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Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.

Aims We investigated N471D WASH complex subunit strumpellin (Washc5) knock‐in and Washc5 knock‐out mice as models for hereditary spastic paraplegia type 8 (SPG8). Methods We generated heterozygous and homozygous N471D Washc5 knock‐in mice and subjected them to a comprehensive clinical, morphological and laboratory parameter screen, and gait analyses. Brain tissue was used for proteomic analysis. Furthermore, we generated heterozygous Washc5 knock‐out mice. WASH complex subunit strumpellin expression was determined by qPCR and immunoblotting. Results Homozygous N471D Washc5 knock‐in mice showed mild dilated cardiomyopathy, decreased acoustic startle reactivity, thinner eye lenses, increased alkaline phosphatase and potassium levels and increased white blood cell counts. Gait analyses revealed multiple aberrations indicative of locomotor instability. Similarly, the clinical chemistry, haematology and gait parameters of heterozygous mice also deviated from the values expected for healthy animals, albeit to a lesser extent. Proteomic analysis of brain tissue depicted consistent upregulation of BPTF and downregulation of KLHL11 in heterozygous and homozygous knock‐in mice. WASHC5‐related protein interaction partners and complexes showed no change in abundancies. Heterozygous Washc5 knock‐out mice showing normal WASHC5 levels could not be bred to homozygosity. Conclusions While biallelic ablation of Washc5 was prenatally lethal, expression of N471D mutated WASHC5 led to several mild clinical and laboratory parameter abnormalities, but not to a typical SPG8 phenotype. The consistent upregulation of BPTF and downregulation of KLHL11 suggest mechanistic links between the expression of N471D mutated WASHC5 and the roles of both proteins in neurodegeneration and protein quality control, respectively.

Aims Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Methods Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro‐engineered three‐dimensional micro‐tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock‐in mice. Results Micro‐tissues grown from desmin‐mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild‐type cells. Within 1 week of culture, the majority of R349P desmin‐mutated tissues disintegrated, whereas wild‐type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin‐mutated tissues partially or completely ruptured, whereas wild‐type tissues did not display signs of damage. Conclusions Our results demonstrate that the progressive degeneration of desmin‐mutated micro‐tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age‐related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high‐intensity muscle contractions. We conclude that micro‐tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies. We investigate the effect of the R349P desmin mutation on the formation, differentiation, and contractile function of muscle micro‐tissues grown from satellite cells. Desmin‐mutated micro‐tissues show progressive degeneration over time, which is closely linked to extracellular matrix fibre breakage and associated with unevenly distributed tensile stress and locally increased contractile forces. Our results suggest that the age‐related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high‐intensity muscle contractions. image

BACKGROUND Duchenne muscular dystrophy (DMD) is a hereditary neuromuscular disease leading to progressive muscle wasting. Since there is a need for MRI variables that serve as early sensitive indicators of response to treatment, several quantitative MRI methods have been suggested for disease monitoring. PURPOSE To evaluate the potential of sodium (23 Na) and proton (1 H) MRI methods to assess early pathological changes in skeletal muscle of DMD. STUDY TYPE Prospective clinical study. POPULATION 23 Na and 1 H MRI of the right leg were performed in 13 patients with DMD (age 7.8 ± 2.4) and 14 healthy boys (age 9.5 ± 2.2). FIELD STRENGTH/SEQUENCE 3 T including a multiecho-spin-echo sequence, diffusion-weighted sequences, 1 H spectroscopy, 3-pt Dixon, and 23 Na ultrashort echo time sequences. ASSESSMENT We obtained water T2 maps, fat fraction (FF), pH, and diffusion properties of the skeletal muscle tissue. Moreover, total tissue sodium concentration (TSC) was calculated from the 23 Na sequence. Intracellular-weighted 23 Na signal (ICwS) was derived from 23 Na inversion-recovery imaging. STATISTICAL TESTS Results from DMD patients and controls were compared using Wilcoxon rank-sum tests and repeated analysis of variance (ANOVA). Spearman-rank correlations and area under the curve (AUC) were calculated to assess the performance of the different MRI methods to distinguish dystrophic from healthy muscle tissue. RESULTS FF, water T2 , and pH were higher in DMD patients (0.07 ± 0.03, 39.4 ± 0.8 msec, 7.06 ± 0.03, all P

