Molecular pathomechanisms of muscular dystrophies

This study aimed at elucidating molecular pathways behind muscular dystrophies, inherited disorders causing progressive weakness and loss of skeletal muscle, with the perspectives of demonstrating the pathogenicity of newly identified mutations, understanding the biology of muscle diseases, and finding options for their treatment. Tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy type 2J (LGMD2J) are caused by mutations in the C-terminal (M-band) part of the sarcomeric protein titin, whereas LGMD2A results from mutations in the muscle-specific protease calpain 3 (CAPN3). In yeast two-hybrid studies aiming at identifying proteins secondarily affected in the diseases, the multifunctional TRIM-related protein myospryn (CMYA5) was identified as a novel binding partner for both M-band titin and CAPN3. The interactions were confirmed by coimmunoprecipitation, and localization of myospryn at the M-band level was supported by multiple methods. Coexpression studies identified myospryn as a proteolytic substrate for CAPN3, and suggested that myospryn may attenuate its autolytic activation. The biological role of the titin myospryn interaction remained unresolved, and the mouse model of TMD/LGMD2J showed normal myospryn localization. However, since the TMD/LGMD2J mutations disrupt the myospryn binding site in titin, they are likely to have a downstream functional effect on myospryn. LGMD1D is caused by dominant mutations in the ubiquitous co-chaperone DNAJB6. LGMD1D muscle showed a myofibrillar pathology, with cytoplasmic accumulations of DNAJB6, aggregated Z-disc-associated proteins, and autophagic rimmed vacuoles. Expression of DNAJB6 constructs in zebrafish embryos confirmed a toxic effect of the mutant cytoplasmic DNAJB6b isoform, and cell culture studies demonstrated a slower turnover and impaired anti-aggregation activity of mutant DNAJB6. Protein interaction studies indicated an association of DNAJB6 with the chaperone-assisted selective autophagy (CASA) pathway, and a modulatory effect of BAG3 on DNAJB6 pathogenicity in zebrafish suggested that CASA has active role in the pathogenesis of LGMD1D. Welander distal myopathy (WDM) results from a dominant mutation in the prion-related domain (PRD) of the RNA-binding protein TIA1, a regulator of splicing and translation, and a component of stress granules (SGs). RT-PCR analysis of selected TIA1 target genes did not show splicing changes in WDM muscle, suggesting that the pathogenesis does not involve extensive mis-splicing. IF microscopy revealed accumulation of TIA1 and other SG proteins in WDM muscle, while image analysis of transfected cells, and fluorescence recovery after photobleaching (FRAP) studies indicated a mild increase in the SG-forming propensity of mutant TIA1. These findings suggest that increased aggregation of the TIA1 PRD causes muscle pathology in WDM, either directly through inappropriate protein aggregation or indirectly by compromising cellular metabolism.Lihasrappeumat ovat perinnöllisiä sairauksia, jotka johtavat luurankolihasten etenevään heikkouteen ja surkastumiseen. Ne aiheutuvat geenivirheistä useissa lihassyyn rakenneosissa, ja monet sairauteen johtavista molekyylitason mekanismeista tunnetaan huonosti. Tässä väitöskirjassa tutkittiin joidenkin lihasrappeumien tautimekanismeja, tavoitteena osoittaa tunnistettujen geenivirheiden patogeenisuus sekä ymmärtää lihastautien molekyylibiologiaa, esimerkiksi hoitojen kehittämistä ajatellen. Suomalaiseen tautiperintöön kuuluvat tibiaalinen lihasdystrofia (TMD) ja hartia-lantiotyypin lihasrappeuma 2J (LGMD2J) aiheutuvat virheistä titiinissä, sarkomeerien keskeisessä rakenne- ja säätelyproteiinissa. Tutkimuksessa tunnistettiin titiinin viallisen osan sitoutumiskumppaniksi myospryyni (myospryn, CMYA5), joka toimii solun kalvoliikenteessä ja viestinvälityksessä. TMD/LGMD2J-hiirimallin lihaksessa ei havaittu poikkeavaa myospryynin sijoittumista, mutta toiminnallinen vaikutus myospryyniin on todennäköinen. Myospryyni tunnistettiin myös LGMD2A-lihasrappeuman taustalla olevan kalpaiini 3 proteaasin substraatiksi ja mahdolliseksi säätelijäksi. Kuvatut proteiinivuorovaikutukset lisäävät lihaksen molekyylibiologian perustuntemusta ja voivat jatkossa auttaa TMD/LGMD2J- ja LGMD2A-tautien synnyn ymmärtämisessä. Hartia-lantiotyypin lihasrappeuma 1D (LGMD1D) aiheutuu proteiinien laadunvalvontakoneistoon kuuluvan kaitsijaproteiini DNAJB6:n geenivirheistä, joiden seurauksena lihassyihin kertyy proteiinisakkautumia. DNAJB6:n virheellisen muodon todettiin aiheuttavan lihastautia seeprakalan alkiossa tuotettuna, mikä sopii yhteen LGMD1D:n vallitsevan periytymismallin kanssa. Soluviljelmissä havaittiin, että geenivirheet hidastavat DNAJB6:n hajotusta sekä haittaavat kaitsijaproteiinin kykyä estää proteiinien aggregaatiota. Tutkimuksissa onnistuttiin myös vahvistamaan DNAJB6:n epäilty vuorovaikutus hiljattain kuvatun ja lihaksen toiminnalle tärkeäksi todetun CASA-mekanismin (chaperone-assisted selective autophagy, kaitsijaproteiiniavusteinen selektiivinen autofagia) kanssa sekä saatiin viitteitä tämän autofagiareitin suorasta osallisuudesta LGMD1D:n synnyssä. Ruotsissa ja Suomessa melko yleinen Welanderin distaalinen myopatia (WDM) aiheutuu geenivirheistä TIA1:ssä, joka on lähetti-RNA:n silmukoinnin ja proteiinitranslaation säätelijä sekä soluihin stressitilanteissa muodostuvien stressijyvästen (stress granule) rakenneosa. Tutkittujen TIA1:n kohdegeenien silmukoinnissa ei todettu muutoksia WDM-lihaksessa. Sen sijaan lihasnäytteissä nähtiin TIA1:n ja muiden proteiinien kasaumia, ja soluviljelmissä tehdyissä toiminnallisissa kokeissa havaittiin virheellisen TIA1:n muodostavan stressijyväsiä hieman normaaliproteiinia tehokkaammin. Tulosten perusteella WDM vaikuttaa siis johtuvan poikkeavasta stressijyväsproteiinien käyttäytymisestä, joka on viime aikoina havaittu merkittäväksi ilmiöksi myös hermorappeumasairauksien synnyssä

