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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.

BackgroundFamilial 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.ResultsSequencing 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.ConclusionThis 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.

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 evidences switch the hypothesis of amyotrophic lateral sclerosis (ALS) from a “neurocentric” to a “multisystemic” or “non-neurocentric” point of view. From 2006, we focused on the study of non-neural cells, patients’ peripheral blood mononuclear cells (PMBCs) and lymphoblastoid cell lines (LCLs). Here, we characterized LCLs of sporadic ALS and patients carrying SOD1, TARDBP and FUS mutations to identify ALS biologically relevant signature, and whether and how mutations differentially affect ALS-linked pathways. Although LCLs are different from motor neurons (MNs), in LCLs we find out some features typical of degenerating MNs in ALS, i.e. protein aggregation and mitochondrial dysfunction. Moreover, different gene mutations otherwise affect ALS cellular mechanisms. TARDBP and FUS mutations imbalance mitochondrial dynamism toward an increased fusion, while sALS and SOD1 mutations mainly affect fission. As regard protein aggregation and/or mislocalization, TARDBP and SOD1 mutations show the presence of aggregates, while FUS mutation does not induce protein aggregation and/or mislocalization. Finally, all LCLs, independently from mutation, are not able to work in a condition of excessive energy request, suggesting that mitochondria from ALS patients are characterized by a significant metabolic defect. Taken together these data indicate that LCLs could be indicated as a valid cellular model in ALS research to study specific pathological pathways or to identify new ones.

Duchenne muscular dystrophy is an X-linked muscle disease characterized by mutations in the dystrophin gene. Many of these can be corrected at the posttranscriptional level by skipping the mutated exon. We have obtained persistent exon skipping in mdx mice by tail vein injection with an adeno-associated viral (AAV) vector expressing antisense sequences as part of the stable cellular U1 small nuclear RNA. Systemic delivery of the AAV construct resulted in effective body-wide colonization, significant recovery of the functional properties in vivo, and lower creatine kinase serum levels, suggesting an overall decrease in muscle wasting. The transduced muscles rescued dystrophin expression and displayed a significant recovery of function toward the normal values at single muscle fiber level. This approach provides solid bases for a systemic use of AAV-mediated antisense-U1 small nuclear RNA expression for the therapeutic treatment of Duchenne muscular dystrophy.

We evaluated the effects of testosterone overload on mitochondrial superoxide dismutase (MnSOD), cytochrome oxidase (COX) and citrate synthase (CS) activities of the rat superficial gastrocnemius both in non-exercised muscle and following moderate endurance training. Basal (bLPO) and stimulated (sLPO) lipid peroxidation was measured as an index of oxidative tissue damage. Furthermore, to assess the relationship between exercise and testosterone-induced metabolic adaptations and contractile protein expression, the distribution of myosin heavy chain (MHC) isoforms was analysed by SDS-PAGE. Samples were obtained from: controls (C), rats treated with testosterone propionate (Tp) (TP, 5 mg kg(-1) i.m. 6 days/week), trained rats (E, 5 days/week) and rats trained and treated with Tp (ETP). MnSOD significantly increased in E and TP in comparison with C and ETP. Training induced a significant increase in COX activity both in E and ETP whereas a statistical reduction was observed in TP in comparison with the other groups. Moreover, testosterone administration was associated with a significant reduction in CS activity which significantly increased in ETP. A reduction in lipid peroxidation was observed in E and ETP in comparison with controls both in basal and stimulated conditions, whereas TP showed a significant increase of bLPO. In trained rats enzymatic changes were correlated with an increase in the proportion of fast oxidative MHC-2A and MHC-2X with decrease of the proportion of fast MHC-2B. In contrast, Tp treatment induced an increase in the proportion of MHC-2B whereas MHC-2A and MHC-2X disappeared. Finally, ETP showed a reduction in MHC-2B and an increase in MHC-1 and MHC-2X. These data suggest that testosterone supplementation seems not to significantly modify the metabolic adaptation induced by exercise in gastrocnemius muscle. Furthermore, testosterone overload to non-exercised rats seems to reduce the mitochondrial function and increase the lipid peroxidation of the muscle.

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. 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. 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. 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(-).