Lost in translation: challenges of current pharmacotherapy for sarcopenia.

A healthy lifespan relies on independent living, in which active skeletal muscle is a critical element. The cost of not recognizing and acting earlier on unhealthy or aging muscle could be detrimental, since muscular weakness is inversely associated with all-cause mortality. Sarcopenia is characterized by a decline in skeletal muscle mass and strength and is associated with aging. Exercise is the only effective therapy to delay sarcopenia development and improve muscle health in older adults. Although numerous interventions have been proposed to reduce sarcopenia, none has yet succeeded in clinical trials. This review evaluates the biological gap between recent clinical trials targeting sarcopenia and the preclinical studies on which they are based, and suggests an alternative approach to bridge the discrepancy.

Mitochondrial Properties in Skeletal Muscle Fiber.

Mitochondria are the primary source of energy production and are implicated in a wide range of biological processes in most eukaryotic cells. Skeletal muscle heavily relies on mitochondria for energy supplements. In addition to being a powerhouse, mitochondria evoke many functions in skeletal muscle, including regulating calcium and reactive oxygen species levels. A healthy mitochondria population is necessary for the preservation of skeletal muscle homeostasis, while mitochondria dysregulation is linked to numerous myopathies. In this review, we summarize the recent studies on mitochondria function and quality control in skeletal muscle, focusing mainly on in vivo studies of rodents and human subjects. With an emphasis on the interplay between mitochondrial functions concerning the muscle fiber type-specific phenotypes, we also discuss the effect of aging and exercise on the remodeling of skeletal muscle and mitochondria properties.

Activation of eIF4E-binding-protein-1 rescues mTORC1-induced sarcopenia by expanding lysosomal degradation capacity.

BACKGROUND: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. METHODS: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. RESULTS: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream activator of autophagosome initiation. We discovered a previously unidentified phenotype of lysosomal failure in TSC1mKO mouse muscle, in which the lysosomal defect was also conserved in the naturally aged mouse muscle, whereas 4EBP1 activation enhanced lysosomal protease activities to compensate for impaired autophagy induced by mTORC1 hyperactivity. Consequently, 4EBP1 activation relieved oxidative stress to prevent toxic aggregate accumulation (0.5-fold reduction compared with TSC1mKO, P < 0.05) in muscle and restored mitochondrial homeostasis and function. CONCLUSIONS: We identify 4EBP1 as a communication hub coordinating protein synthesis and degradation to protect proteostasis, revealing therapeutic potential for activating lysosomal degradation to mitigate sarcopenia.

Muscle 4EBP1 activation modifies the structure and function of the neuromuscular junction in mice.

Dysregulation of mTOR complex 1 (mTORC1) activity drives neuromuscular junction (NMJ) structural instability during aging; however, downstream targets mediating this effect have not been elucidated. Here, we investigate the roles of two mTORC1 phosphorylation targets for mRNA translation, ribosome protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), in regulating NMJ structural instability induced by aging and sustained mTORC1 activation. While myofiber-specific deletion of S6k1 has no effect on NMJ structural integrity, 4EBP1 activation in murine muscle induces drastic morphological remodeling of the NMJ with enhancement of synaptic transmission. Mechanistically, structural modification of the NMJ is attributed to increased satellite cell activation and enhanced post-synaptic acetylcholine receptor (AChR) turnover upon 4EBP1 activation. Considering that loss of post-synaptic myonuclei and reduced NMJ turnover are features of aging, targeting 4EBP1 activation could induce NMJ renewal by expanding the pool of post-synaptic myonuclei as an alternative intervention to mitigate sarcopenia.

A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.

PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.

Paraspinal myopathy-induced intervertebral disc degeneration and thoracolumbar kyphosis in TSC1mKO mice model-a preliminary study.