SUMMARY The pathogenic mechanism by which dominant mutations in VCP cause multisystem proteinopathy (MSP), a rare neurodegenerative disease that presents as fronto-temporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), remains unclear. To explore this, we inactivate VCP in murine postnatal forebrain neurons (VCP conditional knockout [cKO]). VCP cKO mice have cortical brain atrophy, neuronal loss, autophago-lysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. Conditional expression of a single disease-associated mutation, VCP-R155C, in a VCP null background similarly recapitulates features of VCP inactivation and FTLD-TDP, suggesting that this MSP mutation is hypomorphic. Comparison of transcriptomic and proteomic datasets from genetically defined patients with FTLD-TDP reveal that progranulin deficiency and VCP insufficiency result in similar profiles. These data identify a loss of VCP-dependent functions as a mediator of FTLD-TDP and reveal an unexpected biochemical similarity with progranulin deficiency., Graphical abstract, In brief Wani et al. demonstrate that loss of neuronal VCP recapitulates FTLD-TDP pathology in mice. Conditional expression of a VCP-R155C mutation, associated with MSP1, also recapitulates FTLD-TDP consistent with its hypomorphic function. A transcriptomic and proteomic signature of VCP inactivation resembles a transcriptomic signature in brains of patients with GRN insufficiency.

Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set.

Mutations in the sarcomeric protein titin, encoded byTTN, are emerging as a common cause of myopathies. The diagnosis of aTTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence ofTTNvariants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis ofTTN-related myopathies and the pathogenicity ascertainment ofTTNmissense variants. We identified 30 patients with a primaryTTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missenseTTNvariant, or homozygous for oneTTNmissense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizingTTNmissense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.

Background: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype. Methods: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin. Results: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype. Conclusions: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.

Muscle biomechanics relies on active motor protein assembly and passive strain transmission through cytoskeletal structures. The desmin filament network aligns myofibrils at the z-discs, provides nuclear–sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness. As the effect of R349P desmin on axial biomechanics in fully differentiated single muscle fibers is unknown, we used our MyoRobot to compare passive visco-elasticity and active contractile biomechanics in single fibers from fast- and slow-twitch muscles from adult to senile mice, hetero- or homozygous for the R349P desmin mutation with wild type littermates. We demonstrate that R349P desmin presence predominantly increased axial stiffness in both muscle types with a pre-aged phenotype over wild type fibers. Axial viscosity and Ca2+-mediated force were largely unaffected. Mutant single fibers showed tendencies towards faster unloaded shortening over wild type fibers. Effects of aging seen in the wild type appeared earlier in the mutant desmin fibers. Our single-fiber experiments, free of extracellular matrix, suggest that compromised muscle biomechanics is not exclusively attributed to fibrosis but also originates from an impaired intermediate filament network.

RYR1 mutations, the most common cause of non-dystrophic neuromuscular disorders, are associated with the malignant hyperthermia susceptibility (MHS) trait as well as congenital myopathies with widely variable clinical and histopathological manifestations. Recently, bleeding anomalies have been reported in association with certain RYR1 mutations. Here we report a preterm infant born at 32 weeks gestation with arthrogryposis multiplex congenita due to compound heterozygous, previously MHS-associated RYR1 mutations, with additional signs of prenatal hemorrhage. The patient presented at birth with multiple joint contractures, scoliosis, severe thoracic rigidity and respiratory failure. He continued to depend on mechanical ventilation and tube feeding. Muscle histopathology showed a marked myopathic pattern with eccentric cores. Interestingly, the patient had additional unusual prenatal intraventricular hemorrhage, resulting in post-hemorrhagic hydrocephalus as well as epidural hemorrhage affecting the spinal cord. This report adds to the phenotypic variability associated with RYR1 mutations, and highlights possible bleeding complications in affected individuals.

