Astrocytes play crucial roles in the brain and are involved in the neuroinflammatory response. They become reactive in response to virtually all pathological situations in the brain such as axotomy, ischemia, infection, and neurodegenerative diseases (ND). Astrocyte reactivity was originally characterized by morphological changes  and the overexpression of the intermediate filament glial fibrillary acidic protein (GFAP). However, it is unclear how the normal supportive functions of astrocytes are altered by their reactive state. In ND, in which neuronal dysfunction and astrocyte reactivity take place over several years or decades, the issue is even more complex and highly debated, with several conflicting reports published recently. In this review, we discuss studies addressing the contribution of reactive astrocytes to ND. We describe the molecular triggers leading to astrocyte reactivity during ND, examine how some key astrocyte functions may be enhanced or altered during the disease process, and discuss how astrocyte reactivity may globally affect ND progression. Finally we will consider the anticipated developments in this important field. With this review, we aim to show that the detailed study of reactive astrocytes may open new perspectives for ND. Astrocytes are considered reactive when they become hypertrophic and overexpress the intermediate filament GFAP. This “minimal definition” of reactive astrocytes is thus based on the two most universal hallmarks of reactivity, but this does not exclude that additional transcriptional, morphological and functional changes occur in a disease-specific manner, as discussed later in this review. Astrocyte reactivity involves the activation of a transcriptional program triggered by specific signaling cascades (see Section How Do Astrocytes Become Reactive?) that results in long-lasting changes in morphology and function, persisting over several hours, days or even decades. This should be distinguished from “activated astrocytes,” which are stimulated by exposure to neurotransmitters, for example. This transient response involves intracellular Ca2+ on the millisecond to second time scale, and is sometimes accompanied by subtle morphological changes  and Huntington's diseases (HD), as well as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). In particular, we aim to illustrate that astrocyte reactivity is a shared and central feature in ND that requires further characterization.It has been very difficult to distinguish the contribution of astrocytes from that of microglia because they usually become reactive in concert and both are involved in neuroinflammation , which contact synapses. PAP are dynamic and they influence synaptic transmission in physiological conditions . Most involve the expression of mutated amyloid precursor protein (APP), presenilin 1 (PS1), PS2 and/or Tau, and they replicate some neuropathological features and functional alterations of AD as well as memory deficits  repeats in the protein huntingtin (Htt). HD patients present psychiatric, cognitive and motor symptoms, the most characteristic being progressive chorea , and new mouse models are being developed , resulting in dopamine deficiency in the striatum and alteration of the basal ganglia circuitry. This causes major motor symptoms, such as akinesia, bradykinesia, tremor, rigidity and postural instability Agid, , as well+ cells increases after injury . This analysis confirms that the high density of GFAP+ cells is explained by enhanced GFAP expression and cortical atrophy . More recently, new methods have been developed to reduce astrocyte reactivity in vitro, such as exposure to heparin-binding EGF-like growth factor  can detect even mild neuronal dysfunction . Indeed, astrocytes are very well positioned at the tripartite synapse to detect abnormal synaptic activity and microglial cells permanently monitor the brain parenchyma. Once activated by such signals, glial cells further release active molecules to set up a reactive state. For example, purines, pro-inflammatory cytokines and growth factors may be released by reactive astrocytes and, in even larger amounts by activated microglia  in astrocytes, induces their reactivity  may be directly expressed by astrocytes or toxic proteins  can be taken up by astrocytes and activate them , bind to their cognate receptors at the astrocyte membrane and activate various intracellular signaling cascades. These include the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway, the Nuclear Factor of Kappa light polypeptide gene enhancer in B-cells (NF-κB) pathway, the calcineurin (CN) pathways and the Mitogen-Activated Protein Kinase (MAPK) pathway Figure .gfap and S100β  signal through specific cell-surface receptors possessing the glycoprotein 130 receptor (gp130) subunit. Upon binding, they trigger the assembly of multimeric receptors, leading to the phosphorylation and nuclear translocation of STAT3, and the transcription of its target genes  and IL-1β, two NF-κB target genes  and NF-κB , cytokines or extracellular matrix proteins to their specific cell-surface receptors activates the MAPK pathway  are non-coding RNA involved in the post-transcriptional regulation of gene expression. Several studies have linked changes in miRNA expression to astrocyte reactivity   is reduced in the midfrontal cortex of patients with AD . Neuron-astrocyte insert co-cultures were performed to identify the cellular origin of such deficits. They showed that expression of mHtt in astrocytes does not affect their own rate of glucose uptake. However, it impairs glucose uptake in neurons, regardless of their genotype, suggesting that HD astrocytes indirectly regulate neuronal metabolism by diffusible factors . Astrocytic “hyper-metabolism,” characterized by high incorporation of 13C into metabolic intermediates is observed in the brain of 7-month old 3xTg-AD mice  production  and astroglial coupling through gap junction channels is changed in reactive astrocytes in ND  and GABAB (presynaptic) currents are lower in R6/2 and zQ175 mice than in control mice. This results in a lower GABA-mediated tonic inhibition of striatal neurons. Pharmacological manipulation of the GABA transporter-3 (GAT-3), which is preferentially expressed by astrocytes, suggests that HD astrocytes have an impaired capacity to release GABA through GAT-3  3 inflammasome in these cells , neurosteroids, and adhesion molecules involved in neurite outgrowth , neprilysin or matrix metalloproteinase 2 and 9 (MMP9). Neprilysin and IDE are overexpressed in reactive astrocytes in contact with plaques in AD brains , the rate limiting enzyme for Aβ production. Strong BACE1 expression is observed in reactive astrocytes in patients and several mouse models of AD, and following exposure to pro-inflammatory cytokines, which can directly activate the BACE1 promoter  for GFAP or Vim or double KO were initially generated and tested in acute injuries. Disruption of reactive astrocyte cytoskeleton was also studied in ND models and was found to shorten the lifespan of the SOD1G93A mouse model of ALS, ablation of proliferating astrocytes has no effect on disease outcomes  under the GFAP promoter, in presence of the drug ganciclovir  with SOD1G93A mice or they overexpressed a dominant negative form of IκBα (AAV-IκB-SR) in astrocytes by viral gene transfer. The inhibition of the NF-κB pathway in reactive astrocytes did not influence motor neuron survival in culture or in the spinal cord of SOD1G93A mice  was overexpressed by lentiviral gene transfer in the striatum of R6/2 mice to block NF-κB signaling. DN-IKKγ overexpression improved motor performance and prevented shrinkage of striatal neurons in HD mice  and the disease model Table . Some ofOne of the next challenges in the field is to deal with the functional heterogeneity of astrocytes. Indeed, like neurons, astrocytes display remarkable heterogeneity regarding their density, morphology  under the control of the GFAP promoter   in acute models of ischemia and excitotoxicity. Arundic acid, an inhibitor of astrocyte reactivity, normalizes it, but the molecular basis for such changes in NMR signals is unclear  techniques are an attractive alternative to monitor astrocyte reactivity 13C-labeled metabolic substrates such as glucose or acetate. Indeed, acetate is preferentially oxidized by astrocytes and its metabolic fate can be monitored by MRS . 13C-MRS may be translated to the clinics although it remains quite an expensive and sophisticated approach (Ross et al., More cellular selectivity may be achieved by MRS techniques after the infusion of in vitro and in vivo in models of ALS (Rothstein et al., The above-mentioned changes in reactive astrocytes make these cells alternative or complementary therapeutic targets to neurons for ND (Escartin and Bonvento, Therapeutic strategies tested so far for ND have largely focused on neurons and have been mostly unsuccessful to date (Huang and Mucke, Overall, this review illustrates the multifaceted and complex roles of reactive astrocytes during ND. Astrocyte reactivity appears as a conserved response that is initially beneficial but is later corrupted by disease-specific alterations. Huge progress has been made recently as a result of the heightened interest in glial cells, and the development of innovative and cell type-specific approaches. However, these cells remain enigmatic, and many aspects of their physiology need to be clarified. Although the molecular pathways leading to astrocyte reactivity during ND have been described, it is crucial to elucidate what disease-, region- and environmental-specific mechanisms control the functional outcomes associated with astrocyte reactivity Figure . In any The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed “senescence,” can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells’ heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. Despite billions of cell divisions and trillions of cells, humans remain, on average, cancer-free for more than 50 years. One of nature's notable tumor suppressive mechanisms is cellular senescence, a response to nonlethal stress that results in persistent cytostasis. In the absence of normal growth arrest accompanying differentiation, senescence imposes limits on the proliferative capacity of clonal cell lineages. Senescence can be induced by multiple stimuli, including intrinsic cellular processes such as telomere dysfunction and oncogene expression, but also by exogenous factors such as DNA damaging agents or oxidative environments. Abundant published evidence now supports the concept that senescence is a significant impediment to malignancy, and that it is ordinarily very stringent. Indeed, as a number of investigations have shown, many cell types in which one or more senescence pathway components are functionally inactivated remain susceptible to senescence – an indication that robust compensatory mechanisms exist for this important stress response. Despite the resiliency of the senescence response, however, it is prone to failure to varying degrees, depending upon genetic/epigenetic context. Failure of senescence in cells that have undergone oncogene activation, telomere dysfunction, and/or DNA damage can result in changes favoring malignancy and drug resistance. Elucidation of mechanisms that enforce senescence has been sought in expectation that such knowledge should lead to measures that prevent or reverse its failure in susceptible pre-malignant and malignant cell populations. In this review, we focus on telomeres and other mediators of senescence induction as candidate targets for the prevention and treatment of cancers.2Proliferating cells can respond to genotoxic and non-genotoxic stresses in a number of ways, including transient cell-cycle arrest, senescence, and cell death. Senescence is operationally broadly defined as a viable growth arrest characterized by the inability of affected cells to resume proliferation in the presence of appropriate mitogenic factors. While multiple cellular and molecular features, including increased cell size, accumulation of lysosomes, upregulation of cell cycle inhibitors, presence of senescence-associated heterochromatic foci (SAHF), and positive staining for senescence-associated beta-galactosidase  activity, have been associated with senescent cells, no single feature is a universal and specific marker of senescence. Experimental and clinical evidence indicate that an intact senescence response is important for preventing unregulated growth and malignant transformation. In addition, the ability to undergo senescence can determine the efficacy of targeted cancer therapies. As described below, however, senescence is not a discrete mechanism or pathway that can be easily classified as either intact or entirely non-functional. Instead, it is a process that can result from many different inputs with degrees of sensitivity dictated by intrinsic as well as extrinsic factors.2.1in vitro. Telomerase activity and telomere length elongation in cancer are associated with up-regulation of both hTR and hTERT, while overexpression of hTR has been shown to boost telomerase activity and more dramatically extend telomere length in cells that express endogenous or ectopic hTERT in vitro, illustrating the fact that both components are required for a functional telomerase holoenzyme. Although hTERT was initially considered as the limiting component of telomerase, evidence from biochemistry, promoter studies, mouse models, and human tumors has demonstrated contexts where hTR limits telomerase enzyme levels and telomere maintenance In vitro, attrition of TTAGGG repeats upon successive divisions in cells lacking sufficient telomerase activity ultimately results in DNA damage responses including growth arrest, followed by cell enlargement, chromatin condensation, and vacuolization – characteristic features of senescent cells. Multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are composed of cells that display signs of telomere dysfunction-induced senescence In the absence of externally or oncogenically induced stresses, telomerase repression may be the only physiological impediment to indefinite replication. Replicative senescence, as originally described by Hayflick in cultures of cells from non-malignant tissues, is due to natural repression of telomerase and the resulting DNA damage response that occurs when the number of telomeric TTAGGG repeat sequences on the ends of chromosomes becomes too few to support the assembly of stable telomere complexes In the absence of a telomere maintenance mechanism, telomeres shorten with each round of cellular replication to eventually reach critically short lengths that are unable to support stable formation of shelterin protein complexes that protect telomeres from DNA damage surveillance mechanisms 2.2In contrast to replicative senescence, oncogene-induced senescence (OIS) often occurs independently of telomere status in vivoThe presence of senescent cells induced by oncogenic signaling has been documented in several precancerous tissues of both human and mouse, indicating that OIS occurs 2.3in vitro and in vivo  as those activated by telomere dysfunction and oncogene activation, it is noteworthy therapeutically that cancer cells lacking functional tumor suppressors such as p53 or pRB often retain the capacity to undergo external agent-induced senescence. While in some cases, senescence-inducing drugs clearly act through the generation of DNA damage, in other cases the mechanisms involve only alterations in DNA structure and function . Still other compounds may induce senescence through direct or indirect stimulation of stress sensitive kinases, creating imbalances in mitogen signaling pathways Conventional genotoxic radiotherapy and chemotherapeutic regimens are potent inducers of senescence in cancer cells The chief clinical advantage of targeting senescence rather than outright cell death as a desired endpoint is that drug doses required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. In prostate cancer cell lines, for example, a dose of 25 nM doxorubicin is sufficient to induce senescence, while doses >250 nM are necessary to cause cell death 3Gene expression profiling has revealed that there is limited overlap among genes exhibiting altered expression in cells induced to undergo senescence by telomere shortening, oncogene induction, or external agents 3.1in vivoThe importance of p53 in senescence was determined by inhibiting p53 function with dominant negative mutants, specific p53 antisense mRNA, oligonucleotides or viral oncoproteins (such as SV40 T antigen or HPV16 E6); such treatments were sufficient to substantially extend the lifespan of several cell types in culture WAF1e.g., by viral oncoproteins or anti-sense oligonucleotides) independently and synergistically extends replicative life span Senescence effects of p53 are mediated in part through increased expression of the CDK inhibitor p213.2RB family functions that distinguish reversible cell cycle arrest from irreversible senescence-associated changes. Despite the similarities among RB family proteins, defects in pRB, but not in p107 or p130, have been associated with human cancers. This suggests that pRB has unique tumor suppressor properties not attributable to p107 or p130. In support of this concept, pRB has been shown to be preferentially associated with E2F targets involved in DNA replication during OIS, and suppression of pRB, but not p107 or p130, allowed continued DNA synthesis after induction of oncogenic RAS While the causes of p16 induction remain to be defined, its mechanism of action has been well characterized. The binding of p16 to cyclin dependent kinases (CDKs) 4 and 6 induces allosteric conformational changes that disrupt the interaction of these kinases with D-type cyclins Distinctions in pRB-associated phenotypes may be due to differences in the functionality of different phosphorylated forms of pRB . Althoug3.3pRB can play an active role in the formation of senescence-induced heterochromatic foci (SAHF) in human cells. Originally characterized in senescent fibroblasts Rb family members, p130 and p107, which have not been demonstrated to be tumor suppressors, bind preferentially to target promoters in the absence of growth factors and in proliferating cells, respectively, and these repressor complexes are displaceable by E2F-1. HP1, which interacts with pRB, is associated with these distinct repressor complexes and follows a similar pattern of stability/displaceability. Efficient growth arrest by p16/pRB in MEFs is dependent on H3-K9Me, which provides a binding site for HP1. Differences in the stability of repressor complexes at promoters may underlie the different roles of pRB versus p130 and p107 in cell cycle regulation and tumor suppression The chromatin-based role of pRB in transcriptional repression is complicated, with multiple factors cooperating for transcriptional repression of specific promoters. For example, repression of cyclin A has been shown to be dependent on SWI/SNF chromatin remodeling, whereas other forms of transcriptional repression have been shown to be dependent on histone deacetylases or polycomb repressor components Although the mechanisms responsible for formation and propagation of heterochromatin remain to be characterized, H3-K9Me, trimethylation of histone H4 lysine 20 (H4-K20Me), and recruitment of HP1 appear to be involved de novo formation of new marks Molecular details of SAHF formation and stability remain to be determined. In addition to pRB, H3-K9Me, and HP1, SAHFs are known to be enriched in a histone H2A variant, macroH2A, previously associated with silenced chromatin 4A protective role of senescence has been inferred in murine models of lung adenomas, T-cell lymphomas, prostate tumors, and pituitary tumors Direct evidence that senescence is tumor suppressive has been generated using inducible murine models. In a model for p53-dependent liver cancer, the effect of p53 restoration was studied in established liver carcinomas Correlative data indicate that stress-induced senescence is tumor suppressive in human tissues as well. In malignant cells, senescence is a well-documented consequence of various chemotherapeutic regimens 5Unlike cells that are removed by other tumor suppressor mechanisms such as programmed cell death, senescent cells can persist and continue to actively interact with surrounding cells and tissues for extended periods of time. Defective elimination of senescent cells may lead to unregulated accumulation in aging tissues and at sites of age-associated pathologies including cancer via cytokine signaling loops The SASP was originally documented indirectly through the ability of senescent cells to influence the biology of other cells in vitro and in vivo. For example, MMP3 secretion by senescent cells perturbed the proliferation and differentiation of cultured normal breast epithelial cells In some contexts, however, presumably when senescent cells cannot be properly cleared by the immune system, negative consequences can occur 6e.g., telomerase activation) or compromise cellular ability to sense or transduce stress-related signals  have the potential to promote immortalization and resistance to therapeutically induced senescence. Evidence obtained using cell sorting and videomicroscopy showed that fully senescent non-malignant human keratinocytes are capable of spontaneously yielding mitotically competent progeny in vitro occur during oncogenesis in vivo.Contrary to popular belief, the senescence growth arrest is not always irreversible. Since senescence is a response to stress, genomic and epigenomic aberrations that ameliorate stress , effectively conferring drug