1,720,978 research outputs found
Transcriptional reprogramming of muscle fibers by chronic electrical stimulation
Full text linkBackground: Skeletal muscle fibers have a remarkable capacity to adjust their molecular, functional, and metabolic properties in response to developmental and environmental stimuli. A central role for neuromuscular activity in determining skeletal muscle fibers composition was demonstrated by cross-innervation and electrical stimulation experiments in adult skeletal muscle. A fast to slow transition can be induced by chronic low-frequency electrical stimulation (CLFS). In literature, CLFS studies have focused on the effects produced after days or weeks of stimulation. Here, I present the first transcriptome study that identifies the earliest genetic changes in this process.
Methods: The fast EDL muscle was subjected to CLFS for 6 and 12 hours. First, microarray experiments were performed using whole EDL muscle. Then, in order to reduce biological noise caused by different cell types, I applied microgenomic analyses at the level of single fibers (SF), isolated according to the protocol recently developed in my laboratory. Microarray experiments have been produced with updated platforms (Agilent SurePrint G3 Mouse GE 8x60K).
Results and discussion: The expression profiles of whole muscle after 6 and 12 hours of electrical stimulation identified about two hundred differentially expressed (DE) genes. The functional categories of blood vessel development and transcription regulation were most enriched. Genomic analyses of isolated fibers identified more than a thousand DE genes after 12 hours of stimulation. The functional category of blood vessel development was enriched also at the SF level, suggesting that myofibers are able to interact with other cell types in order to stimulate the angiogenesis process. In general, muscle structural genes were equally expressed between stimulated and unstimulated muscles, indicating that changes in fiber type require prolonged stimulation. On the contrary, genes involved in transcription, chromatin-remodeling genes and several myofibril genes acting as signaling molecule were early activated after CLFS.Introduzione: Le fibre del muscolo scheletrico possiedono una notevole capacità di modificare le loro proprietà molecolari, funzionali e metaboliche in risposta a stimoli di crescita e ambientali. Esperimenti di cross-innervazione e stimolazione elettrica hanno dimostrato il ruolo fondamentale dell’attività neuromuscolare nel determinare il tipo di fibra. La stimolazione cronica a bassa frequenza (CLFS) è in grado di indurre il processo di trasformazione di una fibra veloce verso un fenotipo lento. Fino ad ora, questi processi sono stati studiati dopo lunghi periodi di stimolazione. In questo lavoro, mi sono proposta di identificare i cambiamenti trascrizionali precoci di questo processo.
Metodi: Il muscolo veloce EDL di topo è stato sottoposto a CLFS per 6 e 12 ore. Inizialmente ho utilizzato il muscolo intero per produrre i profili di espressione Successivamente, per ovviare al problema dell’eterogenea composizione del muscolo, ho utilizzato l’approccio microgenomico, producendo profili di espressione a livello di una singola fibra, isolata utilizzando il protocollo sviluppato nel mio laboratorio. Tutti i profili sono stati ottenuti mediante l’utilizzo di piattaforme Agilent (SurePrint G3 Mouse GE 8x60K).
Risultati e discussione: I profili di espressione con il muscolo intero hanno identificato circa 200 geni differenzialmente espressi (DE). Lo sviluppo dei vasi sanguigni e la regolazione della trascrizione sono risultate le categorie funzionali più arricchite. Le analisi genomiche a livello di singola fibra hanno identificato più di mille geni DE dopo 12 ore di stimolazione. La categoria funzionale riguardante lo sviluppo dei vasi sanguigni risulta arricchita anche con questo approccio, suggerendo un’interazione tra le fibre muscolari e gli altri tipi cellulari al fine di stimolare il processo di angiogenesi. La maggiorparte dei geni strutturali risulta essere ugualmente espressa, suggerendo che i cambiamenti nel tipo di fibra richiedono un tempo prolungato. Al contrario, i geni coinvolti nella trascrizione, nel rimodellamento della cromatina e alcuni geni miofibrillari che agiscono come molecule di segnale, si attivano rapidamente in risposta a CLFS
Autosomal dominant essential tremor: a novel family with anticipation
No full textEssential tremor (ET) is a common progressive movement disorder characterized by a clear genetic predisposition. In the last years, many efforts have been done to map susceptibility loci for ET. Here, we report a clinical and genetic study of a family with ET showing autosomal dominant inheritance and anticipation over three generations. The family has five affected members and exhibits a remarkable anticipation of age at onset of the disease along the generations. We excluded linkage to any of the three loci previously mapped in autosomal dominant ET families. Our data suggest the existence of an additional locus in which a repeat expansion is the possible genetic defect underlying ET
The down-regulation of pank2 gene in zebrafish as a model of Pantothenate Kinase Associated Neurodegeneration.
Full text linkThe increased iron deposition is a hallmark of many neurodegenerative diseases, but its pathogenic role is still unclear. A strong link between iron and neurodegeneration is evident in a set of heterogeneous neurological disorders, known as Neurodegeneration with Brain Iron Accumulation (NBIA). The most common form of inherited NBIA is associated with mutations in hPank2 gene (PKAN). Pank2 is the rate limiting enzyme in CoA biosynthesis and its downregulation in mammalian cells leads to perturbation of cellular iron homeostasis. Here we explore Pank2 biological function in Danio rerio, and propose this system as an important new tool for the study of PKAN disease
Mitochondrial DNA depletion and OXPHOS complex impairment modify Hypoxia signaling pathway activity in zebrafish
Full text linkMitochondria are essential for cell survival and health, utilizing about 90% of the oxygen we breathe for OXPHOS. Previous studies showed a strong relationship between mitochondrial alterations and hypoxia signaling pathway [1]. POLG-related disorders are a group of diseases characterized by the dysfunction of DNA polymer- ase gamma, crucial for mtDNA replication, repair and stability [2]. Danio rerio (zebrafish) is an ideal vertebrate model of human mitochondrial diseases because of its high conservation of physio- logical processes and genomic structure, transgenic lines availability and embryonic transparency. Using zebrafish embryos, we have performed transient knock-down of the polg gene, inducing a dilated cardiomyopathy and an increased heart bit rate. Moreover, we have developed a transgenic line able to show in vivo the activation of hypoxia-inducible factor 1 (Hif1) signaling. Taking advantage of this reporter line, we established that Hypoxia pathway is up-regulated in polg morphants, maybe due to an increase in ROS production. In addition, using a pharmacological approach targeting OXPHOS complexes, we observed that the inhibition of complexes I and II induces a decrease in Hif1 activation, also in hypoxic conditions. In conclusion, the evidence of Hypoxia reporter activation in polg knock-down embryos suggests the existence of cross-talk mechanisms sensing mitochondrial dysfunction and changing hypoxia signaling, as also confirmed by the OXPHOS complex inhibitors analysis
Generation and characterization of a novel zebrafish reporter line for cAMP/CREB signalling.
No full textShort-term expression profiling of electrically stimulated muscle reveals the initial points of fast to slow transition
No full textOXPHOS complex impairment and mitochondrial DNA depletion modify hypoxia signaling pathway activity in zebrafish.
No full textMitochondria are essential for cell survival and health, utilizing about 90% of the oxygen we breath for OXPHOS. Previous studies showed a strong relationship between mitochondrial alterations and hypoxia signaling pathway, an interesting aspect to be investigated. POLG-related disorders are a group of diseases characterized by the dysfunction of DNA polymerase gamma, an enzyme crucial for mtDNA replication, repair and stability. Danio rerio (zebrabish) is an ideal vertebrate model of human mitochondrial diseases because of its high conservation of physiological processes and genomic structure, transgenic lines availability and embryonic transparency. Using zebrabish embryos, we have performed a transient knock-down of the polg gene, inducing a dilated cardiomyopathy and an increased heart bit rate. Moreover, we have developed a transgenic line able to show in vivo the activation of hypoxia-inducible factor 1 (Hif1) signaling. Taking advantage of this reporter line, we established that Hypoxia pathway is up-regulated in polg morphants. In addition, using a pharmacological approach targeting OXPHOS complexes NADH:ubiquinone reductase (Complex I) and Succinate dehydrogenase (Complex II), we have investigated the effect of mitochondrial dysfunctions on the Hypoxia pathway. We established that Hypoxia pathway is signibicantly reduced, both in normoxia and in hypoxia conditions, perhaps due to an increase in ROS production. In conclusion, our data suggest the existence of cross-talk mechanisms sensing mitochondrial dysfunction and changing Hypoxia signaling. In addition, our results on polg transient inactivation incourage the use of zebrabish as a suitable model to perform CRISPR/Cas9-mediated mutagenesis of DNA polymerase gamma, an ongoing project in our laboratory
A Computational Model of the LGI1 Protein Suggests a Common Binding Site for ADAM Proteins
Full text linkMutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process
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