The majority of hereditary and acquired myopathies are clinically characterized by progressive muscle weakness. We hypothesized that ongoing derangement of skeletal muscle cytoarchitecture at the single fiber level may precede and be responsible for the progressive muscle weakness. Here, we analyzed the effects of aging in wild-type (wt) and heterozygous (het) and homozygous (hom) R349P desmin knock-in mice. The latter harbor the ortholog of the most frequently encountered human R350P desmin missense mutation. We quantitatively analyzed the subcellular cytoarchitecture of fast- and slow-twitch muscles from young, intermediate, and aged wt as well as desminopathy mice. We recorded multiphoton second harmonic generation and nuclear fluorescence signals in single muscle fibers to compare aging-related effects in all genotypes. The analysis of wt mice revealed that the myofibrillar cytoarchitecture remained stable with aging in fast-twitch muscles, whereas slow-twitch muscle fibers displayed structural derangements during aging. In contrast, the myofibrillar cytoarchitecture and nuclear density were severely compromised in fast- and slow-twitch muscle fibers of hom R349P desmin mice at all ages. Het mice only showed a clear degradation in their fiber structure in fast-twitch muscles from the adult to the presenescent age bin. Our study documents distinct signs of normal and R349P mutant desmin-related remodeling of the 3D myofibrillar architecture during aging, which provides a structural basis for the progressive muscle weakness.

Summary Myopathies encompass a wide variety of acquired and hereditary disorders. The pathomechanisms include structural and functional changes affecting, e.g., myofiber metabolism and contractile properties. In this study, we observed increased passive tension (PT) of skinned myofibers from patients with myofibrillar myopathy (MFM) caused by FLNC mutations (MFM-filaminopathy) and limb-girdle muscular dystrophy type-2A due to CAPN3 mutations (LGMD2A), compared to healthy control myofibers. Because the giant protein titin determines myofiber PT, we measured its molecular size and the titin-to-myosin ratio, but found no differences between myopathies and controls. All-titin phosphorylation and site-specific phosphorylation in the PEVK region were reduced in myopathy, which would be predicted to lower PT. Electron microscopy revealed extensive ultrastructural changes in myofibers of various hereditary myopathies and also suggested massive binding of proteins to the sarcomeric I-band region, presumably heat shock proteins (HSPs), which can translocate to elastic titin under stress conditions. Correlative immunofluorescence and immunoelectron microscopy showed that two small HSPs (HSP27 and αB-crystallin) and the ATP-dependent chaperone HSP90 translocated to the titin springs in myopathy. The small HSPs, but not HSP90, were upregulated in myopathic versus control muscles. The titin-binding pattern of chaperones was regularly observed in Duchenne muscular dystrophy (DMD), LGMD2A, MFM-filaminopathy, MFM-myotilinopathy, titinopathy, and inclusion body myopathy due to mutations in valosin-containing protein, but not in acquired sporadic inclusion body myositis. The three HSPs also associated with elastic titin in mouse models of DMD and MFM-filaminopathy. Mechanical measurements on skinned human myofibers incubated with exogenous small HSPs suggested that the elevated PT seen in myopathy is caused, in part, by chaperone-binding to the titin springs. Whereas this interaction may be protective in that it prevents sarcomeric protein aggregation, it also has detrimental effects on sarcomere function. Thus, we identified a novel pathological phenomenon common to many hereditary muscle disorders, which involves sarcomeric alterations. Electronic supplementary material The online version of this article (10.1186/s40478-017-0474-0) contains supplementary material, which is available to authorized users.

Zusammenfassung Ziel der Studie Erfassung der Nutzungsakzeptanz, Zugangsmöglichkeiten und -barrieren eines Online-Selbstmanagementprogramms (moodgym) für Depressionen. Methodik Schriftliche Befragungen von n = 181 stationär behandelten Patienten (Prä-Post-Messung, 8 Wochen) und n = 31 Experten. Ergebnisse Moderate bis hohe Nutzungsakzeptanz. Relevante Zugangsbarrieren waren geringe PC-Erfahrungen, Konzentrationsschwierigkeiten und schwere Krankheitsverläufe. Schlussfolgerung Erstmals konnte gezeigt werden, dass Interventionen wie moodgym einen komplementären Behandlungsbaustein in der stationären Versorgung bilden könnten.