resistance e.g., p53, pRB) play critical roles.Determinants of the stability of the senescence response have not been well characterized. Yet, the implications of delayed cell cycle re-entry or permanent senescence for patient prognoses are profound. In the former case, transient senescence may allow tumor cells time to repair or accommodate the chemotherapeutic stress . This drug has been tested against 39 individual serine, threonine, and tyrosine kinases, representing most of the primary protein kinase families, and has shown highly selective inhibition of CDK4 and CDK6 doi:10.1016/j.semcancer.2015.03.007.Supplementary table related to this article can be found, in the online version, at 7Telomerase is almost universally re-expressed in cancer cells and is regulated at multiple levels involving genetic, epigenetic, transcriptional, post-transcriptional, post-translational, and sub-cellular shuttling mechanisms. At the genetic level, both hTR and hTERT genes have been reported to be amplified in some tumors. Increased hTR gene copy number was detected frequently in head and neck and cervical carcinomas Both the hTR and hTERT genes are subject to tumor specific epigenetic regulation. H3-K9Me marks were increased at the hTR promoter in normal fibroblasts and in several ALT cell lines with low hTR expression in comparison with telomerase positive cancer cell lines, suggesting a role in hTR suppression, while H3-K4Ac, H4-K4Ac, H3-K9Ac and H3-K4Me were associated with hTR expression. Similar analysis of the hTERT promoter also revealed H3-K9Me associated with transcriptional suppression and higher levels of H3-K4Ac, H4-K4Ac and H3-K9Ac in cancer cells compared with normal fibroblasts, though H3-K4Me was not strongly associated with expression in the panel tested in situ studies of a wide range of tumor samples In cancer cells lacking chromatin mediated silencing present in normal cells, both genes are subject to regulation by a wide variety of distinct, but overlapping, transcription factors. hTR is generally expressed at low levels in normal cells and is substantially up-regulated in cancer cells as shown both by fluorescence In contrast, the hTERT gene has been intensely studied since its molecular cloning in 1997 . hTERT tvia ETS factors In general, oncogenic growth promoting pathways have usually been found to activate telomerase expression and promoter activity, while pathways controlling growth suppression, cell death and senescence have the opposite effect. For example, growth factor signaling through MAPK pathways increases hTERT expression in part Other hTERT activation pathways that are frequently deregulated in various cancer settings include those influenced by CDK2 and CDK4 Recently, a whole kinome siRNA screen in ovarian cancer cells for regulators of the hTERT promoter revealed at least 68 kinases that participate in pathways upstream of hTERT regulatory transcription factors, underscoring the complexity of the signaling environment 8versus cancer cells, provide degrees of specificity to telomerase-based drugs and reduce the probability of toxicity to normal tissues Intrinsic differences in telomere maintenance between normal and cancer cells provide an attractive therapeutic opportunity. Several direct strategies to exploit the dependence of cancer cells on aberrant telomerase expression for telomere homeostasis and immortality have been reported. The key advantages of targeting telomerase in comparison with most other cancer targets are its relative universality, criticality and specificity for cancer cells, including putative cancer stem cells via apoptosis. They also demonstrated that expression of DN-hTERT inhibited anchorage independent growth and impeded the development of malignancies in xenografted mice in vitro and in vivo using a murine model Many distinct classes of anticancer compounds directed toward telomerase can conceivably be developed because of the varied cellular processes that regulate telomerase expression and activity. For example, cellular telomerase activity can be controlled through changes in gene transcription and alternative splicing of the telomerase components, and by the nuclear translocation, phosphorylation, folding and turnover of individual components that lead to and regulate the rate of telomerase complex assembly and accessibility to telomeres 8.1Studies that utilized antisense oligonucleotides, including chemically modified nucleic acids (PNA) that target hTR, provided proof-of-principle evidence of the effectiveness of this approach as a means of specifically inhibiting telomerase and inducing telomere shortening http://www.geron.com/imetelstat). Subsequent phase II trials have focused on hematologic disease, childhood CNS malignancies and the application of imetelstat in maintenance therapy for non-small cell lung cancer and metastatic breast cancer patients previously treated by standard chemotherapy and surgical debulking. Data emerging from these trials suggest limited benefit in these specific clinical settings. Disappointing progression-free survival results from a phase 2 trial of imetelstat as maintenance therapy following platinum chemotherapy in patients with non-small cell lung cancer have led the Geron Corp. to suspend further development of imetelstat in solid tumors. However, it has been argued that results from the lung cancer trial showed improved outcomes for a small subset of patients with tumors that had short telomere lengths. Among the trials for hematologic disorders, encouraging data from 13 patients treated with imetelstat for essential thrombocythemia were presented at the annual meeting of the American Society for Hematology in December 2012. The ability of imetelstat to reduce platelet counts in essential thrombocythemia is consistent with earlier trials that reported thrombocytopenia as an adverse effect.Following the success of the preclinical investigations of imetelstat, phase 1 clinical trials were initiated in which imetelstat was tested as a single agent in patients with various aggressive liquid and solid tumors (Supplementary Table 1). The limited information that was released following the completion of those studies suggested that imetelstat presented minimal adverse effects, with thrombocytopenia and neutropenia being the dose limiting toxicity While imetelstat, targeting hTR, was the first telomerase inhibitor to undergo trials in patients, modified antisense oligonucleotides targeting hTERT mRNA have also been shown to impede the proliferation of tumor cells. However, in contrast to the lag period observed in preclinical investigations of imetelstat, the proliferative defect induced by hTERT inhibition was immediate, and occurred in the absence of apparent telomere shortening 8.2in vitro and limit tumor formation in xenografted mice Small molecular weight compounds that inhibit telomerase activity have been identified in screens of chemical libraries or synthesized based on the structure of the tea catechin, epigallocatechin-3-gallate , this area of investigation holds considerable promise.Another plausible approach to the inhibition of telomere maintenance is to directly target telomeres, or to manipulate the shelterin proteins that provide telomere secondary structure and telomerase access , and their utility may be context-dependent.Given the heterogeneity present in most cancers, complete arrest/eradication of the various subpopulations of cells in any given cancer will likely require simultaneous targeting of several molecules and mechanisms that contribute to the malignant phenotype. It is therefore important to anticipate complementary as well as antagonistic effects on different cancer hallmarks that might be achieved using agents directed against specific targets. Accordingly, a literature search has been conducted by a “cross-validation” team to identify potential consequences of the use of selected agents targeting protein complexes involved in the maintenance of replicative immortality on other hallmarks of cancer. The results of this survey are listed in 13Developing optimized and truly holistic cancer prevention and treatment regimens will likely incorporate strategies that target replicative immortality. The chief advantage to be gained by the use of these senescence-inducing regimens is elimination of tumor repopulating ability with reduced collateral damage compared to conventional cytotoxic regimens. There are certain questions and risks associated with this strategy that must be addressed before its wholesale adoption in clinical settings. In the case of telomere and telomerase based strategies, replicative senescence may occur more readily in cancer cells bearing short telomeres than in vital normal cells with long telomeres, but telomere lengths in cancer cells may still be long enough to permit sufficient population doublings for invasion and metastases to occur. Moreover, telomere dysfunction promotes the development of chromosomal instability, which in turn can generate mutations that enable cells to become drug resistant and/or activate ALT mechanisms for telomere maintenance and/or become more malignant. High priority should be given to further research into the determinants of senescence stability, as the implications of delayed cell cycle re-entry, permanent cytostasis, or eventual clearance may be profoundly different. Lower doses of genotoxic drugs needed to induce senescence may reduce collateral damage, but allow establishment of dormancy by resistant cells. Conversely, since it is almost impossible to kill all the cells in a tumor even at the highest tolerated doses of chemotherapy, addition of a complementary agent that induces or enhances stable senescence in the cancer cells that manage to retain viability might additively or synergistically increase therapeutic efficacy. The microenvironmental and systemic effects of senescent cells also need further clarification. While senescent cells have been shown to secrete proteins that aid in their own clearance and tissue repair, persistent senescent cells can disrupt niches, causing the depletion of healthy stem cells, and can promote aging and cancerous phenotypes in surrounding tissues. Given that some senescence-associated secretory phenotypes can be pro-tumorigenic, is senescence still a good objective? Can a proper immune response be counted on to clear senescent cells? Ultimately, given the hypermutability and heterogeneity of most human cancers, it is often unlikely that targeting any single gene product or pathway will provide lasting remission. However, developing agents with good mechanistic understanding, that effectively disrupt replicative immortality, may well add to the arsenal available for combinatorial approaches.Dr. Keith has collaborative agreements with AstraZeneca and Geron Corporations, and is founder and director of Senectus Therapeutics.


