The aim of this work was to identify micro-RNAs (miRNAs) involved in the pathological pathways activated in skeletal muscle damage and regeneration by both dystrophin absence and acute ischemia. Eleven miRNAs were deregulated both in MDX mice and in Duchenne muscular dystrophy patients (DMD signature). Therapeutic interventions ameliorating the mdx-phenotype rescued DMD-signature alterations. The significance of DMD-signature changes was characterized using a damage/regeneration mouse model of hind-limb ischemia and newborn mice. According to their expression, DMD-signature miRNAs were divided into 3 classes. 1) Regeneration miRNAs, miR-31, miR-34c, miR-206, miR-335, miR-449, and miR-494, which were induced in MDX mice and in DMD patients, but also in newborn mice and in newly formed myofibers during postischemic regeneration. Notably, miR-206, miR-34c, and miR-335 were up-regulated following myoblast differentiation in vitro. 2) Degenerative-miRNAs, miR-1, miR-29c, and miR-135a, that were down-modulated in MDX mice, in DMD patients, in the degenerative phase of the ischemia response, and in newborn mice. Their down-modulation was linked to myofiber loss and fibrosis. 3) Inflammatory miRNAs, miR-222 and miR-223, which were expressed in damaged muscle areas, and their expression correlated with the presence of infiltrating inflammatory cells. These findings show an important role of miRNAs in physiopathological pathways regulating muscle response to damage and regeneration.

Mutations in the PINK1 gene cause autosomal recessive Parkinson's disease. The PINK1 gene encodes a protein kinase that is mitochondrially cleaved to generate two mature isoforms. In addition to its protective role against mitochondrial dysfunction and apoptosis, PINK1 is also known to regulate mitochondrial dynamics acting upstream of the PD-related protein Parkin. Recent data showed that mitochondrial Parkin promotes the autophagic degradation of dysfunctional mitochondria, and that stable PINK1 silencing may have an indirect role in mitophagy activation. Here we report a new interaction between PINK1 and Beclin1, a key pro-autophagic protein already implicated in the pathogenesis of Alzheimer's and Huntington's diseases. Both PINK1 N-and C-terminal are required for the interaction, suggesting that full-length PINK1, and not its cleaved isoforms, interacts with Beclin1. We also demonstrate that PINK1 significantly enhances basal and starvation-induced autophagy, which is reduced by knocking down Beclin1 expression or by inhibiting the Beclin1 partner Vps34. A mutant, PINK1 W437X , interaction of which with Beclin1 is largely impaired, lacks the ability to enhance autophagy, whereas this is not observed for PINK1 G309D , a mutant with defective kinase activity but unaltered ability to bind Beclin1. These findings identify a new function of PINK1 and further strengthen the link between autophagy and proteins implicated in the neurodegenerative process. Parkinson's disease (PD) is a frequent neurodegenerative disorder resulting from massive degeneration of the dopaminergic neurons in the substantia nigra. Although most cases are sporadic, several genes are known to cause familial PD, especially with early onset. 1 Mutations in the PINK1 gene are the second most frequent cause of autosomal recessive PD after those in the Parkin gene. 2,3 The PINK1 gene encodes a serine-threonine kinase with an N-terminal mitochondrial import sequence, first characterized as a protein aimed at maintaining mitochondrial integrity and preventing apoptosis in response to cellular stressors. 2,[4][5][6][7][8] This neuroprotective role is partly exerted through phosphorylation of the mitochondrial chaperon, TRAP1, although cytoplasm-restricted PINK1 was also shown to protect against MPTP damage. 9,10 The full-length PINK1 (PINK1-FL) is processed within mitochondria to generate two mature proteins; 4,11 all three isoforms localize both to the mitochondria and cytosol, their relative ratio being regulated by several factors. [10][11][12][13] Increasing data have demonstrated that absence of functional PINK1 induces abnormalities of mitochondrial morphology. 6,14,15 In several studies (mostly in Drosophila), PINK1 was shown to promote fission acting upstream of the Fis1-Drp1 machinery, and the mitochondrial phenotype observed in PINK1 knockout flies or silenced cells was associated to reduced fission. 16,17 Subsequent studies in mammalian cell systems contradicted these results, demonstrating that mutant or silenced PINK1 resulted in incre...

The type of interaction between tumor-associated antigens and specialized antigen-presenting cells such as dendritic cells (DCs) is critical for the type of immunity that will be generated. MUC1, a highly O-glycosylated mucin, is overexpressed and aberrantly glycosylated in several tumor histotypes. This results in the expression of tumor-associated glycoforms and in MUC1 carrying the tumor-specific glycan Tn (GalNAcA1-O-Ser/Thr). Glycopeptides corresponding to three tandem repeats of MUC1, enzymatically glycosylated with 9 or 15 mol of GalNAc, were shown to specifically bind and to be internalized by immature monocyte-derived DCs (iDCs). Binding required calcium and the GalNAc residue and was competed out by GalNAc polymer and Tn-MUC1 or Tn-MUC2 glycopeptides. The macrophage galactose-type C-type lectin (MGL) receptor expressed on iDCs was shown to be responsible for the binding. Confocal analysis and ELISA done on subcellular fractions of iDCs showed that the Tn-MUC1 glycopeptides colocalized with HLA class I and II compartments after internalization. Importantly, although Tn-MUC1 recombinant protein was bound and internalized by MGL, the glycoprotein entered the HLA class II compartment, but not the HLA class I pathway. These data indicate that MGL expressed on iDCs is an optimal receptor for the internalization of short GalNAcs carrying immunogens to be delivered into HLA class I and II compartments. Such glycopeptides therefore represent a new way of targeting the HLA class I and II pathways of DCs. These results have possible implications in designing cancer vaccines. [Cancer Res 2007;67(17):8358-67]

Background-Fabry cardiomyopathy is diagnosed by detection of left ventricular hypertrophy (LVH) in patients with␣-Galactosidase A deficiency. Conventional noninvasive tools are unable to provide a preclinical diagnosis allowing prompt institution of enzymatic therapy. Methods and Results-We studied three groups of patients: 10 patients with causal mutations for Fabry disease and LVH, 10 mutation-positive patients without LV, and 10 healthy relatives without causal mutations and no LVH. All patients with LVH and 6 patients with Fabry disease without LVH with complex repetitive ventricular arrhythmias underwent biventricular endomyocardial biopsy to assess cardiac involvement. In all patients 2-dimensional echocardiography with tissue Doppler analysis in the pulsed Doppler mode was performed: systolic (Sa), early diastolic (Ea), and late diastolic (Aa) velocities were measured, and the Ea/Aa ratio and the dimensionless parameter E/Ea were computed at both corners of the mitral annulus. Histology and electron microscopy studies showed glycosphingolipid deposits in all cases. All mutation-positive patients had significant reduction of Sa, Ea, and Aa velocities at both corners of the mitral annulus compared with normal control subjects. Ea/Aa ratio was significantly lower and E/Ea ratio significantly higher in mutation-positive patients than in control subjects. Patients with LVH showed significantly lower contraction and relaxation tissue Doppler velocities, lower Ea/Aa ratio, and higher E/Ea ratio in comparison with mutation-positive patients with no LVH. Conclusions-Fabry cardiomyopathy is characterized by reduced myocardial contraction and relaxation tissue Doppler velocities, detectable even before development of LVH. Tissue Doppler imaging can provide a preclinical diagnosis of Fabry cardiomyopathy, allowing early institution of enzyme replacement therapy.

BackgroundAssistive and robotic training devices are increasingly used for rehabilitation of the hemiparetic arm after stroke, although applications for the wrist and hand are trailing behind. Furthermore, applying a training device in domestic settings may enable an increased training dose of functional arm and hand training. The objective of this study was to assess the feasibility and potential clinical changes associated with a technology-supported arm and hand training system at home for patients with chronic stroke.MethodsA dynamic wrist and hand orthosis was combined with a remotely monitored user interface with motivational gaming environment for self-administered training at home. Twenty-four chronic stroke patients with impaired arm/hand function were recruited to use the training system at home for six weeks. Evaluation of feasibility involved training duration, usability and motivation. Clinical outcomes on arm/hand function, activity and participation were assessed before and after six weeks of training and at two-month follow-up.ResultsMean System Usability Scale score was 69 % (SD 17 %), mean Intrinsic Motivation Inventory score was 5.2 (SD 0.9) points, and mean training duration per week was 105 (SD 66) minutes. Median Fugl-Meyer score improved from 37 (IQR 30) pre-training to 41 (IQR 32) post-training and was sustained at two-month follow-up (40 (IQR 32)). The Stroke Impact Scale improved from 56.3 (SD 13.2) pre-training to 60.0 (SD 13.9) post-training, with a trend at follow-up (59.8 (SD 15.2)). No significant improvements were found on the Action Research Arm Test and Motor Activity Log.ConclusionsRemotely monitored post-stroke training at home applying gaming exercises while physically supporting the wrist and hand showed to be feasible: participants were able and motivated to use the training system independently at home. Usability shows potential, although several usability issues need further attention. Upper extremity function and quality of life improved after training, although dexterity did not. These findings indicate that home-based arm and hand training with physical support from a dynamic orthosis is a feasible tool to enable self-administered practice at home. Such an approach enables practice without dependence on therapist availability, allowing an increase in training dose with respect to treatment in supervised settings.Trial registrationThis study has been registered at the Netherlands Trial Registry (NTR): NTR3669.