ObjectiveTo assess the clinical, radiologic, myopathologic, and proteomic findings in a patient manifesting a multisystem proteinopathy due to a homozygous valosin-containing protein gene (VCP) mutation previously reported to be pathogenic in the heterozygous state.MethodsWe studied a 36-year-old male index patient and his father, both presenting with progressive limb-girdle weakness. Muscle involvement was assessed by MRI and muscle biopsies. We performed whole-exome sequencing and Sanger sequencing for segregation analysis of the identified p.Arg159His VCP mutation. To dissect biological disease signatures, we applied state-of-the-art quantitative proteomics on muscle tissue of the index case, his father, 3 additional patients with VCP-related myopathy, and 3 control individuals.ResultsThe index patient, homozygous for the known p.Arg159His mutation in VCP, manifested a typical VCP-related myopathy phenotype, although with a markedly high creatine kinase value and a relatively early disease onset, and Paget disease of bone. The father exhibited a myopathy phenotype and discrete parkinsonism, and multiple deceased family members on the maternal side of the pedigree displayed a dementia, parkinsonism, or myopathy phenotype. Bioinformatic analysis of quantitative proteomic data revealed the degenerative nature of the disease, with evidence suggesting selective failure of muscle regeneration and stress granule dyshomeostasis.ConclusionWe report a patient showing a multisystem proteinopathy due to a homozygous VCP mutation. The patient manifests a severe phenotype, yet fundamental disease characteristics are preserved. Proteomic findings provide further insights into VCP-related pathomechanisms.

Background Mutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p. R350P. Methods and results Transverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 +/- 2.9%, HET-sham: 64.5 +/- 4.7%, WT-TAC: 63.5 +/- 4.9%, HET-TAC: 55.7 +/- 5.4%; p < 0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 +/- 2385 mu l/min, HET sham: 10391 +/- 1349 mu l/min, WT-TAC: 8097 +/- 1903 mu l/min, HET-TAC: 5793 +/- 2517 mu l/min; p< 0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 +/- 406, HET-sham: 13526 +/- 781, WT-TAC: 11155 +/- 3315, HET-TAC: 8649 +/- 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC. Conclusion Pressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure.

Background Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. Methods We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). Findings 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1β expression- but also showed activation of Wnt/β-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. Interpretation Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

Muscle biomechanics is determined by active motor-protein assembly and passive strain transmission through cytoskeletal structures. The extrasarcomeric desmin filament network aligns myofibrils at the z-discs, provides nuclear-sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness compatible with a pre-aged phenotype in the disease. Since the specific effect of R349P desmin on axial biomechanics in fully differentiated muscle fibers is unknown, we used our automated MyoRobot biomechatronics platform to compare passive and active biomechanics in single fibers derived from fast- and slow-twitch muscles from adult to senile mice hetero- or homozygous for this desmin mutation with wild-type littermates. Experimental protocols involved caffeine-induced Ca2+-mediated force transients, pCa-force curves, resting length-tension curves, visco-elasticity and ‘slack-tests’. We demonstrate that the presence of R349P desmin predominantly increased single fiber axial stiffness in both muscle types with a pre-aged phenotype over wild-type fibers. Axial viscosity was unaffected. Likewise, no systematic changes in Ca2+-mediated force properties were found. Notably, mutant single fibers showed faster unloaded shortening over wild-type fibers. Effects of ageing seen in the wild-type always appeared earlier in the mutant desmin fibers. Impaired R349P desmin muscle biomechanics is clearly an effect of a compromised intermediate filament network rather than secondary to fibrosis.

