findings also suggest that NMJ instability, as indicated by elevated CAF serum concentration, seems to precede the loss of muscle size and alterations in muscle architecture normally associated with sarcopenia., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Space analogs, such as bed rest, are used to reproduce microgravity-induced morphological and physiological changes and can be used as clinical models of prolonged inactivity. Nevertheless, nonuniform decreases in muscle mass and function have been frequently reported, and peripheral nerve adaptations have been poorly studied, although some of these mechanisms may be explained. Ten young healthy males (18-33 yr) underwent 10 days of horizontal bed rest. Peripheral neurophysiological assessments were performed bilaterally for the dominant (DL) and nondominant upper and lower limbs (N-DL) on the 1st and 10th day of bed rest, including ultrasound of the median, deep peroneal nerve (DPN), and common fibular nerve (CFN) , as well as a complete nerve conduction study (NCS) of the upper and lower limbs. Consistently, reduced F waves, suggesting peripheral nerve dysfunction, of both the peroneal (DL: P = 0.005, N-DL: P = 0.013) and tibial nerves (DL: P = 0.037, N-DL: P = 0.005) were found bilaterally, whereas no changes were observed in nerve ultrasound or other parameters of the NCS of both the upper and lower limbs. In these young healthy males, only the F waves, known to respond to postural changes, were significantly affected by short-term bed rest. These preliminary results suggest that during simulated microgravity, most changes occur at the muscle or central nervous system level. Since the assessment of F waves is common in clinical neurophysiological examinations, caution should be used when testing individuals after prolonged immobility.

Key Points: Few days of unloading are sufficient to induce a decline of skeletal muscle mass and function; notably, contractile force is lost at a faster rate than muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. We provide strong evidence of two mechanisms only hypothesized until now for the rapid muscle force loss in only 10 days of bed rest. Our results show that an initial neuromuscular junction instability, accompanied by alterations in the innervation status and impairment of single fibre sarcoplasmic reticulum function contribute to the loss of contractile force in front of a preserved myofibrillar function and central activation capacity. Early onset of neuromuscular junction instability and impairment in calcium dynamics involved in excitation-contraction coupling are proposed as eligible determinants to the greater decline in muscle force than in muscle size during unloading., Abstract: Unloading induces rapid skeletal muscle atrophy and functional decline. Importantly, force is lost at a much higher rate than muscle mass. We aimed to investigate the early determinants of the disproportionate loss of force compared to that of muscle mass in response to unloading. Ten young participants underwent 10 days of bed rest (BR). At baseline (BR0) and at 10 days (BR10), quadriceps femoris (QF) volume (VOL) and isometric maximum voluntary contraction (MVC) were assessed. At BR0 and BR10 blood samples and biopsies of vastus lateralis (VL) muscle were collected. Neuromuscular junction (NMJ) stability and myofibre innervation status were assessed, together with single fibre mechanical properties and sarcoplasmic reticulum (SR) calcium handling. From BR0 to BR10, QFVOL and MVC decreased by 5.2% (P = 0.003) and 14.3% (P < 0.001), respectively. Initial and partial denervation was detected from increased neural cell adhesion molecule (NCAM)-positive myofibres at BR10 compared with BR0 (+3.4%, P = 0.016). NMJ instability was further inferred from increased C-terminal agrin fragment concentration in serum (+19.2% at BR10, P = 0.031). Fast fibre cross-sectional area (CSA) showed a trend to decrease by 15% (P = 0.055) at BR10, while single fibre maximal tension (force/CSA) was unchanged. However, at BR10 SR Ca 2+ release in response to caffeine decreased by 35.1% (P < 0.002) and 30.2% (P < 0.001) in fast and slow fibres, respectively, pointing to an impaired excitation-contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size during unloading., (© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Sarcopenia, the age-related decline in muscle mass and strength/function, is a prototypical geroscience condition. The dissection of muscle-specific molecular pathways through analyses of tissue biopsies has provided valuable insights into the pathophysiology of sarcopenia. However, such an approach is unsuitable for capturing the dynamic nature of the