Recent evidence has revealed the importance of reciprocal functional interactions between different types of mononuclear cells in coordinating the repair of injured muscles. In particular, signals released from the inflammatory infiltrate and from mesenchymal interstitial cells ) appear to instruct muscle stem cells (satellite cells) to break quiescence, proliferate and differentiate. Interestingly, conditions that compromise the functional integrity of this network can bias muscle repair toward pathological outcomes that are typically observed in chronic muscular disorders, that is, fibrotic and fatty muscle degeneration as well as myofiber atrophy. In this review, we will summarize the current knowledge on the regulation of this network in physiological and pathological conditions, and anticipate the potential contribution of its cellular components to relatively unexplored conditions, such as aging and physical exercise. The regenerative capacity of skeletal muscle relies on muscle stem cells (satellite cells (SCs)), which proliferate in response to exercise to facilitate muscle growth and remodeling, or following myotrauma to repair the injured muscle.This review summarizes the current knowledge on the role played by the cellular network composed by SCs, FAPs and the inflammatory infiltrate  during physiological and pathological perturbations of muscle homeostasis.23 although, the direct contribution of SCs to myofiber hypertrophy and maintenance remains controversial.26Maintenance of muscle mass depends on the integrity of the regenerative machinery, which is composed of SCs and other mononucleated cell types,SCs are located beneath the basal lamina – the anatomical niche – and their activity is regulated by interactions with cellular components of the 'functional niche' – FAPs, immune as well as vessel-derived cells  and 2.α)+9 or stem cell antigen 1 (Sca1)+10 +10 . When is muscles . In addi11 For instance, IL-4 released by eosinophils has been shown to be a key mediator of FAP fate  or subtle  perturbations of skeletal muscle homeostasis may both stimulate FAP-mediated activation of SCs. Inactivation of IL-4-mediated signaling or eosinophil chemotaxis stimulates the adipogenic differentiation of FAPs.15 Likewise, interactions between FAPs and myofibers or SCs also regulate the FAP-mediated adipogenesis.10 Finally, FAP activity is regulated by growth factors , hepatocyte growth factor (HGF)), follistatin and nitric oxide (NO), which are secreted from FAPs themselves, endothelial cells and M1/M2 macrophages38  activates the signal transducer and activator of transcription 3 (STAT3) in SC.46 As for the latter, FAPs may influence the metabolic activity of the muscle since ectopic adipocytes are associated with impaired insulin sensitivity, metabolic syndrome and type 2 diabetes (T2D).49 In this regard, it is intriguing to speculate as to what extent systemic circulating factors may alter FAP differentiation, including high glucose conditions shown to induce adipogenesis in muscle-derived stem cells.50 Moreover, deregulated FAP activity can contribute to the increased intramuscular adipose tissue (IMAT) observed in aged muscles51 and in patients with RA,52 COPD53 and cancer cachexia .54 Thus, FAPs may hold a dual role in which they provide important paracrine stimuli to support SC function in healthy muscle, while contributing to ectopic adipose accumulation in pathological conditions56 , systemic factors and local concentrations of soluble cues . Dysfunctional FAP regulation by alterations of these regulatory conditions may severely deteriorate muscle health, affecting both muscle function and metabolism.itions56  and 5, t61Most neuromuscular disorders are initially alleviated by the regenerative potential of skeletal muscles. For instance, in Duchenne muscular dystrophy (DMD), compensatory regeneration at earlier stages of disease tends to counter the degeneration of dystrophin-deficient myofibers. While, at least in mouse models of muscular dystrophy, this reactive regeneration resembles muscle repair following injury, as the disease progresses the asynchronous waves of regeneration and the changes in niche caused by chronic regeneration eventually bias the repair toward pathogenic fibrosis and fat deposition.62 is typically followed by the sequential activation of FAPs and SCs.64 Progressive impairment of the interplay between SCs, FAPs and immune system is emerging as a key event in switching regeneration from compensatory to pathogenic.66 In particular, FAPs appear to play a central role in this switch.67Optimal regeneration entails a sequence of events that ensures temporally coordinated interactions between SCs, FAPs and cells of the immune system. An initial activation of resident immune cells and the inflammatory infiltrate,16 This alternative phenotype is regulated at the epigenetic level by a network formed by muscle-specific microRNA  that target key subunits of the SWI/SNF chromatin-remodeling complex. In particular, expression of myomiRs correlates with the ability of FAPs to support SC-mediated myogenesis and to adopt a myogenic phenotype at the expense of the fibro-adipogenic lineage.16 This outcome is typical of compensatory regeneration at early stages of disease progression and likely reflects the action of cues from a regeneration-conducive SC niche that resolve FAP bipotency into the pro-myogenic phenotype. Saccone et al.16 showed that myomiRs selectively target two variants of the BAF60 subunit of SWI/SNF complex (BAF60A and B), which activate chromatin remodeling at fibrotic and adipogenic loci. This leads to the selection of the alternative variant – BAF60C – which promotes chromatin remodeling at muscle loci68 and mediates FAP commitment to the myogenic lineage16  reversible HDAC-mediated repression of myomiR allows the expression of BAF60A and B variants and supports maintenance of bipotency in FAPs. In dystrophic muscles, constitutive HDAC activity represses BAF60C and myomiR and favors the expression of BAF60A and B, which direct the fibro-adipogenic phenotype of FAPs. Interestingly, this dynamic HDAC-mediated regulation of phenotypic and functional bipotency of FAPs is observed at early, but not late stages of disease progression in mdx mice, and accounts for the restriction of the beneficial effects of HDAC inhibitors at early but not late stages of disease.12Importantly, this intracellular network is controlled by histone deacetylases (HDACs),β2 (TGFβ2), which are induced by elevated canonical Wnt signaling in dystrophic muscles and affect the behavior of SCs.71Future studies should establish how FAP bipotency, and relative resolution, is regulated by cues from the regenerative microenvironment and should elucidate the signals that controls reciprocal interactions between FAPs, immune system and SCs within physiological and pathological contexts. For instance, it would be interesting to evaluate the contribution of FAPs to the excessive levels of transforming growth factor-α+ cells in healthy and diseased muscles,14 the functional and molecular characterization of human FAPs can provide novel interesting targets for the development of pharmacological treatment of muscular diseases.As the human counterpart of FAPs has been identified as PDGFR74 In rodents, the aging muscle and SC niche has been shown to disrupt SC function and myofiber regenerative capacity.78 Local changes in the SC milieu include a number of events that can alter signaling in SCs. For instance, constitutively elevated activation of p38 kinase, STAT3 activity and reduced Notch signaling has been observed in aged SCs.82 Likewise, increased TGFβ activity and induction of the cyclin-dependent kinase inhibitors associated to inhibition of cellular proliferation, such as p15, p16 and p21, have been reported as potential triggers of SC senescence.81Aging is associated with an accelerated loss of skeletal muscle mass (sarcopenia) and with a reduced regenerative capacity of the musculature, leading to a loss of strength and function.74 that originate from FAP differentiation. In a mouse model of young and old mice sharing the circulatory system (heterochronic parabiosis model) aged SCs were rejuvenated by exposure to a young systemic environment suggesting that the tissue-specific stem cells retain their proliferative potential, but that the aged systemic environment prevents full activation.76 These findings have been sparsely investigated by in vitro studies on human primary cells, leading to contradictory results.82 Age-induced changes in the systemic milieu include reduced local capillary network and endothelial cell apoptosis/senescence, which can lead to reduced secretion of SC stimulatory factors, impaired chemotaxis of immune cells and collectively a more negative balance between positive and negative regulators of SC activity. Recent evidence points to the importance of systemic concentrations of the circulating proteins such as oxytocin83 or growth differentiation factor 11 (GDF11),84 although it is currently controversial whether GDF11 levels decrease or increase with aging, as well as the relative efficacy of GDF11 supplementation in countering the functional decline of aged muscle and SCs.85The events described above likely depend on extensive changes in the SC niche, including deregulated activity and number of FAPs or additional cellular components, such as fibroblasts and adipocytes,+ SCs.25 However, despite the lack of direct effects on muscle fiber size, ablation of Pax7+ cells during sarcopenia generated increased levels of collagen deposition, preferentially in fast muscles,25 which could derive from fibrogenic differentiation of FAPs. In human skeletal muscle in vivo the SC content in type II muscle fibers is selectively reduced with aging, whereas the number of SCs in type I fibers remains similar to young individuals, following the pattern of a selective atrophy of type II muscle fibers.87 Thus, while SC content does decrease during sarcopenia in both rodent and human skeletal muscle, it is not yet entirely defined to what extent the decrease in SC content can account for muscle atrophy or vice versa. Although this selective deterioration of type II fibers and their SC content in human skeletal muscle is partly reversible by resistance training,89 the responsiveness of SCs to a single bout of resistance exercise is reduced with aging.88 Even lifelong (endurance) exercise does not seem to prevent the decrement in type II fiber size or SC content compared to type I fibers.90 However, the amount of adipose infiltration in the old untrained muscle was larger than in the trained groups . It is