Preliminary insights into RNA in CSF of pediatric SMA patients after 6 months of nusinersen.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare autosomal-recessive neurodegenerative disorder caused by mutations in survival motor neuron 1 (SMN1) gene, and consequent loss of function of SMN protein, which results in progressive loss of lower motor neurons, and muscular wasting. Antisense oligonucleotide (ASO) nusinersen (Spinraza®) modulates the pre-mRNA splicing of the SMN2 gene, allowing rebalance of biologically active SMN. It is administered intrathecally via lumbar puncture after removing an equal amount of cerebrospinal fluid (CSF). Its effect was proven beneficial and approved since 2017 for SMA treatment. Given the direct effect of nusinersen on RNA metabolism, the aim of this project was to evaluate cell-free RNA (cfRNA) in CSF of SMA patients under ASOs treatment for biomarker discovery. METHODS: By RNA-sequencing approach, RNA obtained from CSF of pediatric SMA type 2 and 3 patients was processed after 6 months of nusinersen treatment, at fifth intrathecal injection (T6), and compared to baseline (T0). RESULTS: We observed the deregulation of cfRNAs in patients at T6 and we were able to classify these RNAs into disease specific, treatment specific and treatment dependent. Moreover, we subdivided patients into "homogeneous" and "heterogeneous" according to their gene expression pattern. The "heterogeneous" group showed peculiar activation of genes coding for ribosomal components, meaning that in these patients a different molecular effect of nusinersen is observable, even if this specific molecular response was not referable to a clinical pattern. CONCLUSIONS: This study provides preliminary insights into modulation of gene expression dependent on nusinersen treatment and lays the foundation for biomarkers discovery.

New CACNA1A deletions are associated to migraine phenotypes.

BACKGROUND: Familial hemiplegic migraine type 1 (FHM1) is a form of migraine with aura caused by heterozygous mutations in 4 genes: CACNA1A, ATP1A2, SNC1A and PRRT2, but further heterogeneity is expected. Here have been described clinical and molecular features in patients suffering from migraine with Aura (MA), without (MO) and hemiplegic migraine attacks. Next Generation Sequencing by TruSeq Custom Amplicon for CACNA1A and ATP1A2 gene has been performed. All genetic variants have been confirmed by Sanger sequencing and all samples were also analyzed with MLPA assay for ATP1A2-CACNA1A genes to detect duplication or deletion. All MLPA data were verified by Real Time PCR. RESULTS: Sequencing analysis showed 3 point mutations, two novel variants and one already described in literature. Moreover, MLPA analysis showed 3 deletions in 9 sporadic hemiplegic migraine (18%), in 3 patients with non-hemiplegic migraine (4.1%) and in 3 patients affected by episodic ataxia (20%). Two sporadic patients showed a deletion in exons 41-43, while the rest of HM patients (5) showed a deletion in the terminal part of the CACNA1A gene. About episodic ataxia, we have identified deletions in exon 12-15 and in exon 47. Finally, in migraine patients, we have found different subjects affected by different phenotypes deleted in exon 47. CONCLUSION: This work highlights the importance to complement analysis as direct sequencing with quantitative analysis (MLPA). In fact, intragenic CACNA1A rearrangements have been detected. Our work demonstrated that deletions in CACNA1A gene may be associated also to different migraine phenotypes.

Amyotrophic lateral sclerosis and skeletal muscle: an update.

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disease characterized by degeneration of upper and lower motor neurons (MNs), generalized weakness and muscle atrophy. The "neurocentric" view of ALS assumes that the disease primarily affects motor neurons, while muscle alterations only represent a consequence, in the periphery, of motor neuron loss. However, this outlook was recently challenged by evidence suggesting that non-neural cells such as microglia, astrocytes, peripheral blood mononuclear cells (PBMCs) and skeletal muscle fibres participate in triggering motor neuron degeneration, and this stressed the concept that alterations in different cell types may act together to exacerbate the disease. In this review, we will summarize the most recent findings on the alterations of skeletal muscle fibres found in ALS, with particular attention to the relationship between mutant SOD1 and skeletal muscle. We will analyze changes in muscle function, in the expression of myogenic regulatory factors, and also mitochondrial dysfunction, SOD1 aggregation and proteasome activity.

Autologous transplantation of muscle-derived CD133+ stem cells in Duchenne muscle patients.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive muscle disease due to defect on the gene encoding dystrophin. The lack of a functional dystrophin in muscles results in the fragility of the muscle fiber membrane with progressive muscle weakness and premature death. There is no cure for DMD and current treatment options focus primarily on respiratory assistance, comfort care, and delaying the loss of ambulation. Recent works support the idea that stem cells can contribute to muscle repair as well as to replenishment of the satellite cell pool. Here we tested the safety of autologous transplantation of muscle-derived CD133+ cells in eight boys with Duchenne muscular dystrophy in a 7-month, double-blind phase I clinical trial. Stem cell safety was tested by measuring muscle strength and evaluating muscle structures with MRI and histological analysis. Timed cardiac and pulmonary function tests were secondary outcome measures. No local or systemic side effects were observed in all treated DMD patients. Treated patients had an increased ratio of capillary per muscle fibers with a switch from slow to fast myosin-positive myofibers.