To assess the clinical, radiologic, myopathologic, and proteomic findings in a patient manifesting a multisystem proteinopathy due to a homozygous valosin-containing protein gene (We studied a 36-year-old male index patient and his father, both presenting with progressive limb-girdle weakness. Muscle involvement was assessed by MRI and muscle biopsies. We performed whole-exome sequencing and Sanger sequencing for segregation analysis of the identified p.Arg159HisThe index patient, homozygous for the known p.Arg159His mutation inWe report a patient showing a multisystem proteinopathy due to a homozygous

ZusammenfassungDie neuromuskuläre Forschung hat revolutionäre Erweiterungen des technischen Repertoires erlebt. Die vorliegende Arbeit diskutiert die wesentlichen Entwicklungen auf den Gebieten der genetischen Diagnostik, der “Omics”-Anwendungen vom Epigenom bis zum Proteom, der Genom-Editierung in Tierund Zellmodellen sowie neue optische Bildgebungsverfahren, die sowohl die neuromuskuläre Grundlagenforschung als auch die translationale und klinische Forschung transformieren.

We report our results of 1000 diagnostic WES cases based on 2819 sequenced samples from 54 countries with a wide phenotypic spectrum. Clinical information given by the requesting physicians was translated to HPO terms. WES processes were performed according to standardized settings. We identified the underlying pathogenic or likely pathogenic variants in 307 families (30.7%). In further 253 families (25.3%) a variant of unknown significance, possibly explaining the clinical symptoms of the index patient was identified. WES enabled timely diagnosing of genetic diseases, validation of causality of specific genetic disorders of PTPN23, KCTD3, SCN3A, PPOX, FRMPD4, and SCN1B, and setting dual diagnoses by detecting two causative variants in distinct genes in the same patient. We observed a better diagnostic yield in consanguineous families, in severe and in syndromic phenotypes. Our results suggest that WES has a better yield in patients that present with several symptoms, rather than an isolated abnormality. We also validate the clinical benefit of WES as an effective diagnostic tool, particularly in nonspecific or heterogeneous phenotypes. We recommend WES as a first-line diagnostic in all cases without a clear differential diagnosis, to facilitate personal medical care.

Purpose of review Myofibrillar myopathies (MFMs) are hereditary muscle disorders characterized by distinct histopathological features. This review provides an overview of recent research with respect to new disease genes, clinical phenotypes, insights into pathomechanisms and therapeutic strategies. Recent findings Beyond the known disease genes DES, FLNC, MYOT, CRYAB, ZASP, BAG3, FHL1 and TTN, mutations in PLEC, ACTA1, HSPB8 and DNAJB6 have also been associated with a MFM phenotype. Proteomic analysis revealed new information about the composition of protein aggregates in myotilinopathy and identified a new diagnostic marker. New animal models mirror central aspects of MFM pathology and novel therapeutic strategies for treatment of MFM were evaluated in cell and animal models. Summary MFMs are an expanding and numerically significant group of protein aggregate diseases with marked clinical and genetic heterogeneity. Though no specific therapy is currently available, the generation of patient-mimicking cell and animal models now paves the way for the preclinical evaluation of novel therapeutic strategies.

Background and Purpose— Duchenne muscular dystrophy (DMD) is the most frequent skeletal muscle myopathy. Nearly all patients develop cardiomyopathy in their second decade of life. The purpose of this study was to evaluate the frequency, cause, and outcome of stroke in a German cohort of patients with DMD. Methods— Retrospective analysis of medical records of 54 DMD patients, who lived in a regional facility for handicapped people (Wichernhaus Altdorf, Germany) between 1963 and 2013. Results— Fifty-four DMD patients were followed up for 7.4 years on average. Mean age at admission and discharge from the long-term care facility or death were 11.4 and 18.8 years, respectively. Covering a total observation period of 400 patient-years, we identified 4 DMD patients with juvenile arterial ischemic strokes. Off-label systemic thrombolysis in 2 patients resulted in a nearly complete regression of stroke-related symptoms, but 1 patient died of septic pneumonia and cardiac failure 24 days after thrombolysis therapy. In the other 2 patients, who had their ischemic strokes in 1994 and 1998, severe infarction-related symptoms persisted, and 1 patient died 13 days later. DMD-associated cardiomyopathy without evidence of atrial fibrillation was the only risk factor for ischemic stroke in all patients. Conclusions— This study indicates an increased risk for ischemic strokes in DMD patients. Regular cardiological assessment of all DMD patients is mandatory to evaluate the individual risk profile for cardioembolic events and to adapt therapeutic strategies.