therefore intriguing to speculate that changes in the muscle microenvironment or systemic environment related to inactivity or ageing can condition FAP phenotype and ability to release important paracrine cues to SCs and myofibers to support regeneration and muscle growth.Interestingly, sarcopenia in rodents is not further accelerated during conditional ablation of Pax791 Moreover, patients with T2D show a greater decline in muscle mass, muscle strength and functional capacity with aging.92 A common observation in conditions associated with impaired skeletal muscle insulin sensitivity is accumulation of ectopic lipids within (intracellular) and between (extracellular) skeletal muscle fibers94 , presumably serving as energy source during physical activity,95 although they also exhibit increased insulin sensitivity, as compared to healthy untrained subjects.97 In contrast, the IMAT  is to our knowledge not increased in athletes and is associated with reduced insulin sensitivity in both healthy47 and obese48 subjects as well as in acromegaly patients.49 Although the origin of IMAT is not yet known,56 murine muscle-derived stem cells have been shown to undergo adipogenic differentiation upon exposure to elevated glucose levels in vitro,50 providing a potential link between adipocyte accumulation and the systemic milieu. Moreover, while previous work proposed that IMAT can originate from trans-differentiation of myogenic stem cells,98 increasing evidence suggests that FAPs could constitute the mesenchymal stem cells responsible for IMAT accumulation in skeletal muscle.100 In addition, FAP-derived adipocytes may have reduced insulin sensitivity compared to conventional adipocytes, suggesting that accumulation of FAP-derived adipocytes may contribute to a compromised peripheral insulin sensitivity.101In addition to muscle atrophy, inactivity and ageing are commonly associated with increased adiposity, together leading to metabolic dysfunctions such as dyslipidemia, decreased insulin sensitivity, hyperglycemia and an increased risk of developing diabetes mellitus . Since skeletal muscle is the most abundant tissue of the body for glucose disposal, muscle sensitivity to insulin action is essential in development of whole body insulin resistance and hyperglycemia.102 and IL-4 promoter polymorphisms have been associated with T2D.103 Moreover, IL-4-mediated signaling may prevent adipogenesis in muscle and adipose tissue.104 In white adipose tissue, IL-4 released from tissue-resident eosinophils regulates the presence of M2 macrophages,31 which are positively associated with insulin sensitivity.105 When genetically depleting IL-4 secreting eosinophils the content of the M2 macrophage phenotype in adipose tissue is reduced and this is associated with a substantial decrease in glucose tolerance and insulin sensitivity.31 These changes are less explored in humans, however, increased expression of the inflammatory macrophage phenotype marker; CD11c in skeletal muscle of T2D patients has been reported.106Presently, the role of FAPs and their interplay with SCs, eosinophils or macrophages in relation to development of T2D is unknown. However, dysfunctional regulation of FAPs mediated by changes in IL-4 signaling may influence skeletal muscle homeostasis. Interestingly, IL-4 levels have been reported to be positively associated with insulin sensitivity107 and in chronic diseases that cause cachexia.109 Patients suffering from cachexia or patients affected by metabolic syndrome may display elevated levels of glucocorticoids110 that may influence IL-4 secretion. Interestingly, elevated glucocorticoid levels (such as those reached upon dexamethasone treatment) in mice increase the adipogenic differentiation of FAPs, which is otherwise suppressed through IL-4-mediated signaling.15 The effect of dexamethasone treatment on FAP adipogenesis may relate to suppression of eosinophil release of IL-4111 as suggested by Dong et al.15 Thus, elevated levels of glucocorticoids during disease  could increase adipocyte accumulation in skeletal muscle through adipogenic differentiation of FAPs and hereby negatively impact the insulin sensitivity of skeletal muscle.48 Given that steroids are used in the treatment of many muscular disorders (including DMD), the interactions between glucocorticoids, FAP, SCs and cells of the immune system deserve future investigation.Reduced insulin sensitivity is often associated with aging114 Skeletal muscle wasting is a common phenomenon in cancer patients,119 and cancer-related muscle loss affects up to 80% of patients with advanced cancer, leading to poorer prognosis, reduced treatment response and increased risk of complications during surgery and chemotherapy. Ultimately, cachexia accounts for >20% of all cancer-related deaths.124Cachexia consists of an accelerated muscle loss that is associated with chronic diseases, complicates their recovery and is an independent predictor of morbidity and mortality.κB activity,125 suggesting that NF-κB may contribute to muscle wasting in cancer.125 In muscle biopsies from pancreatic cancer patients with accelerated weight loss, an increased number of Pax7+ cells was observed, indicating that cancer cachexia is associated with an expansion of the myogenic precursor pool.125 Expansion of the Sca1+ cell population that resemble mouse FAPs, was also observed in cachectic muscles from tumor-bearing mice. Interestingly, this Sca1+ population preferentially adopt the myogenic lineage under the influence of tumor environment, by expressing the SC-specific transcription factor Pax7, which is induced by serum factors from cachectic mice and patients, in an NF-κB-dependent manner; however, completion of differentiation was also inhibited by the persistent expression of Pax7.125 Restoring the myogenic potential of these cells by Pax7 downregulation or by ectopic expression of MyoD, promoted cell differentiation and fiber fusion and reversed muscle wasting.125 These results reiterate the concept that Sca1+ cells have a latent myogenic potential that can be induced by environmental signals, for example, the elevated levels of cytokines from systemic inflammation. Since, Pax7 expression is regulated by inflammation-induced signals,126 these data reveal again the important interactions between the immune system and FAPs in the control of muscle regeneration. However, it is important to note that Sca1+ cells might only account for a subpopulation of FAPs, or even a distinct population of cells induced in tumor-bearing conditions. A recent study not only identified stromal cells by expression of fibroblast activation protein α , and observed a depletion of these stromal cells to be underlying the cancer-induced cachexia.127 Specifically, these stromal cells appeared to maintain muscle size through paracrine secretion of follistatin, which in turn reduced the muscle expression of ubiquitin ligases such as atrogin-1 and muscle RING-finger 1 (MuRF1) involved in muscle protein breakdown.127 It remains to be investigated if factors released directly by the tumor, alterations in immune cell content/function or other mechanisms may underlie the depletion of the stromal cells in skeletal muscle during cancer cachexia. If these stromal cells are indeed FAPs, an alternative explanation for the cell content depletion during cachexia may relate to an increased adipogenic differentiation of the FAPs. In this regard the progression of cachexia has been associated with an increased amount of intramuscular lipid droplets,54 and although the source of these is not identified they could originate from FAPs.Since both SCs and FAPs may influence muscle homeostasis and growth, their interactions can be implicated in cachexia. In rodents, cancer cachexia is associated with muscle damage and deregulation of Pax7 expression in SCs and interstitial cells, through increased NF-133 In RA patients, loss of muscle strength is associated with RA duration rather than with chronological age, in contrast to the decline with age observed in the general population.134 This indicates a disease-related effect on muscle strength that surpasses the effect of aging. Increased IMAT, potentially originating from FAPs, is observed in the muscle of RA patients and this reduction in muscle density associates with greater joint destruction.52 Furthermore, type II fiber atrophy in RA patients has been reported,135 but compared to patients with osteoarthritis the absolute numbers of SC136 and their in vitro regenerative potential137 were not different in RA patients.Multiple chronic diseases are also associated with elevated systemic inflammation including RA.138 COPD is often accompanied by pronounced muscle wasting (atrophy of both type I and II fibers has been reported139) and metabolic dysfunction.140 Notably, the loss of muscle mass is an independent predictor of mortality in COPD patients.112 The alterations in immune cell function and the chronic inflammatory condition is believed to be a substantial contributor to the loss of muscle mass along with an impaired regenerative capacity and cellular apoptosis.140 SC content is not altered in the muscles of COPD patients compared to controls; however, primary SCs isolated from COPD patients displayed a delayed activation in culture and decreased expression of myosin heavy chain expression during myotube formation compared to controls.141 Interestingly, the increased number of central nuclei observed in the muscles of COPD patients with preserved muscle mass, as compared to those who lost muscle mass,142 suggests that the ability of the muscle to regenerate could attenuate the extent of COPD-related cachexia.COPD is another frequent cause of disease-related cachexia and premature death worldwide.Collectively, the potential role for SC and FAPs in relation to muscle loss due to chronic inflammatory diseases is a matter of current and future investigations .147 and even non-hypertrophying endurance exercise can induce proliferation of at least some SC populations.7 Although the potential impact of different training modalities on the immune-FAP-SC network are described separately below, it is important to note some remarkable differences, and the relative impact on the network, between physical exercise and the pathological conditions described in the previous paragraphs. For instance, in most pathological conditions FAPs are activated by physical injury, which triggers extensive changes in the microenvironment,  or by elevated systemic concentration of inflammatory cytokines ; by contrast, during training most of these signals are absent – except for the case of strenuous exercise – and