BACKGROUND: Increasing kyphosis of the spine in a human is a well-recognized clinical phenomenon that has been associated with back pain, poor physical performance and disability. The pathophysiology of age-related kyphosis is complex and has been associated with physiological changes in vertebrae, intervertebral disc (IVD) and paraspinal musculature, which current cross-sectional studies are unable to demonstrate. Creating an in vivo, paraspinal myopathic animal model for longitudinal study of these changes under controlled conditions is thus warranted. PURPOSE: To confirm the TSC1 gene knockout effect on paraspinal muscle musculature; to analyze the development of spinal kyphosis, IVD degeneration and vertebra structural changes in a longitudinal manner to gain insights into the relationship between these processes. STUDY DESIGN: A prospective cohort study of 28 female mice, divided into 4 groups-9-month-old TSC1mKO (n=7), 9-month-old control (n=4), 12-month-old TSC1mKO (n=8), and 12-month-old controls (n=9). METHODS: High resolution micro-computed tomography was used to measure sagittal spinal alignment (Cobb's angle), vertebral height, vertebral body wedging, disc height index (DHI), disc wedge index (DWI), histomorphometry of trabecular bone and erector spinae muscle cross-sectional area. Paraspinal muscle specimens were harvested to assess for myopathic features with H&E stain, muscle fiber size, density of triangular fiber and central nucleus with WGA/DAPI stain, and percentage of fibers with PGC-1α stain. Intervertebral discs were evaluated for disc score using FAST stain. RESULTS: Compared to controls, paraspinal muscle sections revealed features of myopathy in TSC1mKO mice similar to human sarcopenic paraspinal muscle. While there was significantly greater presence of small triangular fiber and density of central nucleus in 9-and 12-month-old TSC1mKO mice, significantly larger muscle fibers and decreased erector spinae muscle cross-sectional area were only found in 12-month-old TSC1mKO mice compared to controls. TSC1mKO mice developed accelerated thoracolumbar kyphosis, with significantly larger Cobb angles found only at 12 months old. Structural changes to the trabecular bone in terms of higher bone volume fraction and quality, as well as vertebral body wedging were observed only in 12-month-old TSC1mKO mice when compared to controls. Disc degeneration was observed as early as 9 months in TSC1mKO mice and corresponded with disc wedging. However, significant disc height loss was only observed when comparing 12-month-old TSC1mKO mice with controls. CONCLUSIONS: This study successfully shows the TSC1 gene knockout effect on the development of paraspinal muscle myopathy in a mouse which is characteristic of sarcopenia. The TSC1mKO mice is by far the best model available to study the pathological consequence of sarcopenia on mice spine. With paraspinal muscle myopathy established as early as 9 months, TSC1mKO mice developed disc degeneration and disc wedging. This is followed by kyphosis of the spine at 12 months with concomitant disc height loss and vertebral body wedging due to bone remodeling. Age-related bone loss was not found in our study, suggesting osteoporosis and myopathy-induced vertebral body wedging are likely two independent processes. CLINICAL SIGNIFICANCE: This is the first study to provide key insights on the early and late consequences of paraspinal myopathy on intervertebral disc degeneration, spinal kyphosis, and vertebral body changes. With this new understanding, future studies evaluating therapies for spinal degeneration may be performed to develop time-sensitive interventions.

Sarcopenia: Tilting the Balance of Protein Homeostasis.

Sarcopenia, defined as age-associated decline of muscle mass and function, is a risk factor for mortality and disability, and comorbid with several chronic diseases such as type II diabetes and cardiovascular diseases. Clinical trials showed that nutritional supplements had positive effects on muscle mass, but not on muscle function and strength, demonstrating our limited understanding of the molecular events involved in the ageing muscle. Protein homeostasis, the equilibrium between protein synthesis and degradation, is proposed as the major mechanism underlying the development of sarcopenia. As the key central regulator of protein homeostasis, the mammalian target of rapamycin (mTOR) is proposed to be essential for muscle hypertrophy. Paradoxically, sustained activation of mTOR complex 1 (mTORC1) is associated with a loss of sensitivity to extracellular signaling in the elderly. It is not understood why sustained mTORC1 activity, which should induce muscle hypertrophy, instead results in muscle atrophy. Here, recent findings on the implications of disrupting protein homeostasis on muscle physiology and sarcopenia development in the context of mTOR/protein kinase B (AKT) signaling are reviewed. Understanding the role of these molecular mechanisms during the ageing process will contribute towards the development of targeted therapies that will improve protein metabolism and reduce sarcopenia.

Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma.

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

Increased 4E-BP1 Expression Protects against Diet-Induced Obesity and Insulin Resistance in Male Mice.

