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Regulated removal of proteins and organelles by autophagy–lysosome system is critical for muscle homeostasis. Excessive activation of autophagy-dependent degradation contributes to muscle atrophy and cachexia. Conversely, inhibition of autophagy causes accumulation of protein aggregates and abnormal organelles, leading to myofiber degeneration and myopathy. Defects in lysosomal function result in severe muscle disorders such as Pompe (glycogen storage disease type II (GSDII)) disease, characterized by an accumulation of autophagosomes. However, whether autophagy is detrimental or not in muscle function of Pompe patients is unclear. We studied infantile and late-onset GSDII patients and correlated impairment of autophagy with muscle wasting. We also monitored autophagy in patients who received recombinant α -glucosidase. Our data show that infantile and late-onset patients have different levels of autophagic flux, accumulation of p62-positive protein aggregates and expression of atrophy-related genes. Although the infantile patients show impaired autophagic function, the late-onset patients display an interesting correlation among autophagy impairment, atrophy and disease progression. Moreover, reactivation of autophagy in vitro contributes to acid α -glucosidase maturation in both healthy and diseased myotubes. Together, our data suggest that autophagy protects myofibers from disease progression and atrophy in late-onset patients.

An abnormal structural form of glycogen (with less branching points or amylopectin-like polysaccharide) called polyglucosan (PG) may accumulate in various tissues such as striated and smooth muscles, brain, nerve, liver and skin, and cause a group of nine different genetic disorders manifesting with a variety of clinical phenotypes that affect mainly the nervous system (Lafora disease, adult PG body disease), the heart (glycogen storage disease type XV, hypertrophic cardiomyopathy type 6, PG body myopathy type 1) and the skeletal muscle (glycogen storage disease type IV, glycogen storage disease type VII, PG body myopathy type 2), depending on the organs which are mostly affected by the PG aggregates. The pathological feature of PG storage in tissues is a hallmark of these disorders. Whole-genome sequencing has allowed to obtain a diagnosis in a large number of patients with a previously unrecognized disorder. We describe the clinical, pathological and molecular features of these genetic disorders, for many of which the pathological mechanisms underlying the corresponding mutant gene have been investigated and, at least in part, understood.

In this work, the presence of calcium-dependent calcium channels and their receptors (RyR) has been investigated in Paracentrotus lividus eggs and early embryos, from unfertilized egg to four-blastomere stages. Electrophysiological recordings of RyR single-channel current fluctuations showed that RyRs are functional during the first developmental events with a maximum at zygote stage, c. 40 min after fertilization, corresponding to the first cleavage. The nature of vertebrate-like RyRs active at this stage was established by specific activation/blockade experiments.

Myotonic dystrophy (DM) is caused by a (CTG) n expansion in the 3′-untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby misregulating the alternative splicing. Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy. Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG) n range 90–1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and western blotting analyses of several markers of myogenesis indicated that in vitro differentiation–maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG) n expansions were confirmed by long PCR and RNA fluorescence in situ hybridization. Moreover, the DM1 myotubes showed the splicing alteration of insulin receptor and muscleblind-like 1 ( MBNL1 ) genes associated with the DM1 phenotype. Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62/LC3) markers. Z-VAD treatment significantly reduced the decrease in myonuclei number and in average width in 15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenance–regeneration, through premature apoptotic–autophagic activation, rather than altered myogenesis.

This updated and expanded new edition of a successful book describes genetic diagnostic entities of neuromuscular disorders. Neuromuscular syndromes are presented clinically either as a case study or as an overview from the literature, accompanied by text presenting molecular defects, and differential diagnosis. This collection of neuromuscular disorders features the differential clinical phenotypes related to each genotype and are representative of the whole spectrum of a genetic muscle disorder, helping the clinician and neuromuscular physician to make a diagnosis.