the predominant changes occurring in exercised muscles are of metabolic  or biomechanical (contraction/relaxation cycles) nature. As FAPs occupy an interstitial position they are a great candidate as cell types that sense these changes and transmit them to SCs via specific cues. In this regard, recent work reported that SIRT1, a NAD(+)-dependent HDAC known as redox and nutrient sensor, promotes the metabolic switch from fatty acid oxidation to glycolysis during the SC transition from quiescence to proliferation.148 Moreover, SIRT1 regulates autophagic flux in SCs to cope with the high bioenergetic demands during the activation process.149 Finally, SIRT1 connects changes in SC metabolism with changes in the transcriptional machinery toward myogenic commitment of the SC.148 This reprogramming of cellular metabolism decreases intracellular NAD(+) levels and the activity of the HDAC SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Future studies should establish whether SIRT1 is activated by FAP-derived signals.Skeletal muscle is a highly plastic tissue that adapts to stimuli, by proportionally adjusting mass and strength (resistance training) or aerobic capacity (endurance training) in response to exercise. Both acute and prolonged resistance exercise stimulates the proliferation of SCs in healthy subjects,153 it is debated whether SCs possess a role in myofiber hypertrophy in the adult muscle.155 In earlier studies, irradiation was used to ablate SC activity, whereby overload induced hypertrophy in rodents was prevented,157 indicating a direct linkage between SCs and myofiber hypertrophy. However, a later study opposes this contention by showing an intact hypertrophic response in myofibers of SC-depleted muscles in rodents.26 Although these findings from conditional knockout mice seem to reject the hypothesis that SCs are essential for hypertrophy, more recent results indicate that the lack of SCs can attenuate myofiber hypertrophy in the later phases of an overload period.19 The latter is supported by other studies suggesting an important role for SC in myofiber growth and myonuclei accretion.159 In agreement, IGF-1, which can accentuate resistance training-induced muscle hypertrophy,160 may in part act through increased proliferation and differentiation of SC to support myofiber growth.161 This is supported by the increased expression of IGF-1 splice variants in human SCs following eccentric resistance exercise.162Although the role of SCs during myofiber regeneration has been extensively studied and reviewed,164 and following prolonged 168 resistance exercise in both young and old humans.89 In contrast, SC proliferation following resistance exercise may be impaired during ageing and in patients affected by chronic muscular disorders, with current evidence suggesting that this impairment originates from alterations in cues from the SC niche or the systemic environment.88 However, long-term resistance training can reverse the SC distribution in elderly muscle toward that of young muscle.169 Knowledge about the regulation of FAPs in relation to resistance training and hypertrophy is lacking, but in rodents the involvement of a SC-FAP interplay in successful muscle regeneration after muscle damage has been convincingly demonstrated.28 Although damage/regeneration is not a prerequisite for resistance training adaptations,170 the rodent findings combined with human SC data suggest that SC-FAP interplay may have a central role in resistance training adaptations  and GLUT4177 in skeletal muscle. Although increased energy expenditure through endurance training reduces accumulation of adipose tissue, insulin sensitivity and glucose tolerance are improved independent of weight loss.178 The SC response to endurance training has only been evaluated in a few human studies, and increased SC numbers are reported in most,181 but not all182 of these, mostly in the absence of muscle fiber hypertrophy. Recently, hypertrophy of both type I and IIa fibers was observed following 12 weeks of aerobic training, with a concomitant increase in SC number only in type I fibers.181 Furthermore, non-hypertrophying endurance exercise can induce proliferation of SC populations in hybrid fibers (type I/II) without effect on the SC content of type I or II fibers.7 Generally, these data indicate a role for SC proliferation and turnover in muscle maintenance even in the absence of fiber hypertrophy, which is in line with the suggested role for SC-FAP interplay in regulation of a healthy muscle.Endurance exercise increases insulin sensitivity and glucose tolerance,183 Thus, FAP heterogeneity and the dynamic transition from a physiological to compensatory or pathological subpopulation appears as a key issue to investigate in future studies. In particular, the anatomical derivation of FAPs might reveal important differences. For instance, in the presence of physical insults, the ensuing vessel injury or transient ischemia might direct a composition of FAPs that is phenotypically and functionally different from cells derived by the expansion of resident interstitial FAPs.FAPs are emerging as a 'cellular filter' between external perturbations  and the effectors of the muscle regeneration machinery – the SC. Depending on the nature of the perturbations FAPs appear to adopt specific phenotypic and functional properties indicating a highly heterogeneous cell population.Overall, the elucidation of the interplay between SCs, FAPs, their niche and immune cells might have an impact not only in the discovery of interventions toward restoring muscle function and correcting metabolic dysregulation in pathological conditions, but also to improve muscle anabolism and insulin sensitivity, which commonly decreases during aging, inactivity and certain disease states.


Human autoimmune inflammatory diseases are usually characterized by a chronic course. Antigen-induced experimental animal models for these diseases, on the other hand, typically present an acute or sub-acute course and after spontaneous recovery the animal may become resistant to reinduction of the disease. One explanation for this course of events may be that the antigen that induces the disease has a dual effect; on one hand it may serve as the target for the pathogenic immune attack whereas on the other it may lead to the development of protective immune mechanisms. Deficiency in these protective immune pathways may lead to the development of chronic autoimmune diseases. In the editorial below we will address this phenomenon by describing an example of one such antigen, HSP60 that has been shown to have a dual role in immune-mediated disorders, being involved in the induction and propagation of autoimmune diseases as well as in suppressing them .HSP60 is a highly conserved protein, which was initially characterized as a mitochondrial chaperone. Upon exposure to stress or immune activation, HSP60 is also found in the cytosol, cell surface, extracellular space and biological fluids. HSP60 activates innate and adaptive immune responses, and can function as an endogenous danger signal to the immune system. It binds to cell surface receptors, including CD14 and members of the TLR family [reviewed by Overexpression of HSP60 has been described in tissues of various inflammatory diseases suggesting a possible pathogenic role of this antigen in their pathogenesis. Increased expression of human HSP60 is observed in intestinal ulcer of Behcet's Disease (BD) , in inflPeripheral mononuclear cells (PBMCs) taken from patients with various inflammatory diseases can be activated by HSP60. For instance, HSP60 induces IFNγ secretion from atopic dermatitis patients' PBMCs  and PBMCSeveral supporting evidence for the pathogenicity of HSP60 have also been reported in experimental animal models of inflammatory diseases. HSP60 is upregulated in animal model of glomerulonephritis (NTN) as shown by increased HSP60 secretion in the urine and into the extracellular space of the diseased kidney. In addition, administration of HSP60 aggravates the disease in the NTN model and in experimental autoimmune uveitis in mice , 6.Contrary to its role as a target for pathogenic autoimmune inflammatory processes, HSP60 has been shown to activate immunoregulatory pathways that may lead to suppression of these diseases. The finding that immunization with whole MT HSP60 prior to induction of adjuvant arthritis (AA) leads to protection against the disease attributed many experiments aimed at revealing the mechanism of this phenomenon, using mammalian and MT whole protein or synthetic peptides of the proteins. HSP60 based treatment ameliorated or suppressed several animal models of inflammatory diseases such as AA, collagen induced arthritis, insulin-dependent diabetes mellitus and atherosclerosis. Preventive as well as therapeutic HSP60 peptide treatment included various regimens, modes of administration and adjuvants and resulted in suppression of inflammatory cytokines, elevation of Th2 cytokines, and induction of regulatory T cells (CD4+CD25+Foxp3+) [In vitro studies performed on human PBMCs were also indicative of an anti-inflammatory effect of Prozumab: Treatment of naïve PBMCs resulted in suppression of anti CD3-induced INFγ and IL-6 secretion [Interestingly, the presence of antibodies against peptide 6, a specific peptide derived from MT-HSP60, generated resistance to the induction of AA. We have recently shown that Prozumab, a humanized mAb targeting HSP60, is effective in ameliorating animal models of autoimmune inflammatory diseases such as RA and IBD. ecretion .Immune reactivity against HSP60 has been found to have a disease suppressing effect in human autoimmune inflammatory diseases as well. The presence of self-HSP60-specific T cell responses in JIA patients correlates with a benign disease course. Self-HSP-specific T cell responses have also been reported to be immunoregulatory in various other autoimmune diseases, such as RA and JDM, by the production of anti-inflammatory cytokines such as interleukin (IL)-10, IL-4 and TGF-β . A phaseIn summary HSP60 has been identified as a target for both pathogenic as well as protective immune mechanisms in many experimental and human inflammatory diseases. The upregulation of this protein at the sites of inflammation on one hand and its ability to skew the immune system towards suppression of disease on the other hand may explain the spontaneous remission of many experimental models of these inflammatory diseases. Understanding the putative mechanisms of disease suppression in animal models may lead to novel immunotherapeutic approaches to human autoimmune inflammatory diseases.