Age and sex differences in human skeletal muscle: role of reactive oxygen species.

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17-40 years), adult (41-65 years) and aged (66-91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66-91 year-old vs. the 17-40 year-old men; MnSOD activity was significantly greater in 66-91 year-old vs. 17-40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41-65 and 66-91 year-old vs. the 17-40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.

In vitro antioxidant properties of amifostine (WR-2721, Ethyol).

PURPOSE: Amifostine (WR-2721), a phosphorylated aminothiol pro-drug which is an analogue of cysteamine, is a selective cytoprotective agent for normal tissues from the toxicities associated with chemotherapy and irradiation. Despite a growing number of reports strongly supporting amifostine's clinical efficacy, few authors have focused on the biochemical basis of amifostine's antioxidant activity. METHODS: We report on amifostine's free-radical scavenging activity against superoxide (O(2;(-))), hydroxyl (OH(-)) and lipoperoxyl radicals in an in vitro model, using pure chemical systems. Amifostine was dephosphorylated to its active metabolite, WR-1065, by adding 10% non-heat-inactivated serum; different amifostine concentrations (1, 10, 50, 100 microM and 200 microM) and pH conditions (pH 5, 7.4 and 9) were tested. RESULTS: Independent of the concentration, amifostine exhibited no major activity against O(2;(-)) ions, neither did any pH variations in the experimental model provide any scavenger effects of the drug against O(2;(-)) radicals. On the other hand, the protective effect of amifostine against OH(-) radicals was confirmed, yielding an EC(50) of 255 microM at pH 7.4 and 230 microM at pH 5. Finally, amifostine exhibited scavenging activity against spontaneous lipoperoxidation, but no apparent antioxidant effect on iron ascorbate-induced lipoperoxidation. CONCLUSIONS: With this in vitro study, we are able to confirm the scavenging activity of the chemo- and radioprotector amifostine, whose activity seems to be particularly important from a biological point of view, since it is exerted mainly against highly reactive OH(-).

Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle.

This study was conducted in order to provide evidence for the role of reactive oxygen species (ROS) in human skeletal muscle aging. We used human muscle samples obtained from hospitalized patients in an open study with matched pairs of individuals of different ages. The subjects, ranging in age from 17 to 91 years, were grouped as follows: 17-25-, 26-35-, 36-45-, 46-55-, 56-65-, 66-75-, 76-85-, and 86-91-year-old groups. To investigate the relationship between muscle aging and oxidative damage we measured total and Mn-dependent superoxide dismutase (total SOD, MnSOD), glutathione peroxidase (GSHPx), and catalase (CAT) activities; total reduced and oxidized glutathione (GSHtot, GSH, and GSSG) levels; lipid peroxidation (LPO), and protein carbonyl content (PrC). Total SOD activity decreases significantly with age in the 66-75-year-old group, although MnSOD activity increases significantly in the 76-85-year-old group. The activity of the two H2O2 detoxifying enzymes (GSHPx and CAT) did not change with age, as do GSHtot and GSH levels. GSSG levels increased significantly (76-85- and 86-91-year-old groups) with age. We observed a significant increase in LPO levels (66-75- and 76-85-year-old groups), although the PrC content shows a trend of increase without gaining the statistical significance. These results support the idea that ROS play an important role in the human muscle aging process.

Blood free radical antioxidant enzymes and lipid peroxides following long-distance and lactacidemic performances in highly trained aerobic and sprint athletes.

OBJECTIVE: We have determined the differences of the influence of prolonged exercise or higher intensity lactacidemic exercise, on plasma lipid peroxidation and on erythrocyte antioxidant enzymatic defence system. EXPERIMENTAL DESIGN: We measured plasma indices of lipid peroxidation, conjugated dienes (CD) and malondialdehyde (MDA) and erythrocyte enzymes superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase (CAT). The biochemical evaluations were performed in six healthy control males (C) and twelve athletes: six marathon runners (MR) and six sprint-trained athletes (STA) at rest and after a half-marathon (MR) and a training session of 6 x 150 m (STA). RESULTS: In resting conditions MDA was higher in STA and MR than in C (p < 0.01), while only the MR showed significantly elevated levels of CD (p < 0.05). In STA the enzymatic scavenging capacity showed a significantly higher SOD (p < 0.01) and GSHPx (p < 0.01), while CAT was lower than in controls (p < 0.05). In MR only SOD (p < 0.01) was significantly higher than in C. It increased significantly immediately after half-marathon, while CAT decreased 24 and 48 hours postexercise respectively. In these athletes the lipoperoxidative indices increased in the early postexercise phase, while at 24 and 48 hrs both CD and MDA levels decreased. In STA enzyme activities were not modified by anaerobic performance while CD showed a peak 6 hrs postexercise and the MDA showed a progressive increase until 48 hrs afterwards. CONCLUSIONS: Both strenuous long duration exercise and exhaustive sprint training overwhelm our capacity to detoxify ROS, producing oxidative stress. Thus an adequate supply of antioxidants could be appropriate.