MicroRNA e molecole proteiche come biomarcatori dei sottotipi clinici di SLA

Background: Amyotrophic lateral sclerosis (ALS) is a rare, progressive, neurodegenerative disorder caused by degeneration of upper and lower motoneuron. The disease process leads to progressive muscle atrophy, weakness, fasciculations and spasticity. About two-thirds of ALS patients have a spinal form of the disease (spinal ALS), which involves the limbs. The spinal ALS has an upper or lower limb onset that starts with weakness and muscle atrophy in hands or feet whereas one-thirds of patients have a bulbar onset (bulbar ALS) usually presenting with dysarthria and dysphagia. The spinal ALS leads to death after 3-5 years from onset while the bulbar ALS die about after 2-3 years from onset. Incidence and prevalence of ALS are higher in men than in women. Male e female gender influences the clinical features of the disease: man have a greater likelihood spinal regions onset whereas women have onset at an older age. The effects of physical exercise and rehabilitation in patients with ALS are still debated: if a patient is inactive, the loss of exercise or muscle training leads to muscle atrophy and disuse that contribute to deterioration in addition to the weakness and muscle atrophy caused by denervation. MicroRNAs (miRNAs) are small non-coding RNA molecules (19-24 nucleotides), highly conserved, which regulate genes expression at the post transcriptional level. The up-regulation of a specific miRNA determines decreasing expression of the corresponding protein product. Aim: The aim of this work is the identification of new molecular and biochemical biomarkers to facilitate the study of the various subtypes of ALS. We will study if they provide information on the onset and severity of disease and they might be used as indicators of a therapeutic response in rehabilitation. We will investigate the levels of both muscle-specific (myomiRNAs) and inflammatory/angiogenic microRNAs and other molecular signatures (Myostatin, Follistatin). MicroRNAs will be analized and compared with morphometric study in muscle biopsies of ALS patient and controls. In serum, we will investigate the difference between spinal and bulbar ALS whereas, in muscle we will analyze the difference between male and female patients and in the effect of different age of onset of the disease on microRNAs. Methods: In 14 ALS patients (10 spinal and 4 bulbar) we measure the serum levels of muscle-specific miR-1, miR-206, miR-133a/b, miR-27a and the inflammatory miRNAs miR-155, miR-146a, miR-221 and miR-149*, and the expression of Myostatin and Follistatin, which are two antagonist regulator of the muscle growth. Morphometric analysis of muscle fiber size is done to correlate muscle atrophy with biochemical-molecular parameters. In 16 muscles ALS biopsies (9 male and 7 female) we analyze the levels of myomiRNAs and inflammatory miRNAs. The circulating levels of myomiRNAs are analysed also in 12 ALS patients (8 male and 4 female) after rehabilitation treatment of 8 weeks in IRCCS San Camillo (Venice). Results: In ALS patients the expression of serum miR-206 and miR-133 is significantly (p<0.005) increased and miR-27a is significantly (p<0.005) reduced compared to control, also the miRNA profile is significantly higher between spinal versus bulbar ALS. Myostatin/Follistatin ratio was significantly higher in ALS versus control and are higher in bulbar versus spinal ALS. Bulbar ALS patients present more pronounced muscle atrophy than spinal ALS, as documented by our muscle fiber morphometric analysis. In patients ALS biopsy all miRNAs we measured are strongly up-regulated in ALS patients versus control, with the exception of miR-149*. A strong up-regulation (p<0.0001) of all miRNAs is present in male versus female ALS biopsies and in group of ALS patients with age of onset below 55 versus the group of older patients. In serum after 8 weeks of rehabilitation myomiRNAs result down-regulated indicating an active proliferation of satellite cells in muscle and increased neuromuscular junctions. Conclusion: Muscle mass regulators are particularly down-expressed in bulbar ALS, suggesting a more rapid and diffuse muscle atrophic process. Similar results were obtained on Myostatin/Follistatin ratio. The differences in this regulators between clinical subgroups of ALS patients, in serum and in muscle, may suggest a possible differential role of miRNAs in the pathogenetic accurance of muscle atrophy in motoneurons disease. Furthermore our data suggest that miRNAs are good indicators of muscle recovery in response to rehabilitation conducted for eight weeks consisting of aerobic exercise

Individuazione di biomarcatori specifici di cachessia neoplastica e di cachessia associata a scompenso cardiaco: valutazione comparativa di miRNA, GDF15 e FGF21

Scopo del progetto: L’obiettivo di questo progetto è identificare micro-RNA (miRNA) e citochine associati alla cachessia neoplastica e cardiaca valutando quali sono comuni alle 2 forme di cachessia e quali, invece, sono specifici di ogni singola forma. Materiali e metodi: Sono stati arruolati 25 pazienti neoplastici (CP) e 15 controlli, nonché 27 pazienti con scompenso cardiaco (HF) e 17 relativi controlli sani. Per i pazienti neoplastici e con scompenso cardiaco è stato definito lo stato di cachessia e sono stati valutati i livelli sierici di GDF15 e FGF21.L’RNA (muscolare e plasmatico) dei pazienti CP e dei controlli è stato sottoposto al sequenziamento del miRnoma (NGS). I livelli dei miRNA circolanti risultati modulati mediante NGS e potenzialmente coinvolti nei pathways di “muscle wasting” (miR15b-3p, miR21-5p, miR29a-3p, miR29b-3p, miR133a-3p, miR206, miR486-5p, miR203a-3p) sono stati analizzati tramite RT-PCR in tutti i partecipanti. Risultati: I livelli di GDF15 sono più elevati nei pazienti CP e HF rispetto i controlli (p&lt;0.02). FGF21 risulta più elevato solo nei pazienti con cachessia, sia neoplastica che cardiaca (p=0.017 e p=0.046). I livelli di miR15b-3p sono ridotti nel tessuto muscolare e nel plasma dei pazienti sia CP che HF (p&lt;0.02). Nei pazienti oncologici non cachettici i livelli plasmatici di miR21-5p e miR133a-3p sono più elevati rispetto ai controlli (p&lt;0.02). Inoltre, nei pazienti maschi i livelli plasmatici di miR133a-3p e miR206 sono aumentati sia rispetto al totale dei controlli sia rispetto le pazienti femmine (p&lt;0.05). Infine, i pazienti HF cachettici mostrano una riduzione dei livelli plasmatici di miR29b-3p ed un aumento di miR486-5p (p&lt;0.05) e nelle pazienti femmine HF il miR486-5p aumenta rispetto ai controlli femmine (p=0.027). Conclusioni: Il GDF15 aumenta sia nei pazienti oncologici che con scompenso cardiaco. FGF21 aumenta nei pazienti oncologici e con cachessia neoplastica e da scompenso cardiaco. Il miRNA15b-3p risulta ridotto e mostra una correlazione negativa con il GDF15 in entrambe le coorti di partecipanti. I pazienti CP non cachettici mostrano aumento dei miRNA associati al muscolo che suggeriscono l’attivazione di meccanismi atrofici probabilmente durante la precachessia. Nei pazienti HF con cachessia il profilo di espressione di alcuni miRNA risulta alterato in maniera peculiare rispetto alla cachessia neoplastica. L’alterazione dei miRNA in funzione del genere merita ulteriore approfondimento sia nei soggetti sani che nei malati, Cachessiaoncologici e cardiologici

Postmitotic Expression of SOD1G93A Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation

To determine the role of mutant SOD1 gene (SOD1G93A) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1G93A gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1G93A in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1G93A gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1G93A gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1G93A gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1G93A in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1G93A on myogenic program and disclosed potential signaling, activated by SOD1G93A, that affect the identity of the myogenic cell population

