Inflammation, fibrosis and skeletal muscle regeneration in LGMDR9 are orchestrated by macrophages

Aims: Variable degrees of inflammation, necrosis, regeneration and fibrofatty replacement are part of the pathological spectrum of the dystrophic process in alpha dystroglycanopathy LGMDR9 (FKRP-related, OMIM #607155), one of the most prevailing types of LGMDs worldwide. Inflammatory processes and their complex interplay with vascular, myogenic and mesenchymal cells may have a major impact on disease development. The purpose of our study is to describe the specific immune morphological features in muscle tissue of patients with LGMDR9 to enable a better understanding of the phenotype of muscle damage leading to disease progression. Methods: We have analysed skeletal muscle biopsies of 17 patients genetically confirmed as having LGMDR9 by histopathological and molecular techniques. Results: We identified CD206+ MHC class II+ and STAT6+ immune-repressed macrophages dominating the endomysial infiltrate in areas of myofibre regeneration and fibrosis. Additionally, PDGFRβ+ pericytes were located around MHC class II+ activated capillaries residing in close proximity to areas of fibrosis and regenerating fibres. Expression of VEGF was found on many regenerating neonatal myosin+ fibres, myofibres and CD206+ macrophages also co-expressed VEGF. Conclusion: Our results show characteristic immune inflammatory features in LGMDR9 and more specifically shed light on the predominant role of macrophages and their function in vascular organisation, fibrosis and myogenesis. Understanding disease-specific immune phenomena potentially inform about possibilities for anti-fibrotic and anti-inflammatory therapeutic strategies, which may complement Ribitol replacement and gene therapies for LGMDR9 that may be available in the future

Ataxia, Intellectual Disability, and Ocular Apraxia with Cerebellar Cysts: A New Disease?

Cerebellar cysts are rare findings in pediatric neuroimaging and rather characteristic for dystroglycanopathies and GPR56-related encephalopathy. We aim to report on seven children with cerebellar cysts showing absence of weakness and ruling out mutations within eight dystroglycanopathy genes and GPR56. Data about neurological and ophthalmological features, outcome, and creatine kinase values were collected from clinical histories and follow-up examinations. All MR images were qualitatively evaluated for infra- and supratentorial abnormalities. A SNP 6.0-Array was performed in three children. The POMT1, POMT2, POMGnT1, FKRP, FKTN, LARGE, ISPD, B3GALNT2, and GPR56 genes were screened in all patients by Sanger sequencing. Seven children from five families were studied. Ataxia, intellectual disability, and language impairment were found in all patients, ocular motor apraxia in five, and severe myopia in three. None of the patients had weakness, only three a minimally increased creatine kinase value. Qualitative neuroimaging evaluation showed cerebellar cysts and dysplasia in the cerebellar hemispheres and vermis in all children. Additional findings were an enlarged fourth ventricle in all children, vermian hypoplasia and brain stem morphological abnormalities in five. The SNP array showed no pathogenetic imbalances in all children evaluated. In all patients, no mutations were found in POMT1, POMT2, POMGnT1, FKRP, FKTN, LARGE, ISPD, B3GALNT2, and GPR56. The peculiar combination of the same clinical and neuroimaging findings in our patients highly suggests that this phenotype may represent a novel entity, possibly falling within the spectrum of dystroglycanopathies

Regionalized Pathology Correlates with Augmentation of mtDNA Copy Numbers in a Patient with Myoclonic Epilepsy with Ragged-Red Fibers (MERRF-Syndrome)

Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89–100%. However, mtDNA copy numbers were increased 3–7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that “futile” stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome

Установление границ охранной зоны линейного сооружения – магистральный газопровод "НГПЗ - Парабель"

Составлено графическое описание местоположения границ зон с особыми условиями использования территорий границ охранной зоны линейного сооружения – магистральный газопровод "НГПЗ - Парабель".A graphic description of the location of the boundaries of the zones with special conditions for the use of the territories of the boundaries of the protection zone of the linear structure – "the NGPZ-Parabel" gas pipeline has been compiled

