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LAMA2 mutations cause the most frequent congenital muscular dystrophy subtype MDC1A and a variety of milder phenotypes, characterized by total or partial laminin-α2 deficiency. In both severe and milder cases brain MRI invariably shows abnormal white matter signal intensity. We report clinical, histopathological, imaging and genetic data on two siblings with very subtle, and at first undetected, reduction in laminin-α2 expression, and brain MRI showing minor non-specific abnormalities. Clinical features in the female proband were characterized by muscle weakness involving neck and axial muscles, and pelvic girdle and distal lower limb muscles, reduced tendon reflexes and pes cavus. Clinical features in a younger brother were similar, and remained stable in both siblings during the follow up. Whole exome sequencing (WES) detected two heterozygous truncating LAMA2 mutations. Brain MRI in combination with laminin-α2 immunohistochemistry might not be sufficient and WES might be the only means to reach a diagnosis.

Background Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease. Methods Whole exome sequencing was performed on a family diagnosed with nonspecific myopathic changes by means of histological analysis and immunohistochemistry of muscle biopsy samples. Results A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK was completely absent in these patients. Conclusions Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues.

Background: Congenital muscular dystrophies (CMD) with reduced glycosylation of alpha-dystroglycan (α-DG) are a heterogeneous group of conditions associated with mutations in six genes encoding proven or putative glycosyltransferases. Objectives: The aim of the study was to establish the prevalence of mutations in the six genes in the Italian population and the spectrum of clinical and brain MRI findings. Methods: As part of a multicentric study involving all the tertiary neuromuscular centers in Italy, FKRP , POMT1 , POMT2 , POMGnT1 , fukutin , and LARGE were screened in 81 patients with CMD and α-DG reduction on muscle biopsy (n = 76) or with a phenotype suggestive of α-dystroglycanopathy but in whom a muscle biopsy was not available for α-DG immunostaining (n = 5). Results: Homozygous and compound heterozygous mutations were detected in a total of 43/81 patients (53%), and included seven novel variants. Mutations in POMT1 were the most prevalent in our cohort (21%), followed by POMT2 (11%), POMGnT1 (10%), and FKRP (9%). One patient carried two heterozygous mutations in fukutin and one case harbored a new homozygous variant in LARGE. No clear-cut genotype-phenotype correlation could be observed with each gene, resulting in a wide spectrum of clinical phenotypes. The more severe phenotypes, however, appeared to be consistently associated with mutations predicted to result in a severe disruption of the respective genes. Conclusions: Our data broaden the clinical spectrum associated with mutations in glycosyltransferases and provide data on their prevalence in the Italian population.

Exosomes, natural carriers of mRNAs, non-coding RNAs and proteins between donor and recipient cells, actively contribute to cell-cell communication. We investigated the potential pro-fibrotic role of exosomes released by muscle-derived fibroblasts of Duchenne muscular dystrophy (DMD) patients, and of miRNAs carried by exosomes. By fibrosis focused array analysis we found that exosomes from DMD fibroblasts, had significantly higher levels of miR-199a-5p, a miRNA up-regulated in fibrotic conditions, compared to control exosomes, while levels in myoblast-derived exosomes were not increased. In control fibroblasts, exposure to DMD fibroblast-derived exosomes induced a myofibroblastic phenotype with increase in α-smooth actin, collagen and fibronectin transcript and protein expression, soluble collagen production and deposition, cell proliferation, and activation of Akt and ERK signaling, while exposure to control exosomes did not. Transfecting control fibroblasts or loading control exosomes with miR-199a-5p mimic or inhibitor induced opposing effects on fibrosis-related mRNAs and proteins, on collagen production and Akt and ERK pathways. Finally, injection of DMD fibroblast-derived exosomes into mouse tibialis anterior muscle after cardiotoxin-induced necrosis, produced greater fibrosis than control exosomes. Our findings indicate that exosomes produced by local fibroblasts in the DMD muscle are able to induce phenotypic conversion of normal fibroblasts to myofibroblasts thereby increasing the fibrotic response. This conversion is related to transfer of high levels of miR-199a-5p and to reduction of its target caveolin-1; both, therefore, are potential therapeutic targets in muscle fibrosis.

