Gapmer Antisense Oligonucleotides Suppress the Mutant Allele of COL6A3 and Restore Functional Protein in Ullrich Muscular Dystrophy

Dominant-negative mutations in the genes that encode the three major a chains of collagen type VI, COL6A1, COL6A2, and COL6A3, account for more than 50% of Ullrich congenital muscular dystrophy patients and nearly all Bethlem myopathy patients. Gapmer antisense oligonucleotides (AONs) are usually used for gene silencing by stimulating RNA cleavage through the recruitment of an endogenous endonuclease known as RNase H to cleave the RNA strand of a DNA-RNA duplex. In this study, we exploited the application of the allele-specific silencing approach by gapmer AON as a potential therapy for Collagen-VI-related congenital muscular dystrophy (COL6-CMD). A series of AONs were designed to selectively target an 18-nt heterozygous genomic deletion in exon 15 of COL6A3 at the mRNA and pre-mRNA level. We showed that gapmer AONs can selectively suppress the expression of mutant transcripts at both pre-mRNA and mRNA levels, and that the latter strategy had a far stronger efficiency than the former. More importantly, we found that silencing of the mutant transcripts by gapmer AONs increased the deposition of collagen VI protein into the extracellular matrix, thus restoring functional protein production. Our findings provide a clear proof of concept for AON allele-specific silencing as a therapeutic approach for COL6-CM

Changes in MEG resting-state networks are related to cognitive decline in type 1 diabetes mellitus patients

OBJECTIVE: Integrity of resting-state functional brain networks (RSNs) is important for proper cognitive functioning. In type 1 diabetes mellitus (T1DM) cognitive decrements are commonly observed, possibly due to alterations in RSNs, which may vary according to microvascular complication status. Thus, we tested the hypothesis that functional connectivity in RSNs differs according to clinical status and correlates with cognition in T1DM patients, using an unbiased approach with high spatio-temporal resolution functional network.; METHODS: Resting-state magnetoencephalographic (MEG) data for T1DM patients with (n=42) and without (n=41) microvascular complications and 33 healthy participants were recorded. MEG time-series at source level were reconstructed using a recently developed atlas-based beamformer. Functional connectivity within classical frequency bands, estimated by the phase lag index (PLI), was calculated within eight commonly found RSNs. Neuropsychological tests were used to assess cognitive performance, and the relation with RSNs was evaluated.; RESULTS: Significant differences in terms of RSN functional connectivity between the three groups were observed in the lower alpha band, in the default-mode (DMN), executive control (ECN) and sensorimotor (SMN) RSNs. T1DM patients with microvascular complications showed the weakest functional connectivity in these networks relative to the other groups. For DMN, functional connectivity was higher in patients without microangiopathy relative to controls (all p<0.05). General cognitive performance for both patient groups was worse compared with healthy controls. Lower DMN alpha band functional connectivity correlated with poorer general cognitive ability in patients with microvascular complications.; DISCUSSION: Altered RSN functional connectivity was found in T1DM patients depending on clinical status. Lower DMN functional connectivity was related to poorer cognitive functioning. These results indicate that functional connectivity may play a key role in T1DM-related cognitive dysfunction

Effectiveness and Limitations of Unsupervised Home-Based Balance Rehabilitation with Nintendo Wii in People with Multiple Sclerosis

Balance training represents a critical part of the rehabilitation process of individuals living with multiple sclerosis (MS) since impaired postural control is a distinctive symptom of the disease. In recent years, the use of the Nintendo Wii system has become widespread among rehabilitation specialists for this purpose, but few studies have verified the effectiveness of such an approach using quantitative measures of balance. In this study, we analyzed the postural sway features of a cohort of twenty-seven individuals with MS before and after 5 weeks of unsupervised home-based balance training with the Wii system. Center of pressure (COP) time-series were recorded using a pressure platform and processed to calculate sway area, COP path length, displacements, and velocities in mediolateral (ML) and anteroposterior (AP) directions. Although the results show a significant reduction in sway area, COP displacements, and velocity, such improvements are essentially restricted to the ML direction, as the Wii platform appears to properly stimulate the postural control system in the frontal plane but not in the sagittal one. Available Wii games, although somewhat beneficial, appear not fully suitable for rehabilitation in MS owing to scarce flexibility and adaptability to MS needs and thus specific software should be developed

Histopathological Defects in Intestine in Severe Spinal Muscular Atrophy Mice Are Improved by Systemic Antisense Oligonucleotide Treatment