Obesity is a major risk factor driving the global type II diabetes pandemic. However, the molecular factors linking obesity to disease remain to be elucidated. Gender differences are apparent in humans and are also observed in murine models. Here, we link these differences to expression of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), which, upon HFD feeding, becomes significantly reduced in the skeletal muscle and adipose tissue of male but not female mice. Strikingly, restoring 4E-BP1 expression in male mice protects them against HFD-induced obesity and insulin resistance. Male 4E-BP1 transgenic mice also exhibit reduced white adipose tissue accumulation accompanied by decreased circulating levels of leptin and triglycerides. Importantly, transgenic 4E-BP1 male mice are also protected from aging-induced obesity and metabolic decline on a normal diet. These results demonstrate that 4E-BP1 is a gender-specific suppressor of obesity that regulates insulin sensitivity and energy metabolism.

A Pilot Study of Ceramic Powder Far-Infrared Ray Irradiation (cFIR) on Physiology: Observation of Cell Cultures and Amphibian Skeletal Muscle.

The purpose of this research was to assess the potential for far-infrared ray irradiation from ceramic powder to improve exercise performance at room temperature. We designed experiments with murine myoblast cells (C2C12) to study the effect of cFIR irradiation on cell viability and lactate dehydrogenase release under H2O2-mediated oxidative stress and evaluated intracellular levels of nitric oxide and calmodulin. We also used electro-stimulation of amphibian skeletal muscle. Our results show that cFIR strengthened C2C12 under oxidative stress and delayed onset of fatigue induced by muscle contractions. We discuss possible mechanisms including anti-oxidation and prevention of acid build-up in muscle tissue based, and expect to see more applications of cFIR in the future.

TOR and ageing: a complex pathway for a complex process.

Studies in invertebrate model organisms have led to a wealth of knowledge concerning the ageing process. But which of these discoveries will apply to ageing in humans? Recently, an assessment of the degree of conservation of ageing pathways between two of the leading invertebrate model organisms, Saccharomyces cerevisiae and Caenorhabditis elegans, was completed. The results (i) quantitatively indicated that pathways were conserved between evolutionarily disparate invertebrate species and (ii) emphasized the importance of the TOR kinase pathway in ageing. With recent findings that deletion of the mTOR substrate S6K1 or exposure of mice to the mTOR inhibitor rapamycin result in lifespan extension, mTOR signalling has become a major focus of ageing research. Here, we address downstream targets of mTOR signalling and their possible links to ageing. We also briefly cover other ageing genes identified by comparing worms and yeast, addressing the likelihood that their mammalian counterparts will affect longevity.

Emerging roles of E2Fs in cancer: an exit from cell cycle control.

Mutations of the retinoblastoma tumour suppressor gene (RB1) or components regulating the RB pathway have been identified in almost every human malignancy. The E2F transcription factors function in cell cycle control and are intimately regulated by RB. Studies of model organisms have revealed conserved functions for E2Fs during development, suggesting that the cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation. However, given that some human tumours have concurrent RB1 inactivation and E2F amplification and overexpression, we propose that there are alternative tumour-promoting activities for the E2F family, which are independent of cell cycle regulation.

E2f3a and E2f3b contribute to the control of cell proliferation and mouse development.

The E2f3 locus encodes two Rb-binding gene products, E2F3a and E2F3b, which are differentially regulated during the cell cycle and are thought to be critical for cell cycle progression. We targeted the individual inactivation of E2f3a or E2f3b in mice and examined their contributions to cell proliferation and development. Chromatin immunoprecipitation and gene expression experiments using mouse embryo fibroblasts deficient in each isoform showed that E2F3a and E2F3b contribute to G(1)/S-specific gene expression and cell proliferation. Expression of E2f3a or E2f3b was sufficient to support E2F target gene expression and cell proliferation in the absence of other E2F activators, E2f1 and E2f2, suggesting that these isoforms have redundant functions. Consistent with this notion, E2f3a(-/-) and E2f3b(-/-) embryos developed normally, whereas embryos lacking both isoforms (E2f3(-/-)) died in utero. We also find that E2f3a and E2f3b have redundant and nonredundant roles in the context of Rb mutation. Analysis of double-knockout embryos suggests that the ectopic proliferation and apoptosis in Rb(-/-) embryos is mainly mediated by E2f3a in the placenta and nervous system and by both E2f3a and E2f3b in lens fiber cells. Together, we conclude that the contributions of E2F3a and E2F3b in cell proliferation and development are context dependent.