Limb girdle muscular dystrophies (LGMD) are a large group of muscular disorders, with progressive shoulder and pelvic muscle weakness as the most relevant feature. They are classified as autosomal dominant (LGMD1) and autosomal recessive (LGMD2) forms. Up to now, eight genetically defined LGMD1 subtypes (LGMD1 A-H) have been identified [1, 9]. In 2001, the clinical and morphological phenotype of a novel form of LGMD type 1, affecting 32 subjects in a large Spanish family, was described [5]. According to subsequent molecular studies, the disease was demonstrated to be linked to the novel chromosomal locus 7q32.1–32.2. This genetically distinct form of autosomal dominant-LGMD was classified as LGMD1F [10] (OMIM #608423). Recently, using a whole genome sequencing approach, the causative mutation of the LGMD1F was identified in the termination codon of TNPO3, the gene coding for transportin 3. Molecular results at DNA, RNA and protein levels as well as morphological findings supported the pathogenic role of this mutation in LGMD1F [8]. Investigation by next-generation sequencing in further 4 members of the Spanish family, originating from Italy, confirmed the mutation in TNPO3 [13]. Up to now, beside this Italo-Spanish family, only one sporadic LGMD patient has been identified with a heterozygous point mutation in the TNPO3 gene [13]. In this patient we now report the long term clinical and radiological follow-up, morphological and immunochemical studies on patient muscle biopsy, and molecular studies by Real Time PCR and by cell transfection with the mutant cDNA.

Aims Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α-glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fiber vacuolization and autophagy. Since 2006, ERT is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT-related skeletal muscle changes in 18 modestly to moderately affected LOPD patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short-to-long term. Methods We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed whereas the second biopsy was always performed after at least six months of ERT administration. Results After ERT, 15 out of 18 patients showed improved 6MWT (p=0.0007) and most of them achieved respiratory stabilization. Pre-treatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in 3 patients. Post-ERT GAA enzymatic activity was mildly increased compared with pre-treatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; p

The Biobank was established in 1986 as part of the routine diagnostic activity of the Division of Neuromuscular Diseases and Neuroimmunology, of the Carlo Besta Neurological Institute. It stores muscle tissue, cells and DNA from patients with neuromuscular diseases.The biobank provides samples as a service to the scientific community conducting research on neuromuscular disorders. Samples are from patients affected by different forms of muscular dystrophy, including the severe congenital and Duchenne muscular dystrophies, as well as limb girdle muscular dystrophies, congenital myopathies, distal and myofibrillar myopathies, inflammatory myopathies, and metabolic myopathies. Different types of biomaterials are frequently available from a single patient.The Biobank is founding partner of the EuroBioBank network, the first operating network of biobanks for rare diseases in Europe, and of the Italian Telethon Network of Genetic Biobanks. The involvement of the biobank into both networks has been instrumental for standardization of procedures and activities, implementation of sample access policies, and compliance with ELSI requirements. The biobank, with about 13000 biospecimens stored in total at the time of writing, constitutes a key source of biological samples for researchers worldwide.

Dystroglycan is a transmembrane glycoprotein whose interactions with the extracellular matrix (ECM) are necessary for normal muscle and brain development, and disruptions of its function lead to dystroglycanopathies, a group of congenital muscular dystrophies showing extreme genetic and clinical heterogeneity. Specific glycans bound to the extracellular portion of dystroglycan, α-dystroglycan, mediate ECM interactions and most known dystroglycanopathy genes encode glycosyltransferases involved in glycan synthesis. POMK, which was found mutated in two dystroglycanopathy cases, is instead involved in a glycan phosphorylation reaction critical for ECM binding, but little is known about the clinical presentation of POMK mutations or of the function of this protein in the muscle. Here, we describe two families carrying different truncating alleles, both removing the kinase domain in POMK, with different clinical manifestations ranging from Walker–Warburg syndrome, the most severe form of dystroglycanopathy, to limb-girdle muscular dystrophy with cognitive defects. We explored POMK expression in fetal and adult human muscle and identified widespread expression primarily during fetal development in myocytes and interstitial cells suggesting a role for this protein during early muscle differentiation. Analysis of loss of function in the zebrafish embryo and larva showed that pomk function is necessary for normal muscle development, leading to locomotor dysfuction in the embryo and signs of muscular dystrophy in the larva. In summary, we defined diverse clinical presentations following POMK mutations and showed that this gene is necessary for early muscle development.