Acknowledgments This study is supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London (FM and HZ), the Medical Research Council grant (grant reference MR/L013142/1, FM), SMA-Europe grant (FM and HZ) and Great Ormond Street Hospital Children’s Charity grants (FM and HZ). JEM is supported by Great Ormond Street Hospital Children’s Charity. PS is supported by Bill Marshall Fellowship and The CP Charitable Trust at Great Ormond Street Hospital and UCL. SHP is supported by SMA Trust and Euan MacDonald Centre for Motor Neurone Disease Research.Peer reviewedPublisher PD

Differential effects of phytotherapic preparations in the hSOD1 Drosophila melanogaster model of ALS

The present study was aimed at characterizing the effects of Withania somnifera (Wse) and Mucuna pruriens (Mpe) on a Drosophila melanogaster model for Amyotrophic Lateral Sclerosis (ALS). In particular, the effects of Wse and Mpe were assessed following feeding the flies selectively overexpressing the wild human copper, zinc-superoxide dismutase (hSOD1-gain-of-function) in Drosophila motoneurons. Although ALS-hSOD1 mutants showed no impairment in life span, with respect to GAL4 controls, the results revealed impairment of climbing behaviour, muscle electrophysiological parameters (latency and amplitude of ePSPs) as well as thoracic ganglia mitochondrial functions. Interestingly, Wse treatment significantly increased lifespan of hSDO1 while Mpe had not effect. Conversely, both Wse and Mpe significantly rescued climbing impairment, and also latency and amplitude of ePSPs as well as failure responses to high frequency DLM stimulation. Finally, mitochondrial alterations were any more present in Wse-but not in Mpe-Treated hSOD1 mutants. Hence, given the role of inflammation in the development of ALS, the high translational impact of the model, the known anti-inflammatory properties of these extracts, and the viability of their clinical use, these results suggest that the application of Wse and Mpe might represent a valuable pharmacological strategy to counteract the progression of ALS and related symptom

(1)H-NMR analysis provides a metabolomic profile of patients with multiple sclerosis

OBJECTIVE: To investigate the metabolomic profiles of patients with multiple sclerosis (MS) and to define the metabolic pathways potentially related to MS pathogenesis. METHODS: Plasma samples from 73 patients with MS (therapy-free for at least 90 days) and 88 healthy controls (HC) were analyzed by (1)H-NMR spectroscopy. Data analysis was conducted with principal components analysis followed by a supervised analysis (orthogonal partial least squares discriminant analysis [OPLS-DA]). The metabolites were identified and quantified using Chenomx software, and the receiver operating characteristic (ROC) curves were calculated. RESULTS: The model obtained with the OPLS-DA identified predictive metabolic differences between the patients with MS and HC (R2X = 0.615, R2Y = 0.619, Q2 = 0.476; p < 0.001). The differential metabolites included glucose, 5-OH-tryptophan, and tryptophan, which were lower in the MS group, and 3-OH-butyrate, acetoacetate, acetone, alanine, and choline, which were higher in the MS group. The suitability of the model was evaluated using an external set of samples. The values returned by the model were used to build the corresponding ROC curve (area under the curve of 0.98). CONCLUSION: NMR metabolomic analysis was able to discriminate different metabolic profiles in patients with MS compared with HC. With the exception of choline, the main metabolic changes could be connected to 2 different metabolic pathways: tryptophan metabolism and energy metabolism. Metabolomics appears to represent a promising noninvasive approach for the study of M

VGF peptides as novel biomarkers in Parkinson’s disease

Parkinson’s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN). At disease onset, a diagnosis is often difficult. VGF peptides are abundant in the SN and peripheral circulation; hence, we investigate whether their plasma profile may reflect the brain dopamine reduction. Using antibodies against the VGF C-terminal portion, we analyzed the rat brain and human plasma, with immunohistochemistry and ELISA. Rats were unilaterally lesioned with 6-hyroxydopamine and sacrificed either 3 or 6 weeks later with or without levodopa treatment. Plasma samples were obtained from PD patients, either at the time of diagnosis (group 1, drug naïve, n = 23) or upon dopamine replacement (group 2, 1–6 years, n = 24; group 3, &gt; 6 years, n = 16), compared with age-matched control subjects (group 4, n = 21). Assessment of the olfactory function was carried out in group 2 using the “Sniffin’ Sticks” test. VGF immunoreactivity was present in GABAergic neurons and, on the lesioned side, it was reduced at 3 weeks and abolished at 6 weeks after lesion. Conversely, upon levopoda, VGF labeling was restored. In PD patients, VGF levels were reduced at the time of diagnosis (1504 ± 587 vs. 643 ± 348 pmol/mL, means ± S.E.M: control vs. naïve; p &lt; 0.05) but were comparable with the controls after long-term drug treatment (&gt; 6 years). A linear correlation was demonstrated between VGF immunoreactivity and disease duration, levodopa equivalent dose and olfactory dysfunction. Plasma VGF levels may represent a useful biomarker, especially in the early stages of PD

