Casein kinase 1 inhibitor avoids TDP-43 pathology propagation in a patient-derived cellular model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease without a cure to reverse its progression. Its main hallmark is the nuclear protein TDP-43, which undergoes different post-translational modifications leading to a loss of function in the nucleus and an increase in toxicity in the cytoplasm. Previous reports have indicated that pathogenic TDP-43 exhibits prion-like propagation in various contexts. With the aim of advancing therapeutics focused on preventing the propagation of TDP-43 pathology, we studied the potential role of pathogenic TDP-43 in lymphoblasts from sporadic ALS patients. We used lymphoblastoid cell lines from sporadic ALS patients as a source of pathogenic forms of TDP-43, and healthy human cells (lymphoblasts, myoblasts, neuroblastoma SH-SY5Y, or osteosarcoma U2OS) as recipient cells to investigate the seeding and spread of TDP-43 proteinopathy. Furthermore, we evaluated the potential of targeting TDP-43 phosphorylation with a CK-1 inhibitor to prevent the propagation of the pathology. The results presented herein indicate that pathogenic forms of TDP-43 are secreted into the extracellular medium of sporadic ALS lymphoblasts and could be transported by extracellular vesicles, spreading TDP-43 pathology to healthy cells. Moreover, tunneling nanotubes have also been discovered in pathological cells and may be involved in the transport of TDP-43. Interestingly, targeting TDP-43 phosphorylation with an in-house designed CK-1 inhibitor (IGS2.7) was sufficient to halt TDP-43 pathology transmission, in addition to its known effects on restoring the homeostasis of TDP-43 protein in patients-derived cells.

Dysregulated FOXO1 activity drives skeletal muscle intrinsic dysfunction in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicating metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physiopathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that parallels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.

The CAPN3 p.Lys 254del variant is not always associated with dominant CAPN3-related muscular dystrophy.

INTRODUCTION/AIMS: Limb-girdle muscular dystrophy R1 (LGMDR1) calpain 3-related usually presents as a recessively transmitted weakness of proximal limb-girdle muscles due to pathogenic variants in the CAPN3 gene. Pathogenic variants in this gene have also been found in patients with an autosomal dominantly inherited transmission pattern (LGMDD4). The mechanism underlying this difference in transmission patterns has not yet been elucidated. Camptocormia, progressive limb weakness, myalgia, back pain, and increased CK levels are common clinical features associated with dominant forms. The p.Lys254del pathogenic variant was associated with camptocormia in two LGMDD4 families. This study aimed to present carriers found in recessively transmitted LGMDR1 families bearing the p.Lys254del variant that do not show muscle weakness. METHODS: DNA sequencing was performed on exon 5 of CAPN3 in family members to establish the carrier status of the pathogenic variant. They were evaluated clinically and MRI was performed when available. RESULTS: Two families presented with the p.Lys254del pathogenic variant in a homozygous or compound heterozygous state. Family members carrying only the pathogenic variant in the heterozygous state did not demonstrate the myopathic characteristics described in dominant patients. Camptocormia and other severe clinical symptoms were not observed. DISCUSSION: We conclude that the p.Lys254del pathogenic variant per se cannot be solely responsible for camptocormia in dominant patients. Other undisclosed factors may regulate the phenotype associated with the dominant inheritance pattern in CAPN3 pathogenic variant carriers.

Allosteric Modulation of GSK-3ß as a New Therapeutic Approach in Limb Girdle Muscular Dystrophy R1 Calpain 3-Related.

Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3ß (GSK-3ß), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.

Genotype-phenotype correlations in recessive titinopathies.

