Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Movement disorders can be primarily divided into hypokinetic and hyperkinetic. Most of the hypokinetic syndromes are associated with the neurodegenerative disorder Parkinson’s disease (PD). By contrast, hyperkinetic syndromes encompass a broader array of diseases, including dystonia, essential tremor, or Huntington’s disease. The discovery of effective therapies for these disorders has been challenging and has also involved the development and characterization of accurate animal models for the screening of new drugs. Zebrafish constitutes an alternative vertebrate model for the study of movement disorders. The neuronal circuitries involved in movement in zebrafish are well characterized, and most of the associated molecular mechanisms are highly conserved. Particularly, zebrafish models of PD have contributed to a better understanding of the role of several genes implicated in the disease. Furthermore, zebrafish is a vertebrate model particularly suited for large-scale drug screenings. The relatively small size of zebrafish, optical transparency, and lifecycle, are key characteristics that facilitate the study of multiple compounds at the same time. Several transgenic, knockdown, and mutant zebrafish lines have been generated and characterized. Therefore, it is central to critically analyze these zebrafish lines and understand their suitability as models of movement disorders. Here, we revise the pathogenic mechanisms, phenotypes, and responsiveness to pharmacotherapies of zebrafish lines of the most common movement disorders. A systematic review of the literature was conducted by including all studies reporting the characterization of zebrafish models of the movement disorders selected from five bibliographic databases. A total of 63 studies were analyzed, and the most relevant data within the scope of this review were gathered. The majority (62%) of the studies were focused in the characterization of zebrafish models of PD. Overall, the zebrafish models included display conserved biochemical and neurobehavioral features of the phenomenology in humans. Nevertheless, in light of what is known for all animal models available, the use of zebrafish as a model for drug discovery requires further optimization. Future technological developments alongside with a deeper understanding of the molecular bases of these disorders should enable the development of novel zebrafish lines that can prove useful for drug discovery for movement disorders.

Mutations in the co-chaperone Bcl2-associated athanogene 3 (BAG3) can cause myofibrillar myopathy (MFM), a childhood-onset progressive muscle disease, characterized by the formation of protein aggregates and myofibrillar disintegration. In contrast to other MFM-causing proteins, BAG3 has no direct structural role, but regulates autophagy and the degradation of misfolded proteins. To investigate the mechanism of disease in BAG3-related MFM, we expressed wild-type BAG3 or the dominant MFM-causing BAG3 (BAG3(P209L)) in zebrafish. Expression of the mutant protein results in the formation of aggregates that contain wild-type BAG3. Through the stimulation and inhibition of autophagy, we tested the prevailing hypothesis that impaired autophagic function is responsible for the formation of protein aggregates. Contrary to the existing theory, our studies reveal that inhibition of autophagy is not sufficient to induce protein aggregation. Expression of the mutant protein, however, did not induce myofibrillar disintegration and we therefore examined the effect of knocking down Bag3 function. Loss of Bag3 resulted in myofibrillar disintegration, but not in the formation of protein aggregates. Remarkably, BAG3(P209L) is able to rescue the myofibrillar disintegration phenotype, further demonstrating that its function is not impaired. Together, our knockdown and overexpression experiments identify a mechanism whereby BAG3(P209L) aggregates form, gradually reducing the pool of available BAG3, which eventually results in BAG3 insufficiency and myofibrillar disintegration. This mechanism is consistent with the childhood onset and progressive nature of MFM and suggests that reducing aggregation through enhanced degradation or inhibition of nucleation would be an effective therapy for this disease.

For the past few years there has been an exponential increase in the use of animal models to confirm the pathogenicity of candidate disease-causing genetic variants found in patients. One such animal model is the zebrafish. Despite being a non-mammalian animal, the zebrafish model has proven its potential in recapitulating the phenotypes of many different human genetic disorders. This review will focus on recent advances in the modeling of neurodevelopmental disorders in zebrafish, covering aspects from early brain development to techniques used for modulating gene expression, as well as how to best characterize the resulting phenotypes. We also review other existing models of neurodevelopmental disorders, and the current efforts in developing and testing compounds with potential therapeutic value.

Mutations in FLNC for a long time are known in connection to neuromuscular disorders and only recently were described in association with various cardiomyopathies. Here, we report a new clinical phenotype of filaminopathy in four unrelated patients with early-onset restrictive cardiomyopathy (RCM) in combination with congenital myopathy due to FLNC mutations (NM_001458.4:c.3557C>T, p.A1186V, rs1114167361 in three probands and c.[3547G>C; 3548C>T], p.A1183L, rs1131692185 in one proband). In all cases, concurrent myopathy was confirmed by neurological examination, electromyography, and morphological studies. Three of the patients also presented with arthrogryposis. The pathogenicity of the described missense variants was verified by cellular and morphological studies and by in vivo modeling in zebrafish. Combination of in silico and experimental approaches revealed that FLNC missense variants localized in Ig-loop segments often lead to development of RCM. The described FLNC mutations associated with early-onset RCMP extend cardiac spectrum of filaminopathies and facilitate the differential diagnosis of restrictive cardiac phenotype associated with neuromuscular involvement in children.

Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.

BackgroundInflammatory pseudotumor is a rare clinical condition that can be related to immunoglobulin G4 disease. Only a few cases of spinal inflammatory pseudotumors have been reported in the literature and an association with immunoglobulin G4 disease was not conclusive in any of them. We describe what we believe to be the first biopsy-proven case of an epidural inflammatory pseudotumor related to immunoglobulin G4 disease.Case presentationA 57-year-old Caucasian woman presented to our hospital with severe paraparesis, gait disturbance, and sensory loss secondary to a relapsing epidural mass. Examination of a biopsy specimen revealed a lymphoplasmacytic infiltration with fibrosis and an immunoglobulin G4-positive plasma cell ratio of over 50 %, which are compatible with a diagnosis of immunoglobulin G4-related inflammatory pseudotumor. Our patient was successfully treated with systemic and epidural administration of glucocorticoids.ConclusionImmunoglobulin G4-related disease is an emerging clinical condition in which central nervous system involvement is still uncommon. We describe the case of a patient with an epidural mass with medullar compression, which was proved to be an immunoglobulin G4-related epidural inflammatory pseudotumor. Our findings suggest a new manifestation of immunoglobulin G4-related disease. This disorder should be considered in the differential diagnosis of spinal tumors as a potentially treatable condition with glucocorticoids.

The skeletal ciliopathies are a heterogeneous group of disorders with a significant clinical and genetic variability and the main clinical features are thoracic hypoplasia and short tubular bones. To date, 25 genes have been identified in association with skeletal ciliopathies. Mutations in the KIAA0753 gene have recently been associated with Joubert syndrome (JBTS) and orofaciodigital (OFD) syndrome. We report biallelic pathogenic variants in KIAA0753 in four patients with short-rib type skeletal dysplasia. The manifestations in our patients are variable and ranging from fetal lethal to viable and moderate skeletal dysplasia with narrow thorax and abnormal metaphyses. We demonstrate that KIAA0753 is expressed in normal fetal human growth plate and show that the affected fetus, with a compound heterozygous frameshift and a nonsense mutation in KIAA0753, has an abnormal proliferative zone and a broad hypertrophic zone. The importance of KIAA0753 for normal skeletal development is further confirmed by our findings that zebrafish embryos homozygous for a nonsense mutation in kiaa0753 display altered cartilage patterning.