condition. Furthermore, the muscle sampling procedure may be perceived as burdensome especially by multimorbid, frail older adults. To overcome these limitations, sophisticated statistical methods have been devised for the simultaneous analysis of circulating factors related to the multiple domains of sarcopenia. This approach has shown potential for achieving a more comprehensive appraisal of the condition, unveiling new therapeutic targets, and identifying meaningful biomarkers. Here, we discuss the main pathogenetic pathways of sarcopenia, with a focus on mediators that are currently in the spotlight as biomarkers and potential treatment targets., Competing Interests: Conflict of interest statement Nothing to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mitochondrial dysfunction is a relevant mechanism in cardiac aging. Here, we investigated the effects of late-life enalapril administration at a non-antihypertensive dose on mitochondrial genomic stability, oxidative damage, and mitochondrial quality control (MQC) signaling in the hearts of aged rats. The protein expression of selected mediators (i.e., mitochondrial antioxidant enzymes, energy metabolism, mitochondrial biogenesis, dynamics, and autophagy) was measured in old rats randomly assigned to receive enalapril ( n = 8) or placebo ( n = 8) from 24 to 27 months of age. We also assessed mitochondrial DNA (mtDNA) content, citrate synthase activity, oxidative lesions to protein and mtDNA (i.e., carbonyls and the abundance of mtDNA 4834 deletion), and the mitochondrial transcription factor A (TFAM) binding to specific mtDNA regions. Enalapril attenuated cardiac hypertrophy and oxidative stress-derived damage (mtDNA oxidation, mtDNA 4834 deletion, and protein carbonylation), while increasing mitochondrial antioxidant defenses. The binding of mitochondrial transcription factor A to mtDNA regions involved in replication and deletion generation was enhanced following enalapril administration. Increased mitochondrial mass as well as mitochondriogenesis and autophagy signaling were found in enalapril-treated rats. Late-life enalapril administration mitigates age-dependent cardiac hypertrophy and oxidative damage, while increasing mitochondrial mass and modulating MQC signaling. Further analyses are needed to conclusively establish whether enalapril may offer cardioprotection during aging., Competing Interests: authors declare no conflict of interest.

The well-known age-related mitochondrial dysfunction deeply affects heart because of the tissue's large dependence on mitochondrial ATP provision. Our study revealed in aged rat heart a significant 25% decrease in mtDNA relative content, a significant 29% increase in the 4.8 Kb mtDNA deletion relative content, and a significant inverse correlation between such contents as well as a significant 38% decrease in TFAM protein amount. The TFAM-binding activity to specific mtDNA regions increased at those encompassing the mtDNA replication origins, D-loop and Ori-L. The same mtDNA regions were screened for different kinds of oxidative damage, namely Single Strand Breaks (SSBs), Double Strand Breaks (DSBs), abasic sites (AP sites) and oxidized bases as 7,8-dihydro-8-oxoguanine (8oxoG). A marked increase in the relative content of mtDNA strand damage (SSBs, DSBs and AP sites) was found in the D-loop and Ori-L regions in the aged animals, unveiling for the first time in vivo an age-related, non-stochastic accumulation of oxidative lesions in these two regions that appear as hot spots of mtDNA damage. The use of Formamidopyrimidine glycosylase (Fpg) demonstrated also a significant age-related accumulation of oxidized purines particularly in the D-loop and Ori-L regions. The detected increased binding of TFAM to the mtDNA damage hot spots in aged heart suggests a link between TFAM binding to mtDNA and loss of mitochondrial genome likely through hindrance of repair processes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

We previously reported the ability of dietary supplementation with acetyl-l-carnitine (ALCAR) to prevent age-related decreases of mitochondrial biogenesis in skeletal muscle and liver of old rats. Here, we investigate the effects of ALCAR supplementation in cerebral hemispheres and cerebellum of old rats by analyzing several parameters linked to mitochondrial biogenesis, mitochondrial dynamics and antioxidant defenses. We measured the level of the coactivators PGC-1α and PGC-1β and of the factors regulating mitochondrial biogenesis, finding an age-related decrease of PGC-1β, whereas PGC-1α level was unvaried. Twenty eight-month old rats supplemented with ALCAR for one and two months showed increased levels of both factors. Accordingly, the