Secondary mitochondrial dysfunction is a feature in a wide variety of human protein aggregate diseases caused by mutations in different proteins, both in the central nervous system and in striated muscle. The functional relationship between the expression of a mutated protein and mitochondrial dysfunction is largely unknown. In particular, the mechanism how this dysfunction drives the disease process is still elusive. To address this issue for protein aggregate myopathies, we performed a comprehensive, multi-level analysis of mitochondrial pathology in skeletal muscles of human patients with mutations in the intermediate filament protein desmin and in muscles of hetero- and homozygous knock-in mice carrying the R349P desmin mutation. We demonstrate that the expression of mutant desmin causes disruption of the extrasarcomeric desmin cytoskeleton and extensive mitochondrial abnormalities regarding subcellular distribution, number and shape. At the molecular level, we uncovered changes in the abundancy and assembly of the respiratory chain complexes and supercomplexes. In addition, we revealed a marked reduction of mtDNA- and nuclear DNA-encoded mitochondrial proteins in parallel with large-scale deletions in mtDNA and reduced mtDNA copy numbers. Hence, our data demonstrate that the expression of mutant desmin causes multi-level damage of mitochondria already in early stages of desminopathies. Electronic supplementary material The online version of this article (doi:10.1007/s00401-016-1592-7) contains supplementary material, which is available to authorized users.

p97 (VCP) is a homo-hexameric triple-A ATPase that exerts a plethora of cellular processes. Heterozygous missense mutations of p97 cause at least five human neurodegenerative disorders. However, the specific molecular consequences of p97 mutations are hitherto widely unknown. Our in silico structural models of human and Dictyostelium p97 showed that the disease-causing human R93C, R155H, and R155C as well as Dictyostelium R154C, E219K, R154C/E219K p97 mutations constitute variations in surface-exposed locations. In-gel ATPase activity measurements of p97 monomers and hexamers revealed significant mutation- and species-specific differences. While all human p97 mutations led to an increase in ATPase activity, no changes could be detected for the Dictyostelium R154C mutant, which is orthologous to human R155C. The E219K mutation led to an almost complete loss of activity, which was partially recuperated in the R154C/E219K double-mutant indicating p97 inter-domain communication. By means of co-immunoprecipitation experiments we identified an UBX-domain containing Dictyostelium protein as a novel p97 interaction partner. We categorized all UBX-domain containing Dictyostelium proteins and named the interaction partner UBXD9. Pull-down assays and surface plasmon resonance analyses of Dictyostelium UBXD9 or the human orthologue TUG/ASPL/UBXD9 demonstrated direct interactions with p97 as well as species-, mutation- and ATP-dependent differences in the binding affinities. Sucrose density gradient assays revealed that both human and Dictyostelium UBXD9 proteins very efficiently disassembled wild-type, but to a lesser extent mutant p97 hexamers into monomers. Our results are consistent with a scenario in which p97 point mutations lead to differences in enzymatic activities and molecular interactions, which in the long-term result in a late-onset and progressive multisystem disease.

Muscle biomechanics relies on active motor protein assembly and passive strain transmission through cytoskeletal structures. The desmin filament network aligns myofibrils at the z-discs, provides nuclear&ndash, sarcolemmal anchorage and may also serve as memory for muscle repositioning following large strains. Our previous analyses of R349P desmin knock-in mice, an animal model for the human R350P desminopathy, already depicted pre-clinical changes in myofibrillar arrangement and increased fiber bundle stiffness. As the effect of R349P desmin on axial biomechanics in fully differentiated single muscle fibers is unknown, we used our MyoRobot to compare passive visco-elasticity and active contractile biomechanics in single fibers from fast- and slow-twitch muscles from adult to senile mice, hetero- or homozygous for the R349P desmin mutation with wild type littermates. We demonstrate that R349P desmin presence predominantly increased axial stiffness in both muscle types with a pre-aged phenotype over wild type fibers. Axial viscosity and Ca2+-mediated force were largely unaffected. Mutant single fibers showed tendencies towards faster unloaded shortening over wild type fibers. Effects of aging seen in the wild type appeared earlier in the mutant desmin fibers. Our single-fiber experiments, free of extracellular matrix, suggest that compromised muscle biomechanics is not exclusively attributed to fibrosis but also originates from an impaired intermediate filament network.