Mutations in the DNAJB6 gene have been associated with the autosomal dominant limb girdle muscular dystrophy type 1D (LGMD1D), a disorder characterized by abnormal protein aggregates and rimmed vacuoles in muscle fibers. DNAJB6 is a ubiquitously expressed Hsp40 co-chaperone characterized by a J domain that specifies Hsp70 functions in the cellular environment. DNAJB6 is also a potent inhibitor of expanded polyglutamine (polyQ) aggregation preventing aggregate toxicity in cells. In DNAJB6-mutated patients this anti-aggregation property is significantly reduced, albeit not completely lost. To elucidate the pathogenetic mechanisms underlying the DNAJB6-related myopathy, animal models have been created showing that, indeed, conditional muscular expression of a DNAJB6 mutant in the mouse causes a LGMD1D myofibrillary muscle tissue phenotype. Both mutations and phenotypes reported until recently were rather homogeneous, being exclusively missense mutations of a few amino acids of the protein G/F domain, and with a phenotype characterized by adult-onset slowly progressive muscular dystrophy predominantly affecting proximal muscles. Lately, several novel mutations and new phenotypes of DNAJB6 have been described. These mutations once more affect the G/F domain of DNAJB6 with missense changes and a splice site mutation; and the phenotypes include childhood onset and distal involvement of muscles, or childhood-onset LGMD1D with loss of ambulation in early adulthood and respiratory involvement. Thus, the spectrum of DNAJB6-related phenotypes is widening. Although our knowledge about the role of DNAJB6 in the pathogenesis of muscle diseases has made great progression, several questions remain unsolved, including why a ubiquitous protein affects only, or predominantly, skeletal muscle; why only the G/F domain is involved; and what is the possible role of the DNAJB6a isoform. Clarification of these issues will provide clues to implement possible therapeutic strategies for DNAJB6-related myopathies.

Congenital muscular dystrophies due to defects in genes encoding proteins involved in α-dystroglycan (α-DG) glycosylation are a heterogeneous group of muscle disorders variably associated with central nervous system and eye abnormalities. One of the more severe is muscle-eye-brain disease (MEB). Mutations in genes coding for proven or putative glycosyltransferases (POMT1, POMT2, POMGnT1, fukutin, FKRP, and LARGE), the DPM3 gene encoding a DOL-P-Man synthase subunit, and the DAG1 gene encoding α-dystroglycan, have been associated with altered α-DG glycosylation. We report new POMGnT1 mutations and evaluate protein expression in 3 patients and 2 foetuses with variably severe MEB features. We identify two new point mutations (c.643 C > T, c.1863delC), one new intragenic rearrangement (deletion of exons 2–8), and a new intron retention (between exons 21 and 22) resulting from a known point mutation c.1895 + 1 G > T. Our study provides further evidence that rearrangements of the POMGnT1 gene are relatively common. Importantly, if heterozygous, they can be missed on standard genomic DNA sequencing. POMGNT1 protein analysis in 3 patients showed that the severity of the phenotype does not correlate with protein expression. Cerebral MRI is important for identifying MEB and α-dystroglycanopathy phenotypes in children and foetuses, and hence for directing the genetic analysis.

We report on a patient with muscle pain not associated with muscle weakness. Microscopic examination of the muscle biopsy revealed rod-like cytoplasmic bodies in many fast fibres. By electron microscopy these had a crystalloid structure identical to the hexagonally cross-linked caveolin 3-positive tubular arrays, previously described in patients with similarly benign myopathy. We found that these inclusions were positive for calsequestrin and for the calsequestrin-binding protein junctin, as well as for caveolin 3. However, the genes coding for these proteins were not mutated. For diagnostic purposes calsequestrin and caveolin 3 positivity should be checked when rods are encountered in muscle biopsy for mild myopathy.

Mutations in POMT1 and POMT2 genes were originally identified in Walker–Warburg syndrome (WWS) and subsequently reported in patients with milder phenotypes characterised by mental retardation with or without brain abnormalities and without ocular malformations. As part of a multicentric Italian study we screened the POMT1 and POMT2 genes in 61 congenital muscular dystrophy (CMD) patients with α-dystroglycan reduction on muscle biopsy and/or clinical and radiological findings suggestive of the known forms of CMD with α-dystroglycan deficiency. The aim of the study was to establish how frequently mutations in POMT1 and POMT2 occur in CMD patients in the Italian population and to evaluate the spectrum of associated phenotypes. Thirteen patients showed mutations in POMT1 and five harboured mutations in POMT2 , accounting for a total of 20 different mutations, eight of which were novel (two in POMT1 and six in POMT2 ). Normal brain MRI associated with mental retardation and microcephaly was the most frequent finding in patients with mutations in POMT1 (six out of 13), but was also found in a patient with POMT2 mutations. Predominant cerebellar hypoplasia was also frequent both in patients with POMT1 (three out of 13) and POMT2 (three out of 5) mutations. A MEB phenotype with frontal cortical dysplasia and pons abnormalities was found in two patients with POMT1 and in one with POMT2 mutations, while a WWS phenotype was only found in a case with mutations in POMT1 . Mutations causing frameshifts and stop codons were responsible for the more severe phenotypes. Our results provide further evidence that, as previously reported for FKRP , the array of mutations in POMT1 and POMT2 is ample and the spectrum of associated phenotypes is wider than initially thought.

The combination of pulse-chase experiments with high-performance liquid chromatography and continuous flow scintillation detection was used successfully to determine the effects of chronic diabetes on neurosteroid production in the adult rat spinal cord. The long-term diabetes was induced by treatment of adult rats with streptozotocin. In the first part, the review provides an extensive description of the HPLC combined with continuous flow scintillation detection method, its advantages and appropriateness for the question investigated. Afterwards, the paper shows that progesterone formation is up-regulated in the spinal cord of diabetic rats while the biosynthesis of tetrahydroprogesterone decreased. The down-regulation of tetrahydroprogesterone appeared as a mechanism facilitating progesterone accumulation in the spinal cord of streptozotocin-treated rats. Progesterone is well known to be a potent neuroprotective steroid. Enhancement of its biosynthesis may be an endogenous mechanism triggered by neural cells in the spinal tissue to cope with degenerative effects provoked by chronic diabetes. Since steroid metabolism in the spinal cord is pivotal for the modulation of several neurobiological processes including sensorimotor activities, the data analyzed herein may constitute useful information for the development of efficient strategies against deleterious effects of diabetes on the nervous system.

An autosomal dominant protein aggregate myopathy, characterized by high plasma creatine kinase and calsequestrin-1 (CASQ1) accumulation in skeletal muscle, has been recently associated with a missense mutation in CASQ1 gene. The mutation replaces an evolutionarily-conserved aspartic acid with glycine at position 244 (p.D244G) of CASQ1, the main sarcoplasmic reticulum (SR) Ca2+ binding and storage protein localized at the terminal cisternae of skeletal muscle cells. Here, immunocytochemical analysis of myotubes, differentiated from muscle-derived primary myoblasts, shows that sarcoplasmic vacuolar aggregations positive for CASQ1 are significantly larger in CASQ1-mutated cells than control cells. A strong co-immuno staining of both RyR1 and CASQ1 was also noted in the vacuoles of myotubes and muscle biopsies derived from patients. Electrophysiological recordings and sarcoplasmic Ca2+ measurements provide evidence for less Ca2+ release from the SR of mutated myotubes when compared to that of controls. These findings further clarify the pathogenic nature of the p.D244G variant and point out defects in sarcoplasmic Ca2+ homeostasis as a mechanism underlying this human disease, which could be distinctly classified as "CASQ1-couplonopathy"., peer-reviewed

Within the group of muscular dystrophies, dystroglycanopathies represent an important subgroup of recessively inherited disorders. Their severity varies from the relatively mild forms of adult-onset limb-girdle muscular dystrophy (LGMD), to the severe congenital muscular dystrophies (CMD) with cerebral and ocular involvement. We describe 2 consanguineous children of Pakistani origin, carrying a new homozygous missense mutation c.367G>A (p.Gly123Arg) in the ISPD gene. Mutations in this gene have been recently reported as a common cause of congenital and limb-girdle muscular dystrophy. Patient 1 is an 8-year-old female with an intermediate phenotype between CMD and early LGMD; patient 2 is a 20-month-old male and second cousin of patient 1, showing a CMD phenotype. Cognitive development, brain MRI, eye examination, electrocardiogram and echocardiogram were normal in both patients. To our knowledge, this is the first report on the co-occurrence of both a CMD/early LGMD intermediate phenotype and a CMD within the same family carrying a homozygous ISPD mutation.

Introduction Protein aggregation is a common cause of neuropathology. The protein aggregation myopathy Limb-Girdle Muscular Dystrophy 1D (LGMD1D) is caused by mutations of amino acids Phe89 or Phe93 of DNAJB6, a co-chaperone of the HSP70 anti-aggregation protein. Another DNAJB6 mutation, Pro96Arg, was found to cause a distal-onset myopathy in one family. Results We detail the mutational, neuropathological, neurophysiological, neurological and radiological features of five new DNAJB6-myopathy families. One has the known Phe93Leu mutation and classic late-onset slowly progressive LGMD1D. Two have different mutations of Phe91 causing a variant childhood-onset severe limb-girdle myopathy. One has a Phe100Val mutation and distal-onset myopathy, unique early bulbar involvement, and a gender-modified wide age-of-onset range. The last has childhood-onset severe distal-onset myopathy and the first non-missense DNAJB6 mutation, c.346 + 5G > A, causing a splicing defect that entirely eliminates DNAJB6’s G/F domain (ΔG/F), the domain that harbours all other mutations. Clinical and imaging examinations reveal that muscles considered uninvolved in DNAJB6-myopathy, e.g. lateral gastrocnemii, are affected in our patients with new mutations. Mutational modelling based on the known structure of the bacterial DNAJ2 protein indicates that all past and present mutated residues cluster within 15 Å in the G/F domain and all disturb the interface of this domain with the protein’s J domain that confers the interaction with HSP70. Conclusions Our patients expand the phenotypic spectrum of DNAJB6-myopathy and allow tentative genotype-phenotype specifications. Combining with previous studies, the clinical severity spectrum is as follows: ΔG/F and Phe91 mutations, most severe; Phe100, Pro96, Phe89 mutations, intermediate; and Phe93, least severe. As it stands presently, proximal G/F domain mutations (Phe89, Phe91, Phe93) cause proximal limb-girdle myopathy, while distal G/F mutations (Pro96, Phe100) cause distal-onset myopathy. While all mutations affect the G/F–J interaction, each likely does so in different unknown extents or ways. One mutation, ΔG/F, causes its associated severe distal-onset myopathy phenotype in a clear way, through generation of a G/F domain-lacking DNAJB6 protein. Electronic supplementary material The online version of this article (doi:10.1186/s40478-015-0224-0) contains supplementary material, which is available to authorized users.

Transforming growth factor beta (TGFbeta) is one of the growth factors involved in the neuroendocrine control of the gonadotrophin-releasing hormone (GnRH) neurones. It is produced and released by the astrocytes surrounding GnRH neurones and directly controls their secretory activity. TGFbeta signalling is based on a complex of two receptors that transduces the signal through peculiar intracellular substrates, the Smad proteins, which, upon activation, move into the nucleus, and modify the transcription of TGFbeta responsive genes. The present study aimed to verify whether TGFbeta1 is able to regulate the Smad pathway in GT1-1 cells (i.e. an immortalised neuronal cell line releasing GnRH). We show that: (i) GT1-1 cells express Smad 2, 3, 4, and 7; (ii) TGFbeta1 enhances the phosphorylation of Smad 2 and 3 at short times of exposure (15-30 min); (iii) TGFbeta1 induces the synthesis of the inhibitory Smad 7 at longer times (60-120-240 min); (iv) the conditioned medium of type 1 astrocytes enhances the phosphorylation of Smad 2 and 3 in GT1-1 cells and a TGFbeta1 neutralising antibody counteracts this effect. The results indicate that Smads are targets of TGFbeta1 and that astrocytes are able to modulate Smads proteins in GT1-1 cells through the release of TGFbeta1. Taken together, the data provide new evidence that glial cells are important regulators of the GnRH neuronal activity.

Several growth factors (e.g., transforming growth factors beta and alpha, basic fibroblast growth factor), produced by hypothalamic astrocytes, participate in the control of hypothalamic gonadotrophin-releasing hormone (GnRH) neurons. On this basis, we have hypothesized that steroid hormones, like estrogens and progestagens, influence the GnRH neurons by modulating in glial cells the synthesis and the release of these growth factors. Data reported here indicate that the expression of transforming growth factor beta 1 is modulated in hypothalamic astrocytes by a progesterone derivative (i.e., dihydroprogesterone), while estrogens modulate that of basic fibroblast growth factor. Moreover, it is interesting to highlight that the effect of estrogens on basic fibroblast growth factor is mediated by another growth factor (i.e., transforming growth factor alpha). Altogether, the present findings support the concept that steroid hormones and growth factors act in an integrated manner at the level of hypothalamic astrocytes, thus adding a further piece of knowledge in the understanding of the mechanisms controlling GnRH neurons.

The Sgcb-null mouse, with knocked-down β-sarcoglycan, develops severe muscular dystrophy as in type 2E human limb girdle muscular dystrophy. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little fibrosis in limb muscles. To characterize ECM protein deposition and the progression of muscle fibrosis, we evaluated protein and transcript levels of collagens I, III and VI, decorin, and TGF-β1, in quadriceps and diaphragm, at 2, 4, 8, 12, 26, and 52 weeks in Sgcb-null mice, and protein levels at 12, 26, and 52 weeks in mdx mice. In Sgcb-null mice, severe morphological disruption was present from 4 weeks in both quadriceps and diaphragm, and included conspicuous deposition of extracellular matrix components. Histopathological features of Sgcb-null mouse muscles were similar to those of age-matched mdx muscles at all ages examined, but, in the Sgcb-null mouse, the extent of connective tissue deposition was generally greater than mdx. Furthermore, in the Sgcb-null mouse, the amount of all three collagen isoforms increased steadily, while, in the mdx, they remained stable. We also found that, at 12 weeks, macrophages were significantly more numerous in mildly inflamed areas of Sgcb-null quadriceps compared to mdx quadriceps (but not in highly inflamed regions), while, in the diaphragm, macrophages did not differ significantly between the two models, in either region. Osteopontin mRNA was also significantly greater at 12 weeks in laser-dissected highly inflamed areas of the Sgcb-null quadriceps compared to the mdx quadriceps. TGF-β1 was present in areas of degeneration–regeneration, but levels were highly variable and in general did not differ significantly between the two models and controls. The roles of the various subtypes of macrophages in muscle repair and fibrosis in the two models require further study. The Sgcb-null mouse, which develops early fibrosis in limb muscles, appears more promising than the mdx mouse for probing pathogenetic mechanisms of muscle fibrosis and for developing anti-fibrotic treatments. Highlights • The Sgcb-null mouse develops severe muscular dystrophy, the mdx mouse does not. • Fibrosis developed earlier in Sgcb-null quadriceps and diaphragm than mdx. • Macrophages were commoner in mildly inflamed parts of Sgcb-null quadriceps than mdx. • The Sgcb-null model appears more useful than mdx for studying fibrotic mechanisms. • The Sgcb-null model also appears more useful for developing anti-fibrotic treatments.

Background POMT2 mutations have been identified in Walker-Warburg syndrome or muscle-eye-brain-like, but rarely in limb girdle muscular dystrophy (LGMD). Results Two POMT2 mutations, one null and one missense, were found in a patient with LGMD and mild mental impairment, no brain or ocular involvement, minor histopathological features, and slight reduction of α-dystroglycan (α-DG) glycosylation and α-DG laminin binding. Conclusions Our case, the fourth LGMD POMT2-mutated reported to date, provides further evidence of correlation between level of α-DG glycosylation and phenotype severity. © 2013 Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.

Background: Cognitive impairment has been reported in a significant proportion of patients with congenital muscular dystrophies (CMD), generally associated with brain changes. Objectives: The aim of this study was to establish 1) the overall prevalence of CMD and cognitive impairment in the Italian population; 2) the frequency of individual genetically defined forms; and 3) the presence of distinct phenotypes not associated with mutations in the known genes. Methods: We included all patients with CMD and cognitive impairment followed in all the Italian tertiary neuromuscular centers. Clinical, brain MRI, and morphologic data were collected. Genetic screening of the known genes was performed according to clinical and muscle biopsy findings. Results: Ninety-two of the 160 (58%) patients with CMD followed in our centers had cognitive impairment. α-Dystroglycan (α-DG) reduction on muscle biopsy was found in 73/92 (79%), with 42/73 carrying mutations in the known genes. Another 6/92 (7%) showed a laminin α2 deficiency on muscle biopsy and 5 of the 6 carried mutations in LAMA2. The remaining 13/92 (14%) patients had normal α-DG and laminin α2 expression on muscle. Conclusions: This is the first population study establishing the prevalence of CMD and cognitive impairment and providing a classification on the basis of clinical, MRI, and genetic findings. We also showed that cognitive impairment was not always associated with α-DG or laminin α2 reduction or with structural brain changes.

Extent of muscle fibrosis contributes to disease severity in muscular dystrophies. To investigate whether extracellular matrix (ECM) components contribute to the severe fibrosis observed in Duchenne muscular dystrophy (DMD) skeletal muscle, we quantitated several ECM components (transcripts and proteins) in primary DMD and control myotube cultures. We evaluated the fibrogenic transforming growth factor- beta1 (TGF-beta1); the small pleiotropic proteoglycan decorin, involved in collagen fibrillogenesis and TGF-beta1 modulation; metalloproteinases MMP-2 and MMP-9; tissue inhibitors of metalloproteinase (TIMP) 1, 2 and 3; collagens I and VI; and the tissue factor myostatin that inhibits muscle growth. Dystrophic myotube cultures had significantly lower levels of decorin mRNA, as also observed in DMD muscle biopsies, and significantly higher levels of TGF-beta1, myostatin, and collagens I and VI. MMP-2, TIMP-1 and TIMP-2 transcript levels were also significantly increased in DMD, but MMP-9 and TIMP-3 transcripts were unchanged. By zymography, MMP-2 activity was significantly higher in DMD than control. Protein levels were similar in DMD and controls but myostatin protein was significantly increased in DMD. We have found that transcript expression and protein modulation of several ECM components is altered in DMD muscle cells in vitro, indicating that these cells contribute fundamentally to the pathological process, since the inflammation and degeneration characterizing DMD muscle in vivo are presumably absent in culture. Our findings that myostatin-potent inhibitor of satellite cell activation and muscle renewal--is increased, and that decorin-binder and downregulator of TGFbeta1 and myostatin--is decreased, may have implications for DMD therapy to reduce muscle fibrosis.

The spinal cord (SC) is a biosynthetic center for neurosteroids, including pregnenolone (PREG), progesterone (PROG), and 3alpha/5alpha-tetrahydroprogesterone (3alpha/5alpha-THP). In particular, an active form of cytochrome P450 sidechain cleavage (P450scc) has been localized in sensory networks of the rat SC dorsal horn (DH). P450scc is the key enzyme catalyzing the conversion of cholesterol (CHOL) into PREG, the rate-limiting step in the biosynthesis of all classes of steroids. To determine whether neurosteroidogenesis might be involved in the pivotal role played by the DH in nociception, effects of neurogenic pain provoked by sciatic nerve ligature were investigated on P450scc expression, cellular distribution, and activity in the SC. P450scc mRNA concentration was threefold higher in the DH of neuropathic rats than in controls. The nerve ligature also increased the density of P450sccpositive neuronal perykarya and fibers in the ipsilateral DH. Incubation of spinal tissue homogenates with [3H]CHOL revealed that the amount of newly synthesized [3H]PREG from [3H]CHOLwas 80% higher in the DH of neuropathic rats. Radioimmunoassays showed an increase of PREG and 3alpha/5alpha-THP concentrations in neuropathic rat DH. The upregulation of PREG and 3alpha/5alpha-THP biosynthesis might be involved in endogenous mechanisms triggered by neuropathic rats to cope with the chronic pain state. 3alpha/5alpha-THP formation from PREG can also generate PROG, which decreases sensitivity to pain and protects nerve cells against degeneration. Because apoptotic cell death has been demonstrated in the DH during neuropathic pain, activation of neurosteroidogenesis in spinal tissues might also be correlated to the neuroprotective role of steroids in the SC.

Abnormal secretion of steroids by the adrenals and gonads is one of the disturbances occurring in diabetics but the impact of diabetes on steroid formation in the nervous system has never been studied. However, it is well known that numerous actions of peripheral steroids on the nervous system require their conversion into neuroactive metabolites within the neural tissue. As this in situ steroid synthesis/metabolism is crucial for the control of several neurobiological functions, we investigated the effects of streptozotocin-induced diabetes on the gene expression and activity of 3beta-hydroxysteroid dehydrogenase in the spinal cord, a pivotal structure involved in sensorimotor and neurovegetative mechanisms. 3beta-Hydroxysteroid dehydrogenase is a key enzyme which participates to the biosynthesis of all classes of steroids by converting delta5-3beta-hydroxysteroids such as pregnenolone and dehydroepiandrosterone into delta4-3-ketosteroids as progesterone and androstenedione, respectively. Reverse transcription coupled with quantitative real-time polymerase chain reaction revealed that 3beta-hydroxysteroid dehydrogenase gene was over-expressed in the spinal cord of streptozotocin-treated rats compared with controls. Pulse-chase experiments combined with high performance liquid chromatography and continuous flow detection of newly-synthesized steroids showed an increase of 3beta-hydroxysteroid dehydrogenase activity responsible for a hyper-production of progesterone in the spinal cord of diabetic rats. This up-regulation of progesterone biosynthesis was concomitant with a decrease of its transformation into tetrahydroprogesterone, a process which facilitated progesterone accumulation in the spinal cord of streptozotocin-treated rats. Since progesterone is a potent neuroprotective steroid, increase of its production appeared as an endogenous molecular and biochemical mechanism triggered by spinal nerve cells to cope with degenerative effects of streptozotocin-induced diabetes. Our results constitute the first direct evidence showing an impact of diabetes on steroid biosynthetic and metabolic pathways in the nervous system. The data open new perspectives for the modulation of deleterious effects of diabetes by neuroprotective steroids.

Several growth factors (e.g., transforming growth factors beta and alpha, basic fibroblast growth factor), produced by hypothalamic astrocytes, participate in the control of hypothalamic gonadotrophin-releasing hormone (GnRH) neurons. On this basis, we have hypothesized that steroid hormones, like estrogens and progestagens, influence the GnRH neurons by modulating in glial cells the synthesis and the release of these growth factors. Data reported here indicate that the expression of transforming growth factor beta 1 is modulated in hypothalamic astrocytes by a progesterone derivative (i.e., dihydroprogesterone), while estrogens modulate that of basic fibroblast growth factor. Moreover, it is interesting to highlight that the effect of estrogens on basic fibroblast growth factor is mediated by another growth factor (i.e., transforming growth factor alpha). Altogether, the present findings support the concept that steroid hormones and growth factors act in an integrated manner at the level of hypothalamic astrocytes, thus adding a further piece of knowledge in the understanding of the mechanisms controlling GnRH neurons.

Dystroglycanopathies represent an important subgroup of recessively inherited disorders within the group of muscular dystrophies. Their severity may vary from the mild forms of adult-onset limb-girdle muscular dystrophy (LGMD), to the severe congenital muscular dystrophies with cerebral and ocular involvement. Although mutations in at least 17 genes have been identified so far, about 50% of the cases with dystroglycanopathy still remain unsolved. Recently, mutations in the isoprenoid synthase domain containing (ISPD) gene have been reported as a common cause of congenital muscular dystrophy and LGMD. We report clinical, histopathological, immunochemical, genetic and muscular MRI findings in 2 consanguineous children of Pakistani origin, carrying a new homozygous missense mutation (Gly123Arg) in the ISPD gene. Case 1 is a 8 year-old female with an early limb-girdle phenotype, who lost ambulation at the age of 7.5 years. Case 2 is a 2.5 year-old male and second degree cousin of case 1, showing a congenital muscular dystrophy phenotype. Cognitive development, brain MRI, eye examination, electrocardiogram and echocardiogram were normal in both the patients. Western blot showed greater reduction of alpha-dystroglycan glycosylation in patient 2, and may be responsible for the greater severity of his clinical presentation. To our knowledge, this is the first report on the co-occurrence of both early limb-girdle and congenital muscular dystrophies within the same family carrying a new homozygous ISPD mutation.

The adult-onset form of Pompe disease had a wide clinical spectrum, ranging from asymptomatic patients with increased CK to muscle cramps and pain syndrome or rigid-spine syndrome. In addition clinical severity and disease progression are greatly variable. We report on a family with 3 siblings characterized by an unusual adult-onset Pompe disease including dysphagia and weakness of tongue, axial and limb-girdle muscles, in association with atypical globular inclusions in muscle fibres. Our study confirms the great clinical and histological variability of adult-onset Pompe disease and further supports the need of careful evaluation of bulbar function in patients affected by this pathology.