expression of the two transcription factors NRF-1 and TFAM followed the same trend of PGC-1β. The level of mtDNA, ND1 and the activity of citrate synthase, were decreased with aging and increased following ALCAR treatment. Furthermore, ALCAR counteracted the age-related increase of deleted mtDNA. We also analyzed the content of proteins involved in mitochondrial dynamics (Drp1, Fis1, OPA1 and MNF2) and found an age-dependent increase of MFN2 and of the long form of OPA1. ALCAR treatment restored the content of the two proteins to the level of the young rats. No changes with aging and ALCAR were observed for Drp1 and Fis1. ALCAR reduced total cellular levels of oxidized PRXs and counteracted the age-related decrease of PRX3 and SOD2. Overall, our findings indicate a systemic positive effect of ALCAR dietary treatment and a tissue specific regulation of mitochondrial homeostasis in brain of old rats. Moreover, it appears that ALCAR acts as a nutrient since in most cases its effects were almost completely abolished one month after treatment suspension. Dietary supplementation of old rats with this compound seems a valuable approach to prevent age-related mitochondrial dysfunction and might ultimately represent a strategy to delay age-associated negative consequences in mitochondrial homeostasis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chemotherapy can cause cachexia, which consists of weight loss associated with muscle atrophy. The exact mechanisms underlying this skeletal muscle toxicity are largely unknown and co-therapies to attenuate chemotherapy-induced side effects are lacking. By using a rat model of cisplatin-induced cachexia, we here characterized the mitochondrial homeostasis in tibialis anterior cachectic muscle and evaluated the potential beneficial effects of the growth hormone secretagogues (GHS) hexarelin and JMV2894 in this setting. We found that cisplatin treatment caused a decrease in mitochondrial biogenesis (PGC-1α, NRF-1, TFAM, mtDNA, ND1), mitochondrial mass (Porin and Citrate synthase activity) and fusion index (MFN2, Drp1), together with changes in the expression of autophagy-related genes (AKT/FoxO pathway, Atg1, Beclin1, LC3AII, p62) and enhanced ROS production (PRX III, MnSOD). Importantly, JMV2894 and hexarelin are capable to antagonize this chemotherapy-induced mitochondrial dysfunction. Thus, our findings reveal a key-role played by mitochondria in the mechanism responsible for GHS beneficial effects in skeletal muscle, strongly indicating that targeting mitochondrial dysfunction might be a promising area of research in developing therapeutic strategies to prevent or limit muscle wasting in cachexia.

Extremely interesting for aging research are those individuals able to reach older ages still with functions similar to those of younger counterparts. We examined liver samples from ad libitum-fed old (28-month-old, AL-28) and ad libitum-fed very old (32-month-old, AL-32) rats for a number of markers, relevant for mitochondrial functionality and mitochondrial DNA (mtDNA) content. As for the mtDNA content and the protein amounts of the citrate synthase and the antioxidant peroxiredoxin III there were no significant changes in the AL-32 animals. No significant longevity-related change was found for TFAM amount, but a 50% reduction in the amount of the Lon protease, responsible for turnover of TFAM inside mitochondria, characterized the AL-32 rats. No longevity-related change was observed also for the amounts of the mtDNA repair enzymes OGG1 and APE1, whereas the intra-mitochondrial amount of the cytochrome c protein showed a 50% increase in the AL-32 rats, indicating a likely reduced initiation of the intrinsic apoptotic pathway. Totally unexpected was the doubling of two proteins, very relevant for mitochondrial dynamics, namely MFN2 and DRP1, in the AL-32 rats. This prompted us to the calculation of all individual fusion indexes that grouped together in the AL-32 rats, while in the AL-28 animals were very different. We found a strong positive correlation between the fusion indexes and the respective mtDNA contents in two AL-28 and four AL-32 rats. This supports the idea that the limited prevalence of fusion above a still active fission should have ensured a functional mitochondrial network and should have led to a quite narrow range of high mtDNA contents, likely the best-suitable for extended longevity. Our findings strongly suggest that, among the multiple causes leading to the longevity of the AL-32 rats, the maintenance of an adult-like balance of mitochondrial dynamics seems to be very relevant for the regulation of mtDNA content and functionality., (Copyright © 2016 Elsevier Inc. All rights reserved.)