Mouse development with a single E2F activator.

The E2F family is conserved from Caenorhabditis elegans to mammals, with some family members having transcription activation functions and others having repressor functions. Whereas C. elegans and Drosophila melanogaster have a single E2F activator protein and repressor protein, mammals have at least three activator and five repressor proteins. Why such genetic complexity evolved in mammals is not known. To begin to evaluate this genetic complexity, we targeted the inactivation of the entire subset of activators, E2f1, E2f2, E2f3a and E2f3b, singly or in combination in mice. We demonstrate that E2f3a is sufficient to support mouse embryonic and postnatal development. Remarkably, expression of E2f3b or E2f1 from the E2f3a locus (E2f3a(3bki) or E2f3a(1ki), respectively) suppressed all the postnatal phenotypes associated with the inactivation of E2f3a. We conclude that there is significant functional redundancy among activators and that the specific requirement for E2f3a during postnatal development is dictated by regulatory sequences governing its selective spatiotemporal expression and not by its intrinsic protein functions. These findings provide a molecular basis for the observed specificity among E2F activators during development.

Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis.

Deregulation of the Myc pathway and deregulation of the Rb pathway are two of the most common abnormalities in human malignancies. Recent in vitro experiments suggest a complex cross-regulatory relationship between Myc and Rb that is mediated through the control of E2F. To evaluate the functional connection between Myc and E2Fs in vivo, we used a bitransgenic mouse model of Myc-induced T cell lymphomagenesis and analyzed tumor progression in mice deficient for E2f1, E2f2, or E2f3. Whereas the targeted inactivation of E2f1 or E2f3 had no significant effect on tumor progression, loss of E2f2 accelerated lymphomagenesis. Interestingly, loss of a single copy of E2f2 also accelerated tumorigenesis, albeit to a lesser extent, suggesting a haploinsufficient function for this locus. The combined ablation of E2f1 or E2f3, along with E2f2, did not further accelerate tumorigenesis. Myc-overexpressing T cells were more resistant to apoptosis in the absence of E2f2, and the reintroduction of E2F2 into these tumor cells resulted in an increase of apoptosis and inhibition of tumorigenesis. These results identify the E2f2 locus as a tumor suppressor through its ability to modulate apoptosis.

Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control.

Telomeres are the physical ends of eukaryotic chromosomes. They are important for maintaining the integrity of chromosomes and this function is mediated through a number of protein factors. In Saccharomyces cerevisiae, Cdc13p binds to telomeres and affects telomere maintenance, telomere position effects and cell cycle progression through G(2)/M phase. We identified four genes encoding Pol1p, Sir4p, Zds2p and Imp4p that interact with amino acids 1-252 of Cdc13p using a yeast two-hybrid screening system. Interactions of these four proteins with Cdc13p were through direct protein-protein interactions as judged by in vitro pull-down assays. Direct protein-protein interactions were also observed between Pol1p-Imp4p, Pol1p-Sir4p and Sir4p-Zds2p, whereas no interaction was detected between Imp4p-Sir4p and Zds2p-Imp4p, suggesting that protein interactions were specific in the complex. Pol1p was shown to interact with Cdc13p. Here we show that Zds2p and Imp4p also form a stable complex with Cdc13p in yeast cells, because Zds2p and Imp4p co-immunoprecipitate with Cdc13p, whereas Sir4p does not. The function of the N-terminal 1-252 region of Cdc13p was also analyzed. Expressing Cdc13(252-924)p, which lacks amino acids 1-252 of Cdc13p, causes defects in progressive cell growth and eventually arrested in the G(2)/M phase of the cell cycle. These growth defects were not caused by progressive shortening of telomeres because telomeres in these cells were long. Point mutants in the amino acids 1-252 region of Cdc13p that reduced the interaction between Cdc13p and its binding proteins resulted in varying level of defects in cell growth and telomeres. These results indicate that the interactions between Cdc13(1-252)p and its binding proteins are important for the function of Cdc13p in telomere regulation and cell growth. Together, our results provide evidence for the formation of a Cdc13p-mediated telosome complex through its N-terminal region that is involved in telomere maintenance, telomere length regulation and cell growth control.