PURPOSE: High throughput sequencing analysis has facilitated the rapid analysis of the entire titin (TTN) coding sequence. This has resulted in the identification of a growing number of recessive titinopathy patients. The aim of this study was to (1) characterize the causative genetic variants and clinical features of the largest cohort of recessive titinopathy patients reported to date and (2) to evaluate genotype-phenotype correlations in this cohort. METHODS: We analyzed clinical and genetic data in a cohort of patients with biallelic pathogenic or likely pathogenic TTN variants. The cohort included both previously reported cases (100 patients from 81 unrelated families) and unreported cases (23 patients from 20 unrelated families). RESULTS: Overall, 132 causative variants were identified in cohort members. More than half of the cases had hypotonia at birth or muscle weakness and a delayed motor development within the first 12 months of life (congenital myopathy) with causative variants located along the entire gene. The remaining patients had a distal or proximal phenotype and a childhood or later (noncongenital) onset. All noncongenital cases had at least one pathogenic variant in one of the final three TTN exons (362-364). CONCLUSION: Our findings suggest a novel association between the location of nonsense variants and the clinical severity of the disease.

Calcium Mechanisms in Limb-Girdle Muscular Dystrophy with CAPN3 Mutations.

Limb-girdle muscular dystrophy recessive 1 (LGMDR1), previously known as LGMD2A, is a rare disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness of shoulder, pelvic, and proximal limb muscles that usually appears in children and young adults and results in loss of ambulation within 20 years after disease onset in most patients. The pathophysiological mechanisms involved in LGMDR1 remain mostly unknown, and to date, there is no effective treatment for this disease. Here, we review clinical and experimental evidence suggesting that dysregulation of Ca2+ homeostasis in the skeletal muscle is a significant underlying event in this muscular dystrophy. We also review and discuss specific clinical features of LGMDR1, CAPN3 functions, novel putative targets for therapeutic strategies, and current approaches aiming to treat LGMDR1. These novel approaches may be clinically relevant not only for LGMDR1 but also for other muscular dystrophies with secondary calpainopathy or with abnormal Ca2+ homeostasis, such as LGMD2B/LGMDR2 or sporadic inclusion body myositis.

FRZB and melusin, overexpressed in LGMD2A, regulate integrin ß1D isoform replacement altering myoblast fusion and the integrin-signalling pathway.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is characterised by muscle wasting and progressive degeneration of proximal muscles because of mutations in the CAPN3 gene. However, the underlying pathophysiological mechanisms of muscle degeneration are still not well understood. The objective of this study was to assess the relevance of genes with differential expression in the muscle of LGMD2A patients. For this purpose, we analysed their in vitro expression in primary cultures of human myoblasts and myotubes. Abnormal fusion was observed in the myotubes of these patients, which may be explained by the lack of physiological replacement of integrin ß1D. Owing to this observation, we focused on deregulated genes coding proteins that directly interact with integrin, ITGB1BP2 and CD9, as well as FRZB gene, because of its in vitro upregulation in myotubes. Silencing studies established that these genes are closely regulated, CD9 and FRZB being positive regulators of the expression of ITGB1BP2, and in turn, this gene being a negative regulator of the expression of FRZB. Interestingly, we observed that FRZB regulates integrin ß1D expression, its silencing increasing integrin ß1D expression to levels similar to those in controls. Finally, the administration of LiCl, an enhancer of the Wnt-signalling pathway showed similar experimentally beneficial effects, suggesting FRZB silencing or LiCl administration as potential therapeutic targets, though further studies are required.

Costamere proteins and their involvement in myopathic processes.

Muscle fibres are very specialised cells with a complex structure that requires a high level of organisation of the constituent proteins. For muscle contraction to function properly, there is a need for not only sarcomeres, the contractile structures of the muscle fibre, but also costameres. These are supramolecular structures associated with the sarcolemma that allow muscle adhesion to the extracellular matrix. They are composed of protein complexes that interact and whose functions include maintaining cell structure and signal transduction mediated by their constituent proteins. It is important to improve our understanding of these structures, as mutations in various genes that code for costamere proteins cause many types of muscular dystrophy. In this review, we provide a description of costameres detailing each of their constituent proteins, such as dystrophin, dystrobrevin, syntrophin, sarcoglycans, dystroglycans, vinculin, talin, integrins, desmin, plectin, etc. We describe as well the diseases associated with deficiency thereof, providing a general overview of their importance.