ObjectiveA multicentre observational study was aimed to assess the prevalence of late-onset Pompe disease (LOPD) in a large high-risk population, using the dried blood spot (DBS) as a main screening tool.Design/methods17 Italian neuromuscular centres were involved in the late-onset Pompe early diagnosis (LOPED) study. Inclusion criteria were: (1) age ≥5 years, (2) persistent hyperCKaemia and (3) muscle weakness at upper and/or lower limbs (limb-girdle muscle weakness, LGMW). Acid α-glucosidase (GAA) activity was measured separately on DBS by fluorometric as well as tandem mass spectrometry methods. A DBS retest was performed in patients resulted positive at first assay. For the final diagnosis, GAA deficiency was confirmed by a biochemical assay in skeletal muscle, whereas genotype was assessed by GAA molecular analysis.ResultsIn a 14-month period, we studied 1051 cases: 30 positive samples (2.9%) were detected by first DBS screening, whereas, after retesting, 21 samples were still positive. Biochemical and molecular genetic studies finally confirmed LOPD diagnosis in 17 cases (1.6%). The median time from the onset of symptoms/signs to diagnosis was 5 years. Among those patients, 35% showed presymptomatic hyperCKaemia and 59% showed hyperCKaemia+LGMW, whereas 6% manifested with LGMW.ConclusionsLOPED study suggests that GAA activity should be accurately screened by DBS in all patients referring for isolated hyperCKaemia and/or LGMW. A timely diagnosis was performed in five patients with presymptomatic hyperCKaemia, but two had already manifested with relevant changes on muscle morphology and MRI. Consequently, enzyme replacement therapy was started in 14/17 patients, including the 2 patients still clinically presymptomatic but with a laboratory evidence of disease progression.

The objective of this study was to describe a large Italian cohort of patients with late-onset glycogen storage disease type 2 (GSDII) at various stages of disease progression and to evaluate the clinical effectiveness of alglucosidase alpha enzyme replacement therapy (ERT). Previous studies showed in late-onset patients ERT efficacy against placebo and variable response in uncontrolled studies. Seventy-four juvenile or adult GSDII patients were treated with ERT in a multicenter open label, non-randomized study, from 12 months up to 54 months. Recombinant human alpha glucosidase (rh-GAA) was injected by intravenous route at 20 mg/kg every second week. Patients were divided into three groups according to ERT duration: Group A received treatment for 12–23 months ( n  = 16), Group B for 24–35 months ( n  = 14), and Group C for more than 36 months ( n  = 44). Clinical assessment included a 6-min walk test (6MWT), forced vital capacity (FVC), the Walton and Gardner-Medwin score, the number of hours of ventilation, body mass index, echocardiography and blood creatine kinase (CK). Included in our cohort were 33 males and 41 females (M:F = 0.8:1), with a mean age at first symptoms of 28.3 years (range 2–55 years) and a mean age of 43 years at study entry (range 7–72 years). Seven wheelchair bound patients, as well as 27 patients requiring ventilation support, were included. After treatment we could observe an increase in distance walked on the 6MWT in the large majority of patients (48/58; 83%), with an overall mean increase of 63 m (from 320 ± 161 to 383 ± 178 m). After treatment in the majority of patients FVC was improved or unchanged (45/69; 65%). In ventilated patients we observed an improvement in average number of hours off the ventilator (from 15.6 to 12.1 h). Six patients stopped mechanical ventilation and two others started it. The effect of therapy was not related to ERT duration. Nine of 64 patients (13%) that underwent to echocardiography showed a variable degree of cardiac hypertrophy (left ventriculum or septum), and a positive effect was observed after 36 months of ERT in one adult case. Discontinuation of treatment occurred in four patients: one drop-off case, one patient died for a sepsis after 34 months of treatment and two patients stopped ERT for worsening of general clinical condition. Mild adverse effects were observed in four cases (5%). This study represents the largest cohort of late-onset GSDII patients treated with ERT, and confirm a positive effect of treatment. These results, obtained in a large case series on therapy, indicate a favourable effect of ERT therapy, even in more advanced stage of the disease.