Aim To characterise clinical presentation, laboratory and histopathologic characteristics and assess the treatment and outcome of neuromuscular side-effects of checkpoint therapy. Methods The side-effect registry and the institutional database from ten skin cancer centres were queried for reports on myositis and neuromuscular side-effects induced by checkpoint inhibitors. In total, 38 patients treated with ipilimumab, tremelimumab, nivolumab and pembrolizumab for metastatic skin cancer were evaluated and characterised. Results Myositis was the most frequent neuromuscular adverse event. In 32% of cases, myositis was complicated by concomitant myocarditis. Furthermore, cases of isolated myocarditis, myasthenia gravis, polymyalgia rheumatica, radiculoneuropathy and asymptomatic creatine kinase elevation were reported. The onset of side-effects ranged from the first week of treatment to 115 weeks after the start of therapy. Most of the cases were severe (49% grade III–IV Common Terminology Criteria for Adverse Events), and there were two fatalities (5%) due to myositis and myositis with concomitant myocarditis. Only half of the cases (50%) completely resolved, whereas the rest was either ongoing or had sequelae. Steroids were given in 80% of the resolved cases and in 40% of the unresolved cases. Conclusion Immune-mediated neuromuscular side-effects of checkpoint inhibitors greatly vary in presentation and differ from their idiopathic counterparts. These side-effects can be life threatening and may result in permanent sequelae. Occurrence of these side-effects must be taken into consideration for patient information, especially when considering adjuvant immunotherapy with anti–programmed cell-death protein 1 (PD-1) antibodies and monitoring, which should include regular surveillance of creatine kinase.

Hereditary spastic paraplegias (HSPs) are genetically diverse and clinically characterised by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str; also known as WASHC5), a member of the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex, have been shown to cause a form of HSP known as spastic paraplegia 8 (SPG8). To investigate the molecular functions of wild-type (WT) and N417D Str, we generated Dictyostelium Str− cells and ectopically expressed StrWT-GFP or StrN471D-GFP in Str− and WT cells. Overexpression of both proteins apparently caused a defect in cell division, as we observed a clear increase in multinucleate cells. Real-time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str− cells, but western blots showed a twofold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known, as well as new, Str-interacting proteins, and also proteins that no longer bind to StrN471D. At the cellular level, Str− cells displayed defects in cell growth, phagocytosis, macropinocytosis, exocytosis and lysosomal function. Expression of StrWT-GFP in Str− cells rescued all observed defects. In contrast, expression of StrN471D-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit, Str, in the endolysosomal system, and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Str function in the endolysosomal system. This article has an associated First Person interview with the first author of the paper., Summary: Strumpellin, a core member of the WASH complex, plays a key role in the endolysosomal system. Deficiencies in endo-/exocytosis and lysosome morphology are caused by the N471D strumpellin mutation, which is associated with spastic paraplegia 8.

Mutations of the human VCP gene, which encodes the V: alosin C: ontaining P: rotein (synonyms: p97, TER ATPase), are associated with various multi-systemic protein aggregation diseases. We report on a patient with progressive myopathy and incipient cognitive deficits. A diagnostic muscle biopsy revealed an inclusion body myopathy with protein aggregates. Magnetic resonance imaging and F18-positron-emission-tomography disclosed a fronto-temporal atrophy and glucose hypometabolism of the frontal and temporal lobes, respectively. Based on the clinical findings, a genetic analysis was performed which revealed a heterozygous c.277CT (p.Arg93Cys) mutation of the VCP gene, thus confirming the diagnosis of IBMPFD (I: nclusion B: ody M: yopathie with P: aget Disease of the Bones and F: ronto-temporal D: ementia).Mutationen des humanen VCP-Gens, welches für das Valosin-enthaltende Protein (