Plant microRNAs (miRNAs) are a class of small non-coding RNAs which target and regulate the expression of genes involved in several growth, development, and metabolism processes. Recent researches have shown involvement of miRNAs in the regulation of uptake and utilization of nitrogen (N) and phosphorus (P) and more importantly for plant adaptation to N and P limitation conditions by modifications in plant growth, phenology, and architecture and production of secondary metabolites. Developing strategies that allow for the higher efficiency of using both N and P fertilizers in crop production is important for economic and environmental benefits. Improved crop varieties with better adaptation to N and P limiting conditions could be a key approach to achieve this effectively. Furthermore, understanding on the interactions between N and P uptake and use and their regulation is important for the maintenance of nutrient homeostasis in plants. This review describes the possible functions of different miRNAs and their cross-talk relevant to the plant adaptive responses to N and P limiting conditions. In addition, a comprehensive understanding of these processes at molecular level and importance of biological adaptation for improved N and P use efficiency is discussed. In the past half century, there has been a marked increase in food production allowing a significant decline in food shortages worldwide although the world population has doubled during this time  and phosphorus (P) are key elements for crop production and are major constraints for plant growth, development, and yield since intensive crop production relies heavily on a large input of these fertilizers  recent progress in understanding the mechanism of N and P acquisition, assimilation and mobilization in plant; (ii) elaborate on how plants respond to N and P deficiency and in what ways miRNAs contribute to this physiological adaptation; and (iii) discuss the involvement of plant miRNAs in the crosstalk between N and P under limiting conditions.−3), ammonium (NH+4) or urea from the soil, however, NO−3 is preferred form for most arable plants , nitrate transporter 2 (NRT2), the chloride channel (CLC), and slow anion channel associated homologs (SLAC/SLAH)   involved in Pi loading in xylem vessels in roots  a negative regulator of Pi uptake , whose protein hydrolyses the inactive auxin derivative indole-3-acetic acid alanine and releases bioactive auxin, for root development during high osmotic stress , ATAF (Arabidopsis Transcription Activation Factor), CUC (CUp shaped Cotyledon)  a ubiquitous transcription factor consisting of 3 subunits A, B, and C, some of which bind to promoter regions and regulate expression of the nitrate transporters AtNRT2.1 and AtNRT1.1  plants  suggesting the regulatory role of miR169 in N uptake and remobilization. The chl1 mutant plants showed a reduced expression of AtNRT1.1 and its phenotype mimicked over-expressed MIR169a transgenic plants. These indicate that lower expression of miR169 is an adaptive response of plants under N limiting conditions.MiRNA, miR169a is the only candidate reported so far, regulating the expression of key target N transporters under N limiting conditions. The MiR169 family in Arabidopsis consists of 14 members, among these miR169a is the main contributor to the total miR169 level  and ammonium transporter (AMT1.5) genes were also induced  an important radical scavenger that protects plants from oxidative stress damage  family of transcription factors whose expressions were synergistically associated with anthocyanin biosynthesis  gene which encodes a ubiquitin conjugating E2 enzyme  transcripts  or PHOSPHATE TRANSPORTER1:1 (PHT1:1) genes rescued the early senescence phenotype in nla mutant in response to N starvation. The early senescence phenotype of the nla mutant indeed was caused by excessive Pi accumulation similar to pho2 mutant plants  non-coding RNA transcript, which results in the production of tasi-RNAs  one of the dominant TAS4 siRNAs, targets a set of MYB transcription factors PAP1/MYB75, PAP2/MYB90, and MYB113, which regulate genes in the anthocyanin biosynthesis pathway and leaf senescence  and its target genes, which modulates anthocyanin accumulation. Pi starvation might activate expression of these MYB transcription factors leading to enhanced biosynthesis of anthocyanins. In addition, increased levels of PAP1/MYB75 trigger the production of TAS4-siR81 (−) via the activation of miR828 or TAS4, which in turn suppressed the expression of MYB transcription factors      under Pi limitation could probably be mediated by leaf cytokinin concentrations since low cytokinin levels might cause decrease in nitrate reductase activity. Transcriptional profiling of Arabiopsis and maize shed more light on this aspect. Low Pi supply was shown to reduce expression of nitrate reductase genes in Arabidopsis  and micro-nutrients  (Schlüter et al., Current knowledge of the involvement of various miRNAs for the regulation of N and P uptake, assimilation and utilization and plant adaptation to both N and P limitation conditions has been reviewed here along with how these processes affects the modifications in shoot and root growth, development, and architecture, effects on vegetative and reproductive phase and production of secondary metabolites. Nevertheless, a holistic approach to study the interaction of N and P along with other macro- and micro-nutrient elements and the involvement of miRNAs would be of much benefit and would thus require further research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The hair bundles of cochlear hair cells play a central role in the auditory mechano-electrical transduction (MET) process. The identification of MET components and of associated molecular complexes by biochemical approaches is impeded by the very small number of hair cells within the cochlea. In contrast, human and mouse genetics have proven to be particularly powerful. The study of inherited forms of deafness led to the discovery of several essential proteins of the MET machinery, which are currently used as entry points to decipher the associated molecular networks. Notably, MET relies not only on the MET machinery but also on several elements ensuring the proper sound-induced oscillation of the hair bundle or the ionic environment necessary to drive the MET current. Here, we review the most significant advances in the molecular bases of the MET process that emerged from the genetics of hearing. The ability of vertebrates to maintain their balance and sense sound vibrations is decisive for their survival. Although vertebrates live in various environments, they all make use of the same organelle, the hair bundle, that transduces mechanical information into an electrical signal in sensory hair cells. Hair cells are present in the neuromasts of lateral lines in fish and amphibian larvae, where they detect water movement; in the vestibular end organs, where they detect linear and angular acceleration; and in the auditory organs, where they detect sound pressure waves Fig. . Hair cehttp://hereditaryhearingloss.org). In addition, many more genes are involved in syndromic forms of sensorineural deafness. Pathophysiological studies rely on multidisciplinary approaches that include invasive exploration techniques in animal models. Mouse models offer substantial possibilities for genetic manipulation and have proven to be highly relevant for the understanding of human auditory defects because mutations in mouse orthologues of the genes associated with deafness in humans faithfully mimic the sensory defect in most cases.The first physical description of hair bundle functioning was reported in the late 1970s. However, the small number of hair cells in the inner ear (a few thousands) hampered molecular advances, as opposed to other sensory organs like the eye, which contains more than 100 million photoreceptor cells. In the early 1990s, human genetics, the efficiency of which is independent from the number of hair cells, emerged as the best approach to identify molecules involved in MET. Studies focused largely on the cochlea, the mammalian auditory organ, rather than on the vestibular organs because vestibular defects in humans are often compensated by the visual and proprioceptive systems. In addition, deafness is the most frequent sensory defect at birth (approximately one out of 700 newborns is affected by severe or profound hearing impairment). Currently, more than 120 deafness loci have been characterised, and around 80 genes responsible for isolated (non-syndromic) forms of sensorineural deafness have been identified  because their cochlear sensory epithelium continues to develop after birth. At birth, the first steps of hair bundle growth have already occurred. All the V- or U-shaped hair bundles are aligned, and their vertices point towards the cochlear abneural edge  , 7519, 7Vlgr1 knock-out mice, ankle links are absent and abnormally shaped hair bundles are apparent at P2 [The study of the genes responsible for Usher syndrome has been especially informative for our understanding of hair-bundle development. Usher syndrome (USH) is an autosomal recessive disorder that associates congenital hearing impairment with delayed onset retinitis pigmentosa eventually leading to blindness. This disorder has three clinical subtypes. USH1, the most severe form, is characterised by severe to profound congenital deafness, constant vestibular dysfunction and prepubertal onset retinitis pigmentosa. By contrast, USH2 involves only moderate to severe hearing impairment and no vestibular dysfunction. USH3 is distinguished from USH2 by the progressiveness of the hearing impairment and the occasional presence of vestibular dysfunction  actin-nucleating proteins that promote initiation of actin polymerisation, (2) actin-capping proteins that prevent the barbed end from incorporating actin monomers, (3) actin-bundling proteins that cross-link parallel actin filaments, (4) actin side-binding proteins, (5) actin-monomer-sequestering proteins, (6) actin-severing proteins that split actin filaments and (7) actin molecular motors. Mutations in various actin and actin-interacting proteins of these categories cause defects in stereocilia structure  and faster at the very tip  than in the core of stereocilia [The molecular processes that determine stereocilia differential elongation in different rows are still unknown. However, several studies, with conflicting results, have addressed the issue of steady-state actin renewal in mature hair bundles. A treadmilling process was first proposed to ensure the renewal of actin monomers in stereocilia filaments. When actin fused