Le vescicole extracellulari come nuovi mediatori di segnali muscolari

Il muscolo scheletrico produce e secerne fattori solubili, chiamati miochine in grado di svolgere un‟azione a livello autocrino, paracrino, ed endocrino contribuendo alla riparazione o rigenerazione muscolare. Molte di queste proteine vengono secrete durante l'esercizio fisico e sembrano in parte responsabili dei benefici dell‟attività fisica sulla salute. Quindi, queste evidenze sperimentali hanno dimostrato che il muscolo possiede anche un‟importante attività endocrina. Recentemente, è stato proposto che anche le vescicole extracellulari (EV) possano mediare il cross-talk intercellulare sia in condizioni normali che patologiche attraverso il trasferimento di miRNA specifici. Studi precedenti hanno riportato che mioblasti e miotubi rilasciano EV nell'ambiente extracellulare durante il processo di differenziamento miogenico. Nella prima parte di questa tesi è stato indagato se le cellule C2C12 rilasciano tipologie diverse di EV durante le prime fasi del processo di differenziamento miogenico e se queste possiedono funzioni biologiche. Nella seconda parte, sono stati studiati gli effetti dell‟esercizio fisico sul rilascio di EV da parte del muscolo. In particolare, è stato valutato se l'applicazione di impulsi elettrici (EPS) in miotubi cresciuti in vitro induce la secrezione di EV e infine se il tessuto muscolare rilascia EV nel circolo ematico in seguito ad attività fisica. I dati ottenuti dimostrano che durante le prime fasi del processo di differenziamento miogenico, i miociti rilasciano due tipi di vescicole extracellulari: le microvescicole con un diametro medio di 200 nm, contenenti materiale elettron-denso, e vescicole con caratteristiche esosomiali, caratterizzate da un diametro di circa 50-100 nm contenenti materiale elettron-trasparente. Queste due popolazioni di vescicole presentano anche un diverso contenuto di acidi nucleici, in particolare, quelle caratterizzate da una minor densità hanno elevati livelli di RNA, mentre le vescicole più pesanti mostrano livelli di DNA più abbondanti. Inoltre è stato evidenziato che il rapporto relativo tra queste sub-popolazioni di vescicole cambia nel corso del processo di differenziamento miogenico. Quando utilizzate nel trattamento di cellule target, le EV hanno evidenziato la capacità di influenzare l‟attività delle cellule bersaglio, come miociti e macrofagi, in termini di regolazione genica. Per quel che riguarda l‟effetto della contrazione muscolare sul rilascio di vescicole, è stato dimostrato che la stimolazione mediante EPS induce la secrezione di EV, principalmente esosomi, da parte dei miotubi coltivati in vitro. Inoltre, è stato ipotizzato che il muscolo possa rilasciare EV anche in vivo. I dati riportati mostrano infatti la presenza, nel plasma, di EV positive all‟alfa-sarcoglicano (SGCA) e arricchite in miR-206, caratteristiche che ne suggeriscono l‟origine muscolare. Infine, è stata trovata una correlazione positiva tra la capacità aerobica, misurata come VO2max e i livelli dei miRNA muscolo-specifici (MyomiRNAs) contenuti nelle EV, e una induzione di miR-133b e miR-181a-5p vescicolari dopo esercizio aerobico acuto. Nel loro insieme, i dati riportati in questa tesi suggeriscono che il muscolo rilascia, nell‟ambiente extracellulare, EV potenzialmente coinvolte nella riparazione, rigenerazione, e rimodellamento muscolare e nel cross-talk con altri organi. Ulteriori studi saranno necessari al fine di definire il contenuto e le funzioni delle EV muscolari, sia in condizioni fisiologiche che patologiche. Tali informazioni permetteranno di comprendere il ruolo delle EV rilasciate dal muscolo nella regolazione dell'omeostasi energetica e potrebbero portare allo sviluppo di nuovi biomarcatori

Comprehensive Analyses of miRNA and DNA Methylation in 17beta-Estradiol Exposed Juvenile Rainbow Trout Skeletal Muscle

Reproductive development in female teleosts like rainbow trout starts with release of the sex steroid estrogen, necessary for synthesis of the egg yolk protein vitellogenin. Increase in estrogen secretion during these reproductive phases results in increased energy demands. Skeletal muscle serves as an immediate endogenous source to address energy demands during vitellogenesis and spawning due to which it undergoes rapid degradation resulting in water accumulation, poor fillet and low egg quality. An imbalance in protein turnover is observed not only because of rapid increase in degradative pathways but also due to decreased protein synthesis. These changes in protein metabolism in skeletal muscle were identified within 24 hours of E2 exposure and confirmed in both in vivo and in vitro models. Normally during adult myogenesis myogenic precursor cells proliferate and differentiate further to form a mature muscle fiber resulting in either hyperplasty or hypertrophy or both in fishes. Signaling pathways and MRFs need to function in coordination with epigenetic factors and non-coding RNAs to positively or negatively regulate muscle synthesis. Interestingly, estrogen also influences the expression of miRNA and in turn their target genes. However, the effects of estrogen regulating protein imbalance in rainbow trout skeletal muscle is not completely understood. Hence studies to understand the influence of estrogen were carried out using juvenile, sexually immature fish. Juvenile rainbow trout were treated with either estrogen or vehicle and their skeletal muscle collected after 24 hours and 72 hours of treatment to understand the role of miRNA and DNA methylation in regulation of myogenesis. Findings from the miRNA study revealed diluted effects of estrogen after 72 hours of treatment, hence further studies were carried out with samples from 24-hour post treatment.;miRNAs play a crucial role in regulation of gene expression along with epigenetics. These are small non-coding RNAs with length ranging from 21--23bp mostly functioning as post-transcriptional regulators of gene expression. Generally, they bind to the 3\u27 region of transcribed mRNA with a complementary region called seed sequence. These genes are called target genes of miRNA. A single miRNA can target more than one mRNA. Binding of miRNA to its target gene results in either translational repression or deadenylation or both. Deadenylation results in loss of RNA stability, which leads to its degradation. miRNAs express in tissue specific manner and those that express specifically in muscle are called myomiRNA. Various myomiRNA have been identified and studied for their functions. Estrogen influence the expression of miRNA and in turn their target genes. Therefore, rainbow trout skeletal muscle samples were sequenced for small RNAs to understand the effects of estrogen. Differentially expressed miRNA were identified in estrogen treated samples when compared to control. A total of 36 miRNAs were either upregulated or down regulated in E2 exposed skeletal muscle of which two were novel. Target genes of these differentially expressed miRNAs were identified followed by gene ontology enrichment. These target genes are involved in various biological and molecular functions including their role in signaling pathways, cell cycle, DNA methylation, signal transduction and transcription factor binding. Genes regulated by miRNAs were also involved specifically in proliferation and differentiation of myogenic precursor cells and degradative pathways. E2 induced expression of miR-17 and miR-20, which are important in regulation of cell cycle. Further gene expression analysis of myogeneic regulatory factor, Pax7 specific to MPCs confirms increase in their number with decreased expression of MyoD, specific to differentiation. Similarly, miR-23a revealed its role in maintenance of mitochondrial outer membrane permeability and post-transcriptional regulation of atrophic genes. Decreased expression of miR-23a with increase in permeability was supported by increased gene expressions including fbxo32, caspases 3a and 9 that are involved in atrophy and apoptosis respectively. Expression analysis of one of the novel genes, miR-nov-285, was performed to know its expression pattern in different tissues, highest expression was observed in testis. C-5 methylation of cytosine was one of the GO term that was enriched during the target gene analysis of this novel miRNA. Further regulation of Dnmt1 gene expression by this novel miRNA was confirmed by luciferase assay. All together these observations indicate that E2 influences differential expression of various miRNAs which in turn regulate gene expression contributing to muscle metabolism.;Reduced expression of MyoD was observed in our previous study and direct regulation of this differentiation factor was not observed by any of the differentially expressed miRNA. Since gene expression is also regulated transcriptionally by epigenetic mechanisms including DNA methylation, efforts were made to understand its role in reduced MyoD expression. Generally, DNA methylation reduces gene expression by directly blocking transcription factor binding or by recruiting other epigenetic contributors like histone deacetylases or histone methyltransferases. (Abstract shortened by ProQuest.)

Gene Regulation in Ruminants: A Nutritional Perspective

This chapter will focus on cellular regulatory programs implemented by the ruminant physiology in order to respond to external stimuli such as nutrition as well as important physiological events such as parturition. The increasing adoption of “omics” technologies and bioinformatics in nutrition and physiology in ruminant research have allowed us to delineate a clearer picture on what regulates major biological process at a molecular level such as milk synthesis and meat quality and fatty acid composition as well as pathological conditions such as ketosis, mastitis, and heat stress. The assembly of such plethora of information in a blend among nutritional research, molecular biology, and novel tools to study the response of the genome to nutrition has led to emerging disciplines such as nutritional genomics or “nutrigenomics.

Association of circulating hsa-miRNAs with sarcopenia: the SarcoPhAge study.

peer reviewed[en] OBJECTIVE: To identify a microRNA signature associated to sarcopenia in community-dwelling older adults form the SarcoPhAge cohort. METHODS: In a screening phase by next generation sequencing (NGS), we compared the hsa-miRome expression of 18 subjects with sarcopenia (79.6 ± 6.8 years, 9 men) and 19 healthy subjects without sarcopenia (77.1 ± 6 years, 9 men) at baseline. Thereafter, we have selected eight candidate hsa-miRNAs according to the NGS results and after a critical assessment of previous literature. In a validation phase and by real-time qPCR, we then analyzed the expression levels of these 8 hsa-miRNAs at baseline selecting 92 healthy subjects (74.2 ± 10 years) and 92 subjects with sarcopenia (75.3 ± 6.8 years). For both steps, the groups were matched for age and sex. RESULTS: In the validation phase, serum has-miRNA-133a-3p and has-miRNA-200a-3p were significantly decreased in the group with sarcopenia vs controls [RQ: relative quantification; median (interquartile range)]: -0.16 (-1.26/+0.90) vs +0.34 (-0.73/+1.33) (p < 0.01) and -0.26 (-1.07/+0.68) vs +0.27 (-0.55/+1.10) (p < 0.01) respectively. Has-miRNA-744-5p was decreased and has-miRNA-151a-3p was increased in the group with sarcopenia vs controls, but this barely reached significance: +0.16 (-1.34/+0.79) vs +0.44 (-0.31/+1.00) (p = 0.050) and  +0.35 (-0.22/+0.90) vs  +0.03 (-0.68/+0.75) (p = 0.054). CONCLUSION: In subjects with sarcopenia, serum hsa-miRNA-133a-3p and hsa-miRNA-200a-3p expression were downregulated, consistent with their potential targets inhibiting muscle cells proliferation and differentiation