Ocular myopathy, typically manifesting as progressive external ophthalmoplegia (PEO), is among the most common mitochondrial phenotypes. The purpose of this study is to better define the clinical phenotypes associated with ocular myopathy. This is a retrospective study on a large cohort from the database of the “Nation-wide Italian Collaborative Network of Mitochondrial Diseases”. We distinguished patients with ocular myopathy as part of a multisystem mitochondrial encephalomyopathy (PEO-encephalomyopathy), and then PEO with isolated ocular myopathy from PEO-plus when PEO was associated with additional features of multisystemic involvement. Ocular myopathy was the most common feature in our cohort of mitochondrial patients. Among the 722 patients with a definite genetic diagnosis, ocular myopathy was observed in 399 subjects (55.3%) and was positively associated with mtDNA single deletions and POLG mutations. Ocular myopathy as manifestation of a multisystem mitochondrial encephalomyopathy (PEO-encephalomyopathy, n  = 131) was linked to the m.3243A>G mutation, whereas the other “PEO” patients ( n  = 268) were associated with mtDNA single deletion and Twinkle mutations. Increased lactate was associated with central neurological involvement. We then defined, among the PEO group, as “pure PEO” the patients with isolated ocular myopathy and “PEO-plus” those with ocular myopathy and other features of neuromuscular and multisystem involvement, excluding central nervous system. The male proportion was significantly lower in pure PEO than PEO-plus. This study reinforces the need for research on the role of gender in mitochondrial diseases. The phenotype definitions here revisited may contribute to a more homogeneous patient categorization, useful in future studies and clinical trials.

Climate change is altering consumer–plant interactions in ecosystems worldwide. How consumers alter their spatial distribution, grazing activities, and functional morphology in response to climate stress can determine whether their effects on plants intensify or relax. Few studies have considered multiple consumer response metrics to elucidate the mechanisms underpinning the resulting changes in consumer–plant interactions. Here, we tested how drought stress influences the interaction between the dominant consumer, the fungal-farming periwinkle snail Littoraria irrorata, and a foundational plant, cordgrass Spartina alterniflora, in a southeastern US salt marsh. In a 4 mo field experiment, we maintained moderate snail densities in mesh control chambers and clear plastic climate chambers that simulated drought by elevating temperatures and drying soils. Monitoring revealed that snails more often congregated on cordgrass stems than leaves in climate chambers than in controls. Image analyses indicated that this behavioral shift corresponded to snails inflicting shorter, but more numerous, fungal-infested scars on cordgrass leaves, and causing less plant damage in climate chambers than controls. Coincident with their net reduction in grazing, snails maintained longer radulae, whose central teeth were blunter and lateral teeth were sharper, in climate chambers compared to controls. These results suggest that under drought, snail radu lae may experience less frictional wear and that, at intermediate densities, snail–cordgrass interactions re lax. Together with prior research showing that at high densities, snails can denude cordgrass during drought, we conclude that consumer density, behavior, and morphological responses must be integrated in predictions of how climate change will affect the direction, strength, and stability of consumer–plant interactions.

The objective of this study was to determine the degree of brain involvement in a cohort of myotonic dystrophy type 1 and type 2 (DM1, DM2) patients by brain studies and functional tests and to compare the results of the two groups. DM1, DM2 are multisystemic disorders due to polynucleotide expansions. Previous studies on brain involvement by neuroimaging and functional methods have led to contradictory results. Fifty molecularly defined DM1 patients and 14 DM2 patients, were recruited for the study. Age at recruitment, age at disease onset, disease duration and educational level were recorded. Neuromuscular assessment was done by MIRS. An extensive neuropsychological battery was performed in 48/50 DM1 and in a control group of 44 healthy matched subjects. Forty six of 50 DM1 and 12/14 DM2 underwent brain MRI; 21/50 DM1 and 9/14 DM2 underwent brain perfusion SPECT, with semiquantitative analysis of the results. MRI images were classified by ARWMC (age-related white matter changes) score, in order to quantify recurrence, localization and patterns of distribution of white matter hyperintense lesions (WMHLs) in our two cohorts. MRI results were matched to SPECT and to neuropsychological results. Thirty-seven of 46 DM1 and 10/12 DM2 had abnormal MRI imaging, showing scattered supratentorial, bilateral, symmetrical focal or diffuse WMHLs. A typical temporo-insular diffuse subcortical pattern was seen in DM1 subjects only, with no correlation with cognitive involvement. Major cognitive involvement was seen in the case of diffuse frontal lesions. A relationship with CTG expansion size was documented for DM1 subjects. SPECT showed minimal hypoperfusion in the posterior cortex planes in DM1 and, to a lesser extent, in DM2. Very mild degrees of involvement in the DM2 cohort were seen. Neuroimaging and functional investigations confirmed a more severe involvement of the brain in DM1 compared to DM2. A temporo-insular diffuse lesional pattern, specific for DM1, was found on MRI. This confirms greater expansion size as a risk factor for more extensive brain involvement in DM1.