to the green fluorescent protein (actin-GFP) was overexpressed in cells, the actin core renewal speed was unexpectedly fast (~48 h) , and turreocilia . HoweverTMC1 cause deafness in humans [Tmc1 and Tmc2 have no MET currents [Tmc1, Tmc2, and mutated forms of Tmc1 in the hair cells of these double knock-out mice [2+ ions. This suggests that TMC1 and TMC2 are pore-forming subunits of the MET channel [High-speed imaging of the calcium influx through MET channels in cochlear hair cells has shown that these channels are located at the tips of the short and middle row stereocilia but not in tall row stereocilia. MET channels would therefore be located at the basal ends of the tip links . The moln humans  and innecurrents . In addiout mice  modifies channel , 106. Ho channel . Therefo channel  for comm channel  for revi channel .PCDH15 mutation that affects only the CD2 isoform was also found to lead to profound deafness without vestibular defects in human patients. Because mutant mice for CD1 or CD3 are not hearing-impaired [tetraspan membrane protein of hair cell stereocilia (TMHS), have been shown to participate in molecular complexes associated with the lower and upper tip-link insertion points  and protocadherin-15 (USH1F), respectively , 5, 99. impaired , CD2 wouimpaired . Three ol itself  Several features indicate that the MET machinery is subjected to tension even in the absence of sound stimuli. Stereocilia tips of short and middle rows have a prolate shape that is thought to be caused by the resting tension exerted by the tip link on the plasma membrane Fig. . Direct Myo7a4626SB mice show characteristics similar to the abnormal currents observed in TMC1 and TMC2 defective mutants when hair bundles are pushed in the inhibitory direction, which suggests that the recorded MET currents in Myo7a4626SB mice would not be gated by tip links (see above) [The control of the holding tension on the MET machinery depends on the anchoring of the MET channel and the tip link to the actin cytoskeleton. The tip-link tension can be modulated by sliding of the tip-link upper end anchoring point along actin filaments. This mechanism is thought to contribute to the adaptation process that is reflected in the decline in the transduction current evoked by a step displacement of the hair bundle in vitro , 55, 111e above) , 129. The above) , a mechae above) . The dyne above)  and myosIn the cochlea, hair bundles are covered by an acellular gel composed of several types of collagen and non-collagenous glycoproteins called the tectorial membrane. Like the organ of Corti, the tectorial membrane runs along the cochlear duct. It is attached on its medial side to the spiral limbus, and on the other side, it is in firm contact with the tips of the tallest OHC stereocilia row. Notably, hair bundles of IHCs are free standing under the tectorial membrane. Upon sound stimulation, the shear movement between the basilar membrane and the tectorial membrane drives hair bundle oscillations. Many proteins involved in the composition of the tectorial membrane or required for its attachment to hair cells are encoded by genes associated with deafness. The study of mice mutant for these genes has shed new light on the different roles played by the tectorial membrane in auditory MET.TectaΔENT/ΔENT mice, which causes the tectorial membrane to detach from the surface of the organ of Corti, led to the conclusion that the elasticity of the tectorial membrane has little influence on the amplitude and phase of deflexion of OHC stereocilia at the characteristic frequency. Rather, at this frequency, the tectorial membrane probably behaves mostly as an inert mass on which OHC stereocilia can react, ensuring that the OHCs respond to sound stimulation with the proper gain and timing [Tecta Y1870C mutation pinpointed a second mechanical role of the tectorial membrane. Although OHC MET activity is normal in TectaY1870C/+mice, neural thresholds are markedly high, indicating that the tectorial membrane also plays a critical role in driving the hair bundles of IHCs [Tectb−/− mice). Basilar membrane and neural tunings are both sharper than normal in these mice, suggesting that the tectorial membrane also influences the longitudinal spread of sound-induced excitation along the cochlea [Six non-collagenous glycoproteins have been found in the tectorial membrane: α-tectorin, β-tectorin, otogelin, otogelin-like, CEACAM16, and otolin , 46, 97 d timing . Subsequ of IHCs . Three k of IHCs . A third cochlea . Several cochlea . Notably cochlea , 215 . There y 150 mM , 186. Thy 150 mM  drives te Kcnj10 , 242, Kce Kcnj10 , 116, ane Kcnj10 , 190 K+ er NKCC1 , 52. Los+ ions that flow out of the hair cells in their basolateral region has been suggested, although such a process remains to be established. Mutations in the connexin 26 gene (CX26/GJB2) [Gjb2 in the mouse organ of Corti leads to the degeneration of sensory cells and supporting cells. This phenotype has been attributed to defects in the gap junctions that would be involved in the recycling of K+ ions released at the base of hair cells. In addition, the endocochlear potential builds up but fails to be maintained in these mice, probably as a consequence of the loss of tight junctions between hair cells and their supporting cells [CX30/GJB6) is contiguous with CX26/GJB2 on human chromosome 13 (mouse chromosome 14) and is also expressed in the cochlea [GJB6 have been reported in deaf patients [Gjb6 knock-out mouse model led to the mistaken conclusion that inactivation of Gjb6 alone could lead to deafness [Gjb6 gene, both in humans and in mice, also impaired the expression of the Gjb2 gene [Gjb2 in the same Gjb6 knock-out mouse model restored hearing [Gjb6 knock-out mouse mutant, in which sufficient expression of Gjb2 was preserved. Thus, the cause of deafness after GJB6 deletion is the low expression of GJB2 due to the co-deletion of its putative regulatory element [Gjb6 knock-out mouse model [+ ions: KCNQ4 [KCC3 [KCC4 [KCNQ4 encodes a K+ channel subunit and KCC3 and KCC4 encode K+–Cl− cotransporters. Kcnq4 is located at the base of mature OHCs and mediates a voltage-activated K+ current that is already active at the resting membrane potential [Kcnq4−/− mice, this current is abolished, leading to a slow degeneration of OHCs, which probably results from their chronic depolarisation [+ ions from the hair cells’ pericellular space into supporting cells, where these ions would enter the gap junction recycling pathway. Hair cells undergo degeneration both in Kcc3 knock-out mice and Kcc4 knock-out mice, although degeneration occurs earlier in the former than in the latter [The existence of a recycling, through an intercellular gap junction network, K26/GJB2)  are the ng cells . The con cochlea , 65. Delpatients , 156, 47deafness . In factjb2 gene , 126, an element , 32. In se model  fails tos: KCNQ4 , KCC3 [2Q4 [KCC3 , and KCCC3 [KCC4 . KCNQ4 eotential , 103. Inrisation . Kcc3 ane latter , 28.+ concentration and of the endocochlear potential requires strong apical cell–cell junctions in the epithelia lining the endolymphatic compartment of the cochlea, especially in the mechanically stressed sensory epithelium. Junctions between OHCs and their supporting cells are probably subjected to the highest amount of mechanical stress, due to the motion of the sensory epithelium and forces generated by OHC electromotility. These junctions are composed of an atypical combination of tight junctions and adherens junctions [The maintenance of the high endolymphatic Kunctions  containiunctions , 17, 220cre mouse that enables delayed conditional knocking-out of proteins, offers a unique opportunity to distinguish the role of a particular protein in the mature hair bundle from its possible role during development [cre knock-in lines need to be developed to offer a larger panel of genetic tools at different developmental time points and in specific hair cell types. Studies that apply the same strategy to known components of the MET machinery should clarify their respective roles in the mature hair bundle.There has been for the past 10 years remarkable progress in the identification of proteins and protein complexes that constitute the MET machinery. However, the composition of the central element of this machinery, the MET channel, is still under debate. Various strategies to characterise the molecular identity of this channel have been hindered by the limited amount of available material, the multifunction of particular molecules in the developing and mature hair bundle and by the current inability to reconstitute the MET machinery in a controlled exogenous system , will be no exception .2+ pump PMCA2 , which has also been implicated in mouse and human deafness [The retina is also affected by USH. The search for new binding partners of USH1 proteins in the retina is facilitated by the abundance of photoreceptor cells and may help to find new elements of the cochlear MET machinery. Until recently, the pathogenesis of the retinitis pigmentosa observed in USH1 patients remained elusive because mouse models for USH1 genetic forms do not reproduce the retinal degeneration phenotype of humans. The study of USH1 protein distribution in the macaque retina revealed the structural origin of this discrepancy . In primdeafness , 202. Fudeafness .2+ pump PMCA2 [Tecta and Tectb mutations to uncover the various roles of the tectorial membrane in MET. This mutational approach is to be extended with the arrival of more powerful and faster tools to engineer mouse mutants, such as the clustered regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) system to perform genome sequence specific-editing. The CRISPR/Cas system allows the one-step generation of mice carrying mutations in several genes simultaneously [Human genetics has uncovered numerous molecules involved in hair bundle development and function. Each of these molecules provides a starting point to decipher whole molecular complexes. Clearly, the probability of finding new genes associated with deafness in patients from newly recruited families decreases with time, and as a consequence, this approach may cease to provide new candidates at some point. Moreover, lethal mutations cannot be detected by the human genetics approach, which may make some essential components of the MET machinery difficult to identify with this approach. Thus, complementary strategies need to be developed to complete the picture of the molecular networks in the hair bundle. In addition to the yeast two-hybrid technique that can find new interacting components of a molecular complex step by step , 237, remp PMCA2 , 202). Laneously . This syaneously , and henaneously , 88, and