Murine Myoblasts Exposed to SYUIQ-5 Acquire Senescence Phenotype and Differentiate into Sarcopenic-Like Myotubes, an In Vitro Study.

The musculoskeletal system is one of the most affected organs by aging that correlates well with an accumulation of senescent cells as for other multiple age-related pathologies. The molecular mechanisms underpinning muscle impairment because of senescent cells are still elusive. The availability of in vitro model of skeletal muscle senescence is limited and restricted to a small panel of phenotypic features of these senescent cells in vivo. Here, we developed a new in vitro model of senescent C2C12 mouse myoblasts that, when subjected to differentiation, the resulting myotubes showed sarcopenic features. To induce senescence, we used SYUIQ-5, a quindoline derivative molecule inhibitor of telomerase activity, leading to the expression of several senescent hallmarks in treated myoblasts. They had increased levels of p21 protein accordingly with the observed cell cycle arrest. Furthermore, they had enhanced SA-ßgalactosidase enzyme activity and phosphorylation of p53 and histone H2AX. SYUIQ-5 senescent myoblasts had impaired differentiation potential and the resulting myotubes showed increased levels of ATROGIN-1 and MURF1, ubiquitin ligases components responsible for protein degradation, and decreased mitochondria content, typical features of sarcopenic muscles. Myotubes differentiated from senescent myoblasts cultures release increased levels of MYOSTATIN that could affect skeletal muscle cell growth. Overall, our data suggest that a greater burden of senescent muscle cells could contribute to sarcopenia. This study presents a well-defined in vitro model of muscle cell senescence useful for deeper investigation in the aging research field to discover new putative therapeutic targets and senescence biomarkers associated with the aged musculoskeletal system.

Exploring cellular senescence in the musculoskeletal system: Any insights for biomarkers discovery?

The locomotor system comprises skeletal muscles and bones with active metabolism and cellular turnover. Chronic locomotor system disorders gradually arising with aging are inversely associated with the correct function of bone and muscles. Senescent cells appear more frequently in advanced ages or pathological conditions, and the accumulation of senescent cells in muscle tissue negatively correlates with muscle regeneration, which is crucial for maintaining strength and preventing frailty. Senescence in the bone microenvironment, osteoblasts, and osteocytes affects bone turnover favoring osteoporosis. It is likely that in response to injury and age-related damage over the lifetime, a subset of niche cells accumulates oxidative stress and DNA damage beyond the threshold that primes the onset of cellular senescence. These senescent cells may acquire resistance to apoptosis that, combined with the weakened immune system, results in impaired clearance of senescent cells and their accumulation. The secretory profile of senescent cells causes local inflammation, further spreading senescence in neighboring niche cells and impairing tissue homeostasis. The resulting impairment of turnover/tissue repair in the musculoskeletal system reduces the efficiency of the organ in response to environmental needs that finally lead to functional decline. Management of the musculoskeletal system at the cellular level can benefit the quality of life and reduce early aging. This work discusses current knowledge of cellular senescence of musculoskeletal tissues to conclude with biologically active biomarkers effective enough to reveal the underlying mechanisms of tissue flaws at the earliest possible.

The epilepsy-associated protein PCDH19 undergoes NMDA receptor-dependent proteolytic cleavage and regulates the expression of immediate-early genes.

Protocadherin-19 (PCDH19) is a synaptic cell-adhesion molecule encoded by X-linked PCDH19, a gene linked with epilepsy. Here, we report a synapse-to-nucleus signaling pathway through which PCDH19 bridges neuronal activity with gene expression. In particular, we describe the NMDA receptor (NMDAR)-dependent proteolytic cleavage of PCDH19, which leads to the generation of a PCDH19 C-terminal fragment (CTF) able to enter the nucleus. We demonstrate that PCDH19 CTF associates with chromatin and with the chromatin remodeler lysine-specific demethylase 1 (LSD1) and regulates expression of immediate-early genes (IEGs). Our results are consistent with a model whereby PCDH19 favors maintenance of neuronal homeostasis via negative feedback regulation of IEG expression and provide a key to interpreting PCDH19-related hyperexcitability.

Bone-to-Brain: A Round Trip in the Adaptation to Mechanical Stimuli.