Proteomic analysis identifies key differences in the cardiac interactomes of dystrophin and micro-dystrophin

ΔR4-R23/ΔCT micro-dystrophin (μDys) is a miniaturized version of dystrophin currently evaluated in a Duchenne muscular dystrophy (DMD) gene therapy trial to treat skeletal and cardiac muscle disease. In pre-clinical studies, μDys efficiently rescues cardiac histopathology, but only partially normalizes cardiac function. To gain insights into factors that may impact the cardiac therapeutic efficacy of μDys, we compared by mass spectrometry the composition of purified dystrophin and μDys protein complexes in the mouse heart. We report that compared to dystrophin, μDys has altered associations with α1- and β2-syntrophins, as well as cavins, a group of caveolae-associated signaling proteins. In particular, we found that membrane localization of cavins −1 and − 4 in cardiomyocytes requires dystrophin and is profoundly disrupted in the heart of mdx^{5cv} mice,a model of DMD. Following cardiac stress/damage, membrane-associated cavin-4 recruits the signaling molecule ERK to caveolae, which activates key cardio-protective responses. Evaluation of ERK signaling revealed a profound inhibition, below physiological baseline, in the mdx^{5cv} mouse heart. Expression of μDys in mdx^{5cv} mice prevented the development of cardiac histopathology but did not rescue membrane localization of cavins nor did it normalize ERK signaling. Our study provides the first comparative analysis of purified protein complexes assembled in vivo by full-length dystrophin and a therapeutic micro-dystrophin construct. This has revealed disruptions in cavins and ERK signaling that may contribute to DMD cardiomyopathy. This new knowledge is important for ongoing efforts to prevent and treat heart disease in DMD patients

Mucuna pruriens (Velvet bean) Rescues Motor, Olfactory, Mitochondrial and Synaptic Impairment in PINK1(B9) Drosophila melanogaster Genetic Model of Parkinson's Disease

The fruit fly Drosophila melanogaster (Dm) mutant for PTEN-induced putative kinase 1 (PINK1B9) gene is a powerful tool to investigate physiopathology of Parkinson's disease (PD). Using PINK1B9 mutant Dm we sought to explore the effects of Mucuna pruriens methanolic extract (Mpe), a L-Dopa-containing herbal remedy of PD. The effects of Mpe on PINK1B9 mutants, supplied with standard diet to larvae and adults, were assayed on 3–6 (I), 10–15 (II) and 20–25 (III) days old flies. Mpe 0.1% significantly extended lifespan of PINK1B9 and fully rescued olfactory response to 1-hexanol and improved climbing behavior of PINK1B9 of all ages; in contrast, L-Dopa (0.01%, percentage at which it is present in Mpe 0.1%) ameliorated climbing of only PINK1B9 flies of age step II. Transmission electron microscopy analysis of antennal lobes and thoracic ganglia of PINK1B9 revealed that Mpe restored to wild type (WT) levels both T-bars and damaged mitochondria. Western blot analysis of whole brain showed that Mpe, but not L-Dopa on its own, restored bruchpilot (BRP) and tyrosine hydroxylase (TH) expression to age-matched WT control levels. These results highlight multiple sites of action of Mpe, suggesting that its effects cannot only depend upon its L-Dopa content and support the clinical observation of Mpe as an effective medication with intrinsic ability of delaying the onset of chronic L-Dopa-induced long-term motor complications. Overall, this study strengthens the relevance of using PINK1B9 Dm as a translational model to study the properties of Mucuna pruriens for PD treatment

The brief international cognitive assessment for multiple sclerosis (BICAMS): Normative values with gender, age and education corrections in the Italian population

Background: BICAMS (Brief International Cognitive Assessment for Multiple Sclerosis) has been recently developed as brief, practical and universal assessment tool for cognitive impairment in MS subjects. It includes the Symbol Digit Modalities Test (SDMT), the California Verbal Learning Test-2 (CVLT2) and the Brief Visuospatial Memory Test-Revised (BVMT-R) . In this study we aimed at gathering regression based normative data for the BICAMS battery in the Italian population.Methods: Healthy subjects were consecutively recruited among patient friends and relatives. Corrections for demographics were calculated using multivariable linear regression models. Test-retest reliability was assessed using the Pearson correlation coefficient.Results: The BICAMS battery was administered to 273 healthy subjects (180 women, mean age 38.9 ± 13.0&nbsp;years, mean education 14.9 ± 3.0&nbsp;years). Test-retest reliability was good for all the tests.Conclusions: The study provided normative data of the BICAMS for the Italian population confirming good test-retest reliability which can facilitate the use of the battery in clinical practice, also for longitudinal patient assessments