Fibrogenesis and fibrolysis in Duchenne muscular dystrophy

Die Muskeldystrophie Typ Duchenne ist die häufigste neuromuskuläre Erkrankung im Kindes- und Jugendalter. Sie wird X-chromosomal rezessiv vererbt und tritt bei 1:3500 neugeborenen Knaben auf. Der klinische Verlauf ist durch einen kontinuierlichen Verlust der willkürmotorischen Fähigkeiten und eine deutlich verkürzte Lebenserwartung gekennzeichnet. Ursache ist das Fehlen von Dystrophin aufgrund von Mutationen im DMD-Gen. Dystrophin ist ein subsarcolemmal gelegenes Protein, das eine zentrale Rolle für die Stabilität und die Funktionstüchtigkeit von Muskelfasern spielt. Zu Beginn der Erkrankung kommt es zu einer intensiven Degeneration und Regeneration von Muskelfasern und zu einer inflammatorischen Reaktion. Im Verlauf dominiert die Fibrose der quergestreiften Muskulatur („fibrogene Regeneration“). Der Vergleich zwischen verschiedenen Spezies mit einer dystrophinnegativen Muskeldystrophie ergibt, dass allein durch das Fehlen von Dystrophin weder der Phänotyp noch der histologische Befund erklärt werden kann. Bei der mild betroffenen mdx-Maus entwickelt sich erst spät eine Muskelfibrose, bei dem schwer betroffenen GRMD- Hund ist die Muskulatur frühzeitig fibrotisch degeneriert. Es müssen daher sekundäre Prozesse pathophysiologisch bedeutsam sein, die speziesabhängig unterschiedlich reguliert werden. Eine Organfibrose ist Ausdruck einer Störung zwischen Auf- und Abbau der extrazellulären Matrix (ECM). Als Komponenten der ECM wurden Kollagen I, III, IV, VI, XIV, Elastin, Tenaszin und Fibronektin immunhistochemisch untersucht. Es wurde reifes Bindegewebe mit einem erhöhten Anteil fibrillärer Kollagene I und III und einer vermehrten Expression von Proteinen wie Tenaszin, die in der Muskelentwicklung und Muskelregeneration eine Rolle spielen, nachgewiesen. Die basalmembranständigen Kollagene IV und VI waren entsprechend der vermehrten Regeneration hochreguliert. Die MMP (Matrix-Metalloproteinase)-1 und MMP-2 sind am Abbau und Umbau der ECM beteiligt, MMP-1 besonders in den Abbau fibrillärer Kollagene. Die Expression von MMP-1 und MMP-2 sowie von deren Inhibitoren TIMP (tissue inhibitor of metalloproteinase)-1 und TIMP-2 wurde auf Proteinebene immunhistochemisch und mittels Westernblot untersucht. Die mRNA Expression wurde mit der in situ- Hybridisierung lokalisiert und mit der RT-PCR quantifiziert. Es ergab sich ein einheitliches Muster mit einer Hochregulation von TIMP-1, TIMP-2 und MMP-2. Für MMP-2 wurde mit der Zymographie auch eine gesteigerte Enzymaktivität nachgewiesen. Von MMP-1 war trotz der stark vermehrten Synthese von ECM weder die mRNA noch das Protein vermehrt nachweisbar. Die Ergebnisse dieser Untersuchung sprechen dafür, dass bei der DMD eine Hemmung der Fibrolyse vorliegt. Da bislang keine kurative Behandlungsmöglichkeit zur Verfügung steht, erscheint es sinnvoll antifibrotische Therapieansätze - wie z.B. den Einsatz von TGF-β1 Antagonisten - zu evaluieren.Duchenne muscular dystrophy (DMD) is the most frequent neuromuscular disorder in children and adolescents. It is an X-linked recessive disorder with an incidence of one in 3,500 newborn boys. Progressive weakness results in loss of gait at the age of 10-12 years and life expectancy is limited to 20-25 years of age because of respiratory and/or cardiac failure. DMD is caused by mutations of the DMD-gene. The gene product is the subsarcolemmal protein dystrophin which is important for the stability and function of muscle fibres. Loss of dystrophin begins with marked muscle fibre degeneration and regeneration associated with inflammatory changes and an increase of endo- and perimysial extracellular matrix (ECM). This process results in a vast destruction of striated muscles and excessive deposition of connective tissue (“misdirected” or “fibrogenic” regeneration). The comparison between different species with dystrophin negative muscular dystrophy shows that loss of dystrophin alone does explain neither phenotype nor histological findings. The mildly affected mdx mouse shows remarkable muscle fibrosis only in a late stage of the disease. However, in the severely affected GRMD dog, muscle fibrosis already occurs during the first months of life. One may hypothesize that secondary pathophysiological changes are differentially regulated in various species. Organ fibrosis is the result of disequilibrium of fibrolysis und fibrogenesis. In this work, the ECM components collagen I, III, IV, VI, XIV, elastin, tenascin, and fibronectin were investigated by immunohistochemical staining. As a result, mature connective tissue with an increased content of fibrillary collagens and of proteins, which are involved in myogenesis and regeneration like tenascin, could be demonstrated. Basal membrane associated collagens IV and VI were found increased according to the degree of muscle fibre regeneration. Matrix metalloproteinases (MMP)-1 and MMP-2 are required for degeneration and remodelling of ECM. MMP-1 is especially involved in degeneration of fibrillary collagens. Expression of mRNA and of protein were investigated for MMP-1, MMP-2, and for the tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2. Protein was shown by immunohistochemical staining and Westernblot. The mRNA expression was localized by in situ hybridisation and quantified by RT-PCR. There was a consistent pattern of up regulation of MMP-2, TIMP-1, and TIMP-2. However, in MMP-1 neither mRNA nor protein was increased in spite of a remarkable up regulation of ECM synthesis. These findings suggest that decreased fibrolysis in muscle contributes to the course of the disease in DMD. Because there is no curative therapy available, Studies on antifibrotic drugs like TGFβ-1 antagonist are warranted