Entire CAPN3 gene deletion in a patient with limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A) due to mutations in the CAPN3 gene is one of the most common of autosomal recessive limb-girdle muscular dystrophies. We describe a patient who had a typical LGMD2A phenotype and posterior compartment involvement on MRI. Different genetic analyses were performed, including microarray analysis. There was an apparently homozygous mutation in exon 24, c.2465G>T, p.(*822Leuext62*), and a lack of correlation in the disease segregation analyses. This suggested the presence of a genomic rearrangement. In fact, a heterozygous deletion of the entire CAPN3 gene was found. This novel deletion comprised the terminal region of the GANC gene and the entire CAPN3 gene. This finding points out the need to reconsider and adapt our current strategy of molecular diagnosis in order to detect these types of genomic rearrangements that escape standard mutation screening procedures.

Altered expression of proteins involved in metabolism in LGMDR1 muscle is lost in cell culture conditions.

BACKGROUND: Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy due to mutations in the CAPN3 gene. While the pathophysiology of this disease has not been clearly established yet, Wnt and mTOR signaling pathways impairment in LGMDR1 muscles has been reported. RESULTS: A reduction in Akt phosphorylation ratio and upregulated expression of proteins implicated in glycolysis (HK-II) and in fructose and lactate transport (GLUT5 and MCT1) in LGMDR1 muscle was observed. In vitro analysis to establish mitochondrial and glycolytic functions of primary cultures were performed, however, no differences between control and patients were observed. Additionally, gene expression analysis showed a lack of correlation between primary myoblasts/myotubes and LGMDR1 muscle while skin fibroblasts and CD56- cells showed a slightly better correlation with muscle. FRZB gene was upregulated in all the analyzed cell types (except in myoblasts). CONCLUSIONS: Proteins implicated in metabolism are deregulated in LGMDR1 patients' muscle. Obtained results evidence the limited usefulness of primary myoblasts/myotubes for LGMDR1 gene expression and metabolic studies. However, since FRZB is the only gene that showed upregulation in all the analyzed cell types it is suggested its role as a key regulator of the pathophysiology of the LGMDR1 muscle fiber. The Wnt signaling pathway inactivation, secondary to FRZB upregulation, and GLUT5 overexpression may participate in the impaired adipogenesis in LGMD1R patients.

C3KO mouse expression analysis: downregulation of the muscular dystrophy Ky protein and alterations in muscle aging.

Mutations in CAPN3 gene cause limb-girdle muscular dystrophy type 2A (LGMD2A) characterized by muscle wasting and progressive degeneration of scapular and pelvic musculature. Since CAPN3 knockout mice (C3KO) display features of muscle pathology similar to those features observed in the earliest-stage or preclinical LGMD2A patients, gene expression profiling analysis in C3KO mice was performed to gain insight into mechanisms of disease. Two different comparisons were carried out in order to determine, first, the differential gene expression between wild-type (WT) and C3KO soleus and, second, to identify the transcripts differentially expressed in aging muscles of WT and C3KO mice. The up/downregulation of two genes, important for normal muscle function, was identified in C3KO mice: the Ky gene, encoding a protease implicated in muscle development, and Park2 gene encoding an E3 ubiquitin ligase (parkin). The Ky gene was downregulated in C3KO muscles suggesting that Ky protease may play a complementary role in regulating muscle cytoskeleton homeostasis in response to changes in muscle activity. Park2 was upregulated in the aged WT muscles but not in C3KO muscles. Taking into account the known functions of parkin E3 ligase, it is possible that it plays a role in ubiquitination and degradation of atrophy-specific and damaged proteins that are necessary to avoid cellular toxicity and a cellular stress response in aging muscles.

Senescence plays a role in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of an accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from patients with DM1 and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which related to the accumulation of cellular senescence. The data from transcriptome analyses were corroborated in human myoblasts and blood samples, as well as in mouse and Drosophila models of the disease. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts, whereas the DM1 Drosophila model showed reduced longevity and impaired locomotor activity. Moreover, functional studies highlighted the impact of BMI1 and downstream p16INK4A/RB and ARF/p53/p21CIP pathways in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds Quercetin, Dasatinib, or Navitoclax reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in Drosophila in vivo. Our results identify the accumulation of senescence as part of DM1 pathophysiology and, therefore, demonstrate the efficacy of senolytic compounds in the preclinical setting.

Does the severity of the LGMD2A phenotype in compound heterozygotes depend on the combination of mutations?

INTRODUCTION: Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by a deficiency of calpain-3/p94. Although the symptoms in most LGMD2A patients are generally homogeneous, some variation in the severity and progression of the disease has been reported. METHODS: We describe 2 patients who carry the same combination of compound heterozygous mutations (pG222R/pR748Q) and whose symptoms are exceptionally benign compared to homozygotes with each missense mutation. RESULTS: The benign phenotype observed in association with the combined pG222R and pR748Q mutations suggested that it may result from a compensatory effect of compound heterozygosity rather than the individual mutations themselves. Our analyses revealed that these two mutations exert different effects on the protease activity of calpain-3, suggesting "molecular complementation" in these patients. CONCLUSION: We propose several hypotheses to explain how this specific combination of mutations may rescue the normal proteolytic activity of calpain-3, resulting in an exceptionally benign phenotype.

Frizzled related protein deficiency impairs muscle strength, gait and calpain 3 levels.

BACKGROUND: Limb-girdle muscular dystrophy recessive 1 calpain3-related (LGMDR1), previously known as LGMD2A, is a disease caused by mutations in the CAPN3 gene. It is characterized by progressive weakness and muscle degeneration. Frizzled related protein (FRZB), upregulated in LGMDR1, was identified as a key regulator of the crosstalk between Wnt and integrin signalling pathways. FRZB gene silencing showed a recovery in the expression of some of the costamere protein levels in myotubes. RESULTS: Here, we performed a comprehensive characterization of Frzb-/- mice muscles to study the absence of Frzb in skeletal muscle and eventual links with the molecular characteristics of LGMDR1 patient muscles. Frzb-/- mice showed reduced muscle size and strength. Gait analysis showed that Frzb-/- mice moved more slowly but no impaired regeneration capacity was observed after muscle injury. Additionally, Frzb-/- mice muscle showed an increased number of mesoangioblasts. Lack of Frzb gene in Frzb-/- mice and its increased expression in LGMDR1 patients, showed contrary regulation of Rora, Slc16a1, Tfrc and Capn3 genes. The reciprocal regulation of Frzb and Capn3 genes further supports this axis as a potential target for LGMDR1 patients. CONCLUSIONS: Our data confirm a role for Frzb in the regulation of Rora, Slc16a1, Tfrc, and Capn3 genes in muscle cells. In vivo, reduced muscle strength and gait in the Frzb-/- mice are intriguing features. The reciprocal relationship between FRZB and CAPN3 further supports a key role for this axis in patients with LGMDR1.

[DNA arrays: a general overview and specific applications]. / Matrices de ADN: visión general y aplicaciones específicas.

Difficulties derived from the big complexity of the human genome (because of the great amount of genes) and difficulties at the time of performing a correct molecular diagnosis have led to the utilization of advanced technologies such as DNA arrays. By this technique, it is possible to analyze in a single experiment great sequences of genomic regions, to detect mutations or polymorphisms (characteristic of each individual), and to analyze variations in the expression of genes implicated in different disorders or in the utilization of drugs. Similarly, the use of DNA arrays-derived information has led to a considerable advance in the personalized medicine that is applied to every patient, avoiding unwanted adverse effects and the consecution of the best performance effectiveness for each drug.

Copy number variation analysis increases the diagnostic yield in muscle diseases.

OBJECTIVE: Copy number variants (CNVs) were analyzed from next-generation sequencing data, with the aim of improving diagnostic yield in skeletal muscle disorder cases. METHODS: Four publicly available bioinformatic analytic tools were used to analyze CNVs from sequencing data from patients with muscle diseases. The patients were previously analyzed with a targeted gene panel for single nucleotide variants and small insertions and deletions, without achieving final diagnosis. Variants detected by multiple CNV analysis tools were verified with either array comparative genomic hybridization or PCR. The clinical significance of the verified CNVs was interpreted, considering previously identified variants, segregation studies, and clinical information of the patient cases. RESULTS: Combining analysis of all different mutation types enabled integration of results and identified the final cause of the disease in 9 myopathy cases. Complex effects like compound heterozygosity of different mutation types and compound disease arising from variants of different genes were unraveled. We identified the first large intragenic deletion of the titin (TTN) gene implicated in the pathogenesis of a severe form of myopathy. Our work also revealed a "double-trouble" effect in a patient carrying a single heterozygous insertion/deletion mutation in the TTN gene and a Becker muscular dystrophy causing deletion in the dystrophin gene. CONCLUSIONS: Causative CNVs were identified proving that analysis of CNVs is essential for increasing the diagnostic yield in muscle diseases. Complex severe muscular dystrophy phenotypes can be the result of different mutation types but also of the compound effect of 2 different genetic diseases.

Muscle MRI findings in patients with limb girdle muscular dystrophy with calpain 3 deficiency (LGMD2A) and early contractures.

Limb girdle muscular dystrophy 2A is a common variant secondary to mutations in the calpain 3 gene. A proportion of patients has early and severe contractures, which can cause diagnostic difficulties with other conditions. We report clinical and muscle magnetic resonance imaging findings in seven limb girdle muscular dystrophy 2A patients (four sporadic and three familial) who had prominent and early contractures. All patients showed a striking involvement of the posterior thigh muscles. The involvement of the other thigh muscles was variable and was related to clinical severity. Young patients with minimal functional motor impairment showed a predominant involvement of the adductors and semimembranosus muscles while patients with restricted ambulation had a more diffuse involvement of the posterolateral muscles of the thigh and of the vastus intermedius with relative sparing of the vastus lateralis, sartorius and gracilis. At calf level all patients showed involvement of the soleus muscle and of the medial head of the gastrocnemius with relative sparing of the lateral head. MRI findings were correlated to those found in two patients with the phenotype of limb girdle muscular dystrophy 2A without early contractures and the pattern observed was quite similar. However, the pattern observed in limb girdle muscular dystrophy 2A is different from that reported in other muscle diseases such as Emery-Dreifuss muscular dystrophy and Bethlem myopathy which have a significant clinical overlap with limb girdle muscular dystrophy 2A once early contractures are present. Our results suggest that muscle MRI may help in recognising patients with limb girdle muscular dystrophy 2A even when the clinical presentation overlaps with other conditions, and may therefore, be used as an additional investigation to target the appropriate biochemical and genetic tests.

Limb-girdle muscular dystrophy 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the gene CAPN3 located in the chromosome region 15q15.1-q21.1. To date more than 300 mutations have been described. This gene encodes for a 94-kDa nonlysosomal calcium-dependent cysteine protease and its function in skeletal muscle is not fully understood. It seems that calpain-3 has an unusual zymogenic activation that involves, among other substrates, cytoskeletal proteins. Calpain-3 is thought to interact with titin and dysferlin. Calpain-3 deficiency produces abnormal sarcomeres that lead eventually to muscle fiber death. Hip adductors and gluteus maximus are the earliest clinically affected muscles. No clinical differences have been reported depending on the type of mutation in the CAPN3 gene. The muscle biopsy shows variability of fiber size, interstitial fibrosis, internal nuclei, lobulated fibers, and, in some cases, presence of eosinophils. Recent gene expression profiling studies have shown upregulation of interleukin-32 and immunoglobulin genes, which may explain the eosinophilic infiltration. Two mouse knockout models of CAPN3 have been characterized. There are no curative treatments for this disease. However, experimental therapeutics using mouse models conclude that adeno-associated virus (AAV) vectors seem to be one of the best approaches because of their efficiency and persistency of gene transfer.

Gene expression profiling in limb-girdle muscular dystrophy 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that beta-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies.