Plectin, a highly versatile and multifunctional cytolinker, acts as a central connector of the intermediate filament (IF) and other cytoskeletal systems. In skeletal muscle, plectin orchestrates and anchors the extrasarcomeric desmin filament network to sites of strategic importance and thereby substantially contributes to its fundamental biomechanical properties. Lack of plectin in skeletal muscle leads to a faulty organization of the desmin IFs, thereby inflicting a reduced mechanical stress tolerance and a progressive myopathic process. Accordingly, the morphological hallmark of the skeletal muscle pathology in most plectinopathies, including epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is the accumulation of desmin-positive protein aggregates. To address the consequences of plectin-deficiency on other types of IFs, RNA and protein expression as well as localization of various IF subtypes was evaluated in muscle tissue from wild-type and muscle-specific conditional plectin knockout (MCK-Cre/cKO) mice. Notably, vimentin and syncoilin, as well as several other IF subtypes, were significantly upregulated in MCK-Cre/cKO muscles and accumulated in subsarcolemmal and sarcoplasmic areas. In plectin-deficient mouse myoblasts, increased expression levels of vimentin were accompanied by the formation of thickened IF bundles. Primary human myoblasts, treated with the plectin inhibitor plecstatin-1, displayed increased bundling of desmin and vimentin, thus supporting the notion of drastic structural and organizational changes in the network. Finally, we were able to demonstrate the presence of vimentin-positive protein aggregates in skeletal muscle specimens from EBS-MD patients. Together, these data indicate that the depletion of plectin in muscle unequivocally affected the expression and localization of various types of IFs.

Plectin, a multifunctional cytolinker and intermediate filament stabilizing protein, is essential for muscle fibre integrity and function. Mutations in the human plectin gene (PLEC) cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). The disorganization and aggregation of desmin filaments in conjunction with degenerative changes of the myofibrillar apparatus are key features in the skeletal muscle pathology of EBS-MD. We performed a comprehensive analysis addressing protein homeostasis in this rare protein aggregation disease by using human EBS-MD tissue, plectin knock-out mice and plectin-deficient cells. Protein degradation pathways were analysed in muscles from EBS-MD patients, muscle-specific conditional plectin knockout (MCK-Cre/cKO) mice, as well as in plectin-deficient (Plec-/-) myoblasts by electron and immunofluorescence microscopy. To obtain a comprehensive picture of autophagic processes, we evaluated the transcriptional regulation and expression levels of autophagic markers in plectin-deficient muscles and myoblasts (RNA-Seq, qRT-PCR, immunoblotting). Autophagic turnover was dynamically assessed by measuring baseline autophagy as well as specific inhibition and activation in mCherry-EGFP-LC3B-expressing Plec+/+ and Plec-/- myoblasts, and by monitoring primary Plec+/+ and Plec-/- myoblasts using organelle-specific dyes. Wild-type and MCK-Cre/cKO mice were treated with chloroquine or metformin to assess the effects of autophagy inhibition and activation in vivo. Our study identified the accumulation of degradative vacuoles as well as LC3- and SQSTM1-positive patches in EBS-MD patients, MCK-Cre/cKO mouse muscles and Plec-/- myoblasts. The transcriptional regulation of ~30% of autophagy-related genes was altered, and protein levels of downstream targets of the autophagosomal degradation machinery were elevated in MCK-Cre/cKO muscle lysates (e.g., LAMP2, BAG3 and SQSTM1 to ~160, ~150 and ~140% of controls, respectively; p < 0.05). Autophagosome turnover was compromised in mCherry-EGFP-LC3B-expressing Plec-/- myoblasts (~40% reduction in median red:green ratio, reduced puncta number, smaller puncta; p < 0.01). By labelling autophagic compartments with CYTO-ID dye or lysosomes with LYSO-ID, we found reduced signal intensities in primary Plec-/- cells (p < 0.001). Treatment with chloroquine led to drastic swelling of autophagic vacuoles in primary Plec+/+ myoblasts, while the swelling in Plec-/- cells was moderate, establishing a defect in their autophagic clearance. Chloroquine treatment of MCK-Cre/cKO mice corroborated that loss of plectin coincides with impaired autophagic clearance, while metformin amelioratively induced autophagic flux. Our work demonstrates that the characteristic protein aggregation pathology in EBS-MD is linked to an impaired autophagic flux. The obtained results open a new perspective on the understanding of the protein aggregation pathology in plectin-related disorders and provide a basis for further pharmacological intervention.

Plectinopathy-associated disorders are caused by mutations in the PLECTIN (PLEC) gene encoding Plectin protein. PLEC mutations cause a spectrum of diseases defined by varying degrees of signs, mostly with epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and plectinopathy-related disorder is limb-girdle muscular dystrophy type 2Q (LGMD2Q). Here we report three cases with EBS-MD and LGMD2Q disorders analyzed by exome sequencing followed by mutation confirmation. A complete clinical examination was done by expert specialists and clinical geneticists in Next Generation Genetic polyclinic, Mashhad, Iran (NGC, years 2020_2021),. Genomic DNA was extracted and evaluated through whole-exome sequencing analysis followed by Sanger sequencing for co-segregation analysis of PLEC candidate variants. We found three cases with the plectinopathy-related disease, two patients with limb-girdle muscular dystrophy type 2Q (LGMD2Q), and the other affected proband suffers from epidermolysis bullosa simplex combined with muscular dystrophy (EBS-MD) with variable zygosity mutations for PLEC. Motor development disorder and muscular dystrophy symptoms have different age onset in affected individuals. Patients with EBS demonstrated symptoms such as blistering, skin scars, neonatal-onset, and nail dystrophy. We report plectinopathy-associated disorders to expand clinical phenotypes in different types of PLEC-related diseases. We suppose to design more well-organized research based on comprehensive knowledge about the genetic basis of plectinopathy diseases.

Plectin, a highly versatile and multifunctional cytolinker, has been implicated in several multisystemic disorders. Most sequence variations in the human plectin gene (PLEC) cause epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), an autosomal recessive skin-blistering disorder associated with progressive muscle weakness. In this study, we performed a comprehensive cell biological analysis of dermal fibroblasts from three different patients with EBS-MD, where PLEC expression analyses revealed preserved mRNA levels in all cases, whereas full-length plectin protein content was significantly reduced or completely absent. Downstream effects of pathogenic PLEC sequence alterations included massive bundling of vimentin intermediate filament networks, including the occurrence of ring-like nuclei-encasing filament bundles, elongated mitochondrial networks, and abnormal nuclear morphologies. We found that essential fibroblast functions such as wound healing, migration, or orientation upon cyclic stretch were significantly impaired in the cells of patients with EBS-MD. Finally, EBS-MD fibroblasts displayed reduced adhesion capacities, which could be attributed to smaller focal adhesion contacts. Our study not only emphasizes plectin's functional role in human skin fibroblasts, it also provides further insights into the understanding of EBS-MD-associated disease mechanisms.

Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) is an autosomal recessive disorder caused by pathogenic variants in PLEC1, which encodes plectin. It is characterized by mild mucocutaneous fragility and blistering and muscle weakness. Translational readthrough-inducing drugs, such as repurposed aminoglycoside antibiotics, may represent a valuable therapeutic alternative for untreatable rare diseases caused by nonsense variants. To evaluate whether systemic gentamicin, at a dose of 7.5 mg/kg/d for 14 consecutive days, is clinically beneficial in a patient with EBS-MD. A single patient in Madrid, Spain, received 2 treatment courses with gentamicin on July 2019 and February 2020 with a follow-up period of 120 and 150 days, respectively. In this case report of a woman in her 30s with EBS-MD, before gentamicin treatment, the patient had mucocutaneous involvement, skeletal and respiratory muscle weakness, and myalgia that negatively affected her quality of life. Outcomes were evaluated with extensive laboratory tests and clinical scales. No nephrotoxic or ototoxic effects were detected after intravenous gentamicin administration. Gentamicin treatment was followed by plectin expression in the skin for at least 5 months. Although minimal changes were noted in skeletal muscle function (as measured by the Hammersmith functional motor scale and its expanded version: 6/40 to 7/40 and from 10/66 to 11/66, respectively) and respiratory musculature (maximal inspiratory and expiratory pressures D0 vs D16, MIP: 2.86 vs 3.63 KPa and MEP: 2.93 vs 4.63 KPa), myalgia disappeared (VAS dropped from 6 to 0), and quality of life improved (EuroQoL-5D-3L pain and anxiety dropped from 2 to 1). The findings of this single case report suggest that gentamicin treatment may help suppress PLEC1 premature termination codons and induce plectin expression in EBS-MD primary keratinocytes and skin. Our study suggests that gentamicin may play an important role in treating EBS-MD owing to nonsense variants.