Neuromuscular Diseases Affect Number Representation and Processing: An Exploratory Study

Spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD) both are rare genetic neuromuscular diseases with progressive loss of motor ability. The neuromotor developmental course of those diseases is well documented. In contrast, there is only little evidence about characteristics of general and specific cognitive development. In both conditions the final motor outcome is characterized by an inability to move autonomously: children with SMA never accomplish independent motoric exploration of their environment, while children with DMD do but later lose this ability again. These profound differences in developmental pathways might affect cognitive development of SMA vs. DMD children, as cognition is shaped by individual motor experiences. DMD patients show impaired executive functions, working memory, and verbal IQ, whereas only motor ability seems to be impaired in SMA. Advanced cognitive capacity in SMA may serve as a compensatory mechanism for achieving in education, career progression, and social satisfaction. This study aimed to relate differences in basic numerical concepts and arithmetic achievement in SMA and DMD patients to differences in their motor development and resulting sensorimotor and environmental experiences. Horizontal and vertical spatial-numerical associations were explored in SMA/DMD children ranging between 6 and 12 years through the random number generation task. Furthermore, arithmetic skills as well as general cognitive ability were assessed. Groups differed in spatial number processing as well as in arithmetic and domain-general cognitive functions. Children with SMA showed no horizontal and even reversed vertical spatial-numerical associations. Children with DMD on the other hand revealed patterns in spatial numerical associations comparable to healthy developing children. From the embodied Cognition perspective, early sensorimotor experience does play a role in development of mental number representations. However, it remains open whether and how this becomes relevant for the acquisition of higher order cognitive and arithmetic skills