Besides the classical ones (support/protection, hematopoiesis, storage for calcium, and phosphate) multiple roles emerged for bone tissue, definitively making it an organ. Particularly, the endocrine function, and in more general terms, the capability to sense and integrate different stimuli and to send signals to other tissues, has highlighted the importance of bone in homeostasis. Bone is highly innervated and hosts all nervous system branches; bone cells are sensitive to most of neurotransmitters, neuropeptides, and neurohormones that directly affect their metabolic activity and sensitivity to mechanical stimuli. Indeed, bone is the principal mechanosensitive organ. Thanks to the mechanosensing resident cells, and particularly osteocytes, mechanical stimulation induces metabolic responses in bone forming (osteoblasts) and bone resorbing (osteoclasts) cells that allow the adaptation of the affected bony segment to the changing environment. Once stimulated, bone cells express and secrete, or liberate from the entrapping matrix, several mediators (osteokines) that induce responses on distant targets. Brain is a target of some of these mediator [e.g., osteocalcin, lipocalin2, sclerostin, Dickkopf-related protein 1 (Dkk1), and fibroblast growth factor 23], as most of them can cross the blood-brain barrier. For others, a role in brain has been hypothesized, but not yet demonstrated. As exercise effectively modifies the release and the circulating levels of these osteokines, it has been hypothesized that some of the beneficial effects of exercise on brain functions may be associated to such a bone-to-brain communication. This hypothesis hides an interesting clinical clue: may well-addressed physical activities support the treatment of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases?

A Physically Active Status Affects the Circulating Profile of Cancer-Associated miRNAs.

Circulating miRNAs are ideal diagnostics and prognostics biomarkers in cancer since altered levels of specific miRNAs have been associated to development/progression of several cancers. Physical activity is a recognized preventive strategy against several cancers, but it may also modify the baseline levels of cancer-associated miRNAs and, hence, may act as a confounding pre-analytical variable. This study aimed at understanding whether physical activity-dependent changes in cancer-associated circulating miRNAs profile could act as a confounding variable. A panel comprising 179 miRNAs was assayed in plasma from 20 highly trained and 10 sedentary men. RT-qPCR data were analyzed with the 2-2ΔΔCT methods and normalized on hsa-miR-320d, as determined by bioinformatics analysis. miRNAs associated with the diagnosis of the most prevalent cancers were considered. Only those miRNAs, relevantly associated with cancers, found ≥2-fold up- or downregulated in highly trained subjects compared to sedentary were disclosed. The results reveal that chronic physical activity determined modifications altering the baseline level of several cancer-associated miRNAs and, hence, their diagnostic and prognostic potential. In conclusion, based on our results, a physically active status emerges as an important pre-analytical variable able to alter the basal level of circulating miRNAs, and these alterations might be considered as potentially misleading the analytical output.

SRF and SRFΔ5 Splicing Isoform Recruit Corepressor LSD1/KDM1A Modifying Structural Neuroplasticity and Environmental Stress Response.

Ten to 20% of western countries population suffers from major depression disorder (MDD). Stressful life events represent the main environmental risk factor contributing to the onset of MDD and other stress-related neuropsychiatric disorders. In this regard, investigating brain physiology of stress response underlying the remarkable individual variability in terms of behavioral outcome may uncover stress-vulnerability pathways as a source of candidate targets for conceptually new antidepressant treatments. Serum response factor (SRF) has been addressed as a stress transducer via promoting inherent experience-induced Immediate Early Genes (IEGs) expression in neurons. However, in resting conditions, SRF also represents a transcriptional repressor able to assemble the core LSD1/CoREST/HDAC2 corepressor complex, including demethylase and deacetylase activities. We here show that dominant negative SRF splicing isoform lacking most part of the transactivation domain, namely SRFΔ5, owes its transcriptional repressive behavior to the ability of assembling LSD1/CoREST/HDAC2 corepressor complex meanwhile losing its affinity for transcription-permissive cofactor ELK1. SRFΔ5 is highly expressed in the brain and developmentally regulated. In the light of its activity as negative modulator of dendritic spine density, SRFΔ5 increase along with brain maturation suggests a role in synaptic pruning. Upon acute psychosocial stress, SRFΔ5 isoform transiently increases its levels. Remarkably, when stress is chronically repeated, a different picture occurs where SRF protein becomes stably upregulated in vulnerable mice but not in resilient animals. These data suggest a role for SRFΔ5 that is restricted to acute stress response, while positive modulation of SRF during chronic stress matches the criteria for stress-vulnerability hallmark.

The Role of Protocadherin 19 (PCDH19) in Neurodevelopment and in the Pathophysiology of Early Infantile Epileptic Encephalopathy-9 (EIEE9).

PCDH19 is considered one of the most clinically relevant genes in epilepsy, second only to SCN1A. To date about 150 mutations have been identified as causative for PCDH19-female epilepsy (also known as early infantile epileptic encephalopathy-9, EIEE9), which is characterized by early onset epilepsy, intellectual disabilities, and behavioral disturbances. Although little is known about the physiological role of PCDH19 and the pathogenic mechanisms that lead to EIEE9, in this review, we will present latest researches focused on these aspects, underlining protein expression, its known functions and the mechanisms by which the protein acts, with particular interest in PCDH19 extracellular and intracellular roles in neurons.

The female epilepsy protein PCDH19 is a new GABAAR-binding partner that regulates GABAergic transmission as well as migration and morphological maturation of hippocampal neurons.

The PCDH19 gene (Xp22.1) encodes the cell-adhesion protein protocadherin-19 (PCDH19) and is responsible for a neurodevelopmental pathology characterized by female-limited epilepsy, cognitive impairment and autistic features, the pathogenic mechanisms of which remain to be elucidated. Here, we identified a new interaction between PCDH19 and GABAA receptor (GABAAR) alpha subunits in the rat brain. PCDH19 shRNA-mediated downregulation reduces GABAAR surface expression and affects the frequency and kinetics of miniature inhibitory postsynaptic currents (mIPSCs) in cultured hippocampal neurons. In vivo, PCDH19 downregulation impairs migration, orientation and dendritic arborization of CA1 hippocampal neurons and increases rat seizure susceptibility. In sum, these data indicate a role for PCDH19 in GABAergic transmission as well as migration and morphological maturation of neurons.

Biosynthesis of Astrocytic Trehalose Regulates Neuronal Arborization in Hippocampal Neurons.

Trehalose is a nonreducing disaccharide that has recently attracted much attention because of its ability to inhibit protein aggregation, induce autophagy, and protect against dissections and strokes. In vertebrates, the biosynthesis of trehalose was long considered absent due to the lack of annotated genes involved in this process. In contrast, trehalase (TreH), which is an enzyme required for the cleavage of trehalose, is known to be conserved and expressed. Here, we show that trehalose is present as an endogenous metabolite in the rodent hippocampus. We found that primary astrocytes were able to synthesize trehalose and release it into the extracellular space. Notably, the TreH enzyme was observed only in the soma of neurons, which are the exclusive users of this substrate. A statistical analysis of the metabolome during different stages of maturation indicated that this metabolite is implicated in neuronal maturation. A morphological analysis of primary neurons confirmed that trehalose is required for neuronal arborization.

Biosynthesis of glycerol phosphate is associated with long-term potentiation in hippocampal neurons.

INTRODUCTION: Neurons have a very high energy requirement, and their metabolism is tightly regulated to ensure delivery of adequate substrate to sustain neuronal activity and neuroplastic changes. The mechanisms underlying the regulation of neuronal metabolism, however, are not completely clear. OBJECTIVE: The objective of this study was to investigate the central carbon metabolism in neurons, in order to identify the regulatory pathways governing neuronal anabolism and catabolism. METHODS: Here we first have applied MS-based endometabolomics to elucidate the metabolic dynamics in cultured hippocampal primary neurons. Using nanoLC-ESI-LTQ Orbitrap MS approach followed by statistical analysis, we measure the dynamics of uniformly labeled 13C-glucose entering neurons. We adapted the method by coupling offline patch-clamp setup with MS to confirm findings in vivo. RESULTS: According to non-parametric statistical analysis of metabolic dynamics, in cultured hippocampal neurons, the glycerol phosphate shuttle is active and correlates with the metabolic flux in the pentose phosphate pathway. In the hippocampus, glycerol-3-phosphate biosynthesis was activated in response to long-term potentiation together with the upregulation of glycolysis and the TCA cycle, but was inactive or silenced in basal conditions. CONCLUSIONS: We identified the biosynthesis of glycerol-3-phosphate as a key regulator in mechanisms implicated in learning and memory. Notably, defects in enzymes linked with the glycerol phosphate shuttle have been implicated in neurological disorders and intellectual disability. These results could improve our understanding of the general mechanisms of learning and memory and facilitate the development of novel therapies for metabolic disorders linked with intellectual disability.

Myosin IXa Binds AMPAR and Regulates Synaptic Structure, LTP, and Cognitive Function.

Myosin IXa (Myo9a) is a motor protein that is highly expressed in the brain. However, the role of Myo9a in neurons remains unknown. Here, we investigated Myo9a function in hippocampal synapses. In rat hippocampal neurons, Myo9a localizes to the postsynaptic density (PSD) and binds the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) GluA2 subunit. Myo9a(+/-) mice displayed a thicker PSD and increased levels of PSD95 and surface AMPAR expression. Furthermore, synaptic transmission, long-term potentiation (LTP) and cognitive functions were impaired in Myo9a(+/-) mice. Together, these results support a key role for Myo9a in controlling the molecular structure and function of hippocampal synapses.

The neurobiology of X-linked intellectual disability.

X-linked intellectual disability (XLID) affects 1% to 3% of the population. XLID subsumes several heterogeneous conditions, all of which are marked by cognitive impairment and reduced adaptive skills. XLID arises from mutations on the X chromosome; to date, 102 XLID genes have been identified. The proteins encoded by XLID genes are involved in higher brain functions, such as cognition, learning and memory, and their molecular role is the subject of intense investigation. Here, we review recent findings concerning a representative group of XLID proteins: the fragile X mental retardation protein; methyl-CpG-binding protein 2 and cyclin-dependent kinase-like 5 proteins, which are involved in Rett syndrome; the intracellular signaling molecules of the Rho guanosine triphosphatases family; and the class of cell adhesion molecules. We discuss how XLID gene mutations affect the structure and function of synapses.

Identification, isolation and expansion of myoendothelial cells involved in leech muscle regeneration.

Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells.