Translational Regulation of Utrophin by miRNAs

Background Utrophin is the autosomal homolog of dystrophin, the product of the Duchenne Muscular Dystrophy (DMD) locus. Its regulation is of therapeutic interest as its overexpression can compensate for dystrophin's absence in animal models of DMD. The tissue distribution and transcriptional regulation of utrophin have been characterized extensively, and more recently translational control mechanisms that may underlie its complex expression patterns have begun to be identified. Methodology/Principal Findings Using a variety of bioinformatic, molecular and cell biology techniques, we show that the muscle isoform utrophin-A is predominantly suppressed at the translational level in C2C12 myoblasts. The extent of translational inhibition is estimated to be ~99% in C2C12 cells and is mediated by both the 5′- and 3′-UTRs of the utrophin-A mRNA. In this study we identify five miRNAs (let-7c, miR-150, miR-196b, miR-296-5p, miR-133b) that mediate the repression, and confirm repression by the previously identified miR-206. We demonstrate that this translational repression can be overcome by blocking the actions of miRNAs, resulting in an increased level of utrophin protein in C2C12 cells. Conclusions/Significance The present study has identified key inhibitory mechanisms featuring miRNAs that regulate utrophin expression, and demonstrated that these mechanisms can be targeted to increase endogenous utrophin expression in cultured muscle cells. We suggest that miRNA-mediated inhibitory mechanisms could be targeted by methods similar to those described here as a novel strategy to increase utrophin expression as a therapy for DMD

Differential Requirement for Utrophin in the Induced Pluripotent Stem Cell Correction of Muscle versus Fat in Muscular Dystrophy Mice

Duchenne muscular dystrophy (DMD) is an incurable degenerative muscle disorder. We injected WT mouse induced pluripotent stem cells (iPSCs) into mdx and mdx∶utrophin mutant blastocysts, which are predisposed to develop DMD with an increasing degree of severity (mdx <<< mdx∶utrophin). In mdx chimeras, iPSC-dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Dystrobrevin was observed in dystrophin-positive and, at a lesser extent, utrophin-positive areas. In the mdx∶utrophin mutant chimeras, although iPSC-dystrophin was also supplied to the muscle sarcolemma, mice still displayed poor skeletal muscle histopathology, and negligible levels of dystrobrevin in dystrophin- and utrophin-negative areas. Not only dystrophin-expressing tissues are affected by iPSCs. Mdx and mdx∶utrophin mice have reduced fat/body weight ratio, but iPSC injection normalized this parameter in both mdx and mdx∶utrophin chimeras, despite the fact that utrophin was compromised in the mdx∶utrophin chimeric fat. The results suggest that the presence of utrophin is required for the iPSC-corrections in skeletal muscle. Furthermore, the results highlight a potential (utrophin-independent) non-cell autonomous role for iPSC-dystrophin in the corrections of non-muscle tissue like fat, which is intimately related to the muscle

A Small Peptide Modeled after the NRAGE Repeat Domain Inhibits XIAP-TAB1-TAK1 Signaling for NF-κB Activation and Apoptosis in P19 Cells

In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38MAPK and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38MAPK phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/β phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis

Description and initial clinical study of a digital chest X-ray selenium detector based system (Thoravision) [Descrizione e valutazione clinica preliminare di un sistema radiografico digitale del torace con rivelatore al selenio (Thoravision)]

The authors describe an innovative digital chest imaging system (Thoravision, Philips Medical System) based on a selenium detector directly converting X-rays in electric charges detectable by electrometers, amplified and digitalized to be sent to the imaging processor. The study was performed on a series of up to 7000 digital chest X-ray examinations and a sample of 173 cases was selected on the basis of particular diseases and pathologic patterns. In the latter case, digital and conventional radiographic techniques were compared. According to this analysis, the digital system was slightly superior in detecting anatomical details, small pleural effusions (3 cases) and slight low-contrast interstitial pathologic changes, mainly in apical (12 cases) and retrocardiac (19 cases) lung areas. The conventional screen-film system better showed small hilar calcifications (2 cases). From the protection viewpoint, at equivalent entrance doses, .15-.26 Gy/cm2 of dose-area product (DAP) values were reported with the digital system, using a tension of 150 kV in orthogonal projections, which are considerably lower than .25-.70 Gy/cm2 measured with the conventional technique. Moreover, Thoravision permitted to perform up to 100 exposures/hour, using a phantom, and 15-20 digital chest examinations/hour, versus the average 12-15 with the conventional screen-film system. The initial results are very encouraging from the clinical and cost-effectiveness viewpoints, thanks to higher Thoravision productivity and to exam optimization, even though wider and prolonged experience plus a careful study of diagnostic accuracy, sensitivity and specificity in each group of chest conditions is advisable, to assess the real innovative impact of this digital chest imaging system

Description and initial clinical study of a digital chest X-ray selenium detector based system (Thoravision) [Descrizione e valutazione clinica preliminare di un sistema radiografico digitale del torace con rivelatore al selenio (Thoravision)]

The authors describe an innovative digital chest imaging system (Thoravision, Philips Medical System) based on a selenium detector directly converting X-rays in electric charges detectable by electrometers, amplified and digitalized to be sent to the imaging processor. The study was performed on a series of up to 7000 digital chest X-ray examinations and a sample of 173 cases was selected on the basis of particular diseases and pathologic patterns. In the latter case, digital and conventional radiographic techniques were compared. According to this analysis, the digital system was slightly superior in detecting anatomical details, small pleural effusions (3 cases) and slight low-contrast interstitial pathologic changes, mainly in apical (12 cases) and retrocardiac (19 cases) lung areas. The conventional screen-film system better showed small hilar calcifications (2 cases). From the protection viewpoint, at equivalent entrance doses, .15-.26 Gy/cm2 of dose-area product (DAP) values were reported with the digital system, using a tension of 150 kV in orthogonal projections, which are considerably lower than .25-.70 Gy/cm2 measured with the conventional technique. Moreover, Thoravision permitted to perform up to 100 exposures/hour, using a phantom, and 15-20 digital chest examinations/hour, versus the average 12-15 with the conventional screen-film system. The initial results are very encouraging from the clinical and cost-effectiveness viewpoints, thanks to higher Thoravision productivity and to exam optimization, even though wider and prolonged experience plus a careful study of diagnostic accuracy, sensitivity and specificity in each group of chest conditions is advisable, to assess the real innovative impact of this digital chest imaging system

Modeling socially anhedonic syndromes: genetic and pharmacological manipulation of opioid neurotransmission in mice

Social anhedonia, or the diminished capacity to experience pleasure and reward from social affiliation, is a major symptom of different psychiatric disorders, including some forms of infantile autism and schizophrenia spectrum disorders. The brain opioid hypothesis of social attachment is a promising model for achieving insights into how neurobiological and developmental factors contribute to the regulation of social reward. In this study, genetic knocking-out and naltrexone (NTRX) treatment during the first 4 days of life were used to disrupt opioid neurotransmission in mouse pups and their attachment relationships with the mother. Both permanent (genetic) and transient (pharmacological) manipulations of opioid neurotransmission exerted long-term effects on social affiliation. When juveniles, both μ-opioid receptor knockout mice and NTRX-treated pups showed reduced interest in peers and no preference for socially rewarding environment. These results demonstrate that sociability in juvenile mice is highly dependent on the establishment during infancy of a positive affective relationship with their mothers and that opioid neurotransmission has a major role in the regulation of social hedonic capacity. If the validity of this animal model will be confirmed by future research, translational studies focusing on the interaction between early experience and opioid neurotransmission could provide useful insights for identifying endophenotypes of human psychiatric disorders associated with social anhedonia

Mechanisms underlying the effect of N-terminal 26-230 tau fragment on synaptic dysfunction and neurodegeneration.

Alzheimer's disease is characterized by progressive impairment of brain plasticity Although senile plaques and neurofibrillary tangles in the brain are the pathological hallmarks, their presence seems not to be related to the severity of dementia. In contrast, altered synaptic communication and neuronal loss, caused by other tau or amyloid species represent a direct pathological cause for dementia. Among tau species we focus on tau fragment (26-230) or N-tau. Clevage of tau by apoptotic proteases plays a crucial role in neurodegeneration and in the development of the neurofibrillary tangles as AD progresses. We have found that N-tau adversely affects neurons. Indeed, its overexpression has marked toxic effects on neurons that can be inhibited by the NMDAR antagonist and by inhibitors of NMDAR-associated processes such as MAP kinase and calpain. We have used conditional transgenic mice for N-tau and primary neuronal cultures overexpressing N-tau for decoding the signal transduction pathway that link Ntau expression to NMDAR activity and their upstream and downstream effectors inducing plasticity failure. Data regarding the electrophysiological recording of NMDAR, morphological, qualitative and quantitative analysis of synapse number, dendritic spines and behavioral analysis of N-tau transgenic mice performed to give an accurate and global view of the molecular events involved in N-tau modulation of neuronal plasticity will be presented

SMN affects membrane remodelling and anchoring of the protein synthesis machinery

Disconnection between membrane signalling and actin networks can have catastrophic effects depending on cell size and polarity. The survival motor neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways in which SMN can act, such as mRNA translation, actin network and endocytosis. Here, we found that SMN accumulates at membrane protrusions during the dynamic rearrangement of the actin filaments. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dynamics. We propose that SMN could assembly translational platforms associated with and governed by the plasma membrane. This activity could be crucial in cells that have an exacerbated interdependence of membrane remodelling and local protein synthesis

AN ITALIAN SURVEY OF COMPLIANCE WITH MAJOR GUIDELINES FOR L-THYROXINE OF PRIMARY HYPOTHYROIDISM.

Abstract OBJECTIVE: The adherence by endocrinologists to guideline regarding levothyroxine (LT4) therapy and the compliance of patients may impact the management of hypothyroidism. The aim of this study was to compare the adherence of Italian endocrinologists to the ATA/AACE and ETA guidelines on the management of newly diagnosed primary hypothyroidism and to validate the Italian version of the Morisky-Green Medical Adherence Scale-8 (MMAS-8) questionnaire as applied to the evaluation of the adherence of patients with hypothyroidism to LT4 treatment. METHODS: This was an observational, longitudinal, multicenter, cohort study, involving 12 Italian Units of Endocrinology. RESULTS: The study enrolled 1,039 consecutive outpatients (mean age 48 years; 855 women, 184 men). The concordance of Italian endocrinologists with American Association of Clinical Endocrinologists/American Thyroid Association (AACE/ATA) and European Thyroid Association (ETA) recommendations was comparable (77.1% and 71.7%) and increased (86.7 and 88.6%) after the recommendations on LT4 dose were excluded, considering only the remaining recommendations on diagnosis, therapy, and follow-up. The MMAS-8 was filled out by 293 patients. The mean score was 6.71 with 23.9% low (score &lt;6), 38.6% medium (6 to &lt;8), 37.5% highly (= 8) adherers; the internal validation coefficient was 0.613. Highly adherent patients were not more likely to have good control of hypothyroidism compared with either medium (69% versus 72%, P = .878) or low (69% versus 43%, P = .861) adherers. CONCLUSION: Clinical management of hypothyroidism in Italy demonstrated an observance of international guidelines by Italian endocrinologists. Validation of the Italian version of the MMAS-8 questionnaire provides clinicians with a reliable and simple tool for assessing the adherence of patients to LT4 treatment. ABBREVIATIONS: AACE = American Association of Clinical Endocrinologists; ATA = American Thyroid Association; EDIPO = Endotrial SIE: DIagnosis and clinical management of Primitive hypothyrOidism in Italy; eCRF = electronic case report form; ETA = European Thyroid Association; fT3 = free triiodothyronine; fT4 = free thyroxine; LT4 = levothyroxine; MMAS-8 = Morisky-Green Medical Adherence Scale-8; PH = primary hypothyroidism; T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid-stimulating hormone; US = ultrasonography