Kaitsijamutaatiot neuromuskulaaritaudeissa

Vertaisarvioitu.Kaitsijaproteiinien eli kaperonien ja kokaperonien geenivirheet aiheuttavat neuromuskulaaritauteja vaihtelevilla mekanismeilla. Peittyvästi periytyvät taudit liittyvät tyypillisesti kaitsijaproteiinin toiminnan puutokseen, kun taas vallitsevasti periytyvien taustalla voi olla kaitsijaproteiinikoneiston häiriintyminen (esimerkiksi DNAJB6- ja BAG3-geenivirheet) tai muut solumyrkylliset vaikutukset (sHSP-geenivirheet). Tautimekanismien selviäminen on viime vuosina avannut mahdollisuuksia myös tämän kasvavan tautiryhmän hoitoyrityksille.Peer reviewe

Neuromuscular Diseases Due to Chaperone Mutations: A Review and Some New Results

Skeletal muscle and the nervous system depend on efficient protein quality control, and they express chaperones and cochaperones at high levels to maintain protein homeostasis. Mutations in many of these proteins cause neuromuscular diseases, myopathies, and hereditary motor and sensorimotor neuropathies. In this review, we cover mutations in DNAJB6, DNAJB2, αB-crystallin (CRYAB, HSPB5), HSPB1, HSPB3, HSPB8, and BAG3, and discuss the molecular mechanisms by which they cause neuromuscular disease. In addition, previously unpublished results are presented, showing downstream effects of BAG3 p.P209L on DNAJB6 turnover and localization

Increasing Role of Titin Mutations in Neuromuscular Disorders

Peer reviewe

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis

Objective Inclusion body myositis (IBM) has an unclear molecular etiology exhibiting both characteristic inflammatory T-cell activity and rimmed-vacuolar degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM. Methods We performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n = 24), tibial muscular dystrophy (n = 6), and histopathologically normal group (n = 9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs). Results We observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca2+-induced apoptotic pathways of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows increased expression and altered splicing usage in IBM muscles. Interpretation Our analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles.Peer reviewe

Autophagy Regulates the Liver Clock and Glucose Metabolism by Degrading CRY1

The circadian clock coordinates behavioral and circadian cues with availability and utilization of nutrients. Proteasomal degradation of clock repressors, such as cryptochrome (CRY) 1, maintains periodicity. Whether macroautophagy, a quality control pathway, degrades circadian proteins remains unknown. Here we show that circadian proteins BMAL1, CLOCK, REV-ERB alpha, and CRY1 are lysosomal targets, and that macroautophagy affects the circadian clock by selectively degrading CRY1. Autophagic degradation of CRY1, an inhibitor of gluconeogenesis, occurs in a diurnal window when rodents rely on gluconeogenesis, suggesting that CRY1 degradation is timeimprinted to maintenance of blood glucose. High-fat feeding accelerates autophagic CRY1 degradation and contributes to obesity-associated hyperglycemia. CRY1 contains several light chain 3 (LC3)-interacting region (LIR) motifs, which facilitate the interaction of cargo proteins with the autophagosome marker LC3. Using mutational analyses, we identified two distinct LIRs on CRY1 that exert circadian glycemic control by regulating CRY1 degradation, revealing LIRs as potential targets for controlling hyperglycemia.Peer reviewe

Actininopathy : A new muscular dystrophy caused by ACTN2 dominant mutations

Objective To clinically and pathologically characterize a cohort of patients presenting with a novel form of distal myopathy and to identify the genetic cause of this new muscular dystrophy. Methods We studied 4 families (3 from Spain and 1 from Sweden) suffering from an autosomal dominant distal myopathy. Affected members showed adult onset asymmetric distal muscle weakness with initial involvement of ankle dorsiflexion later progressing also to proximal limb muscles. Results In all 3 Spanish families, we identified a unique missense variant in the ACTN2 gene cosegregating with the disease. The affected members of the Swedish family carry a different ACTN2 missense variant. Interpretation ACTN2 encodes for alpha actinin2, which is highly expressed in the sarcomeric Z-disk with a major structural and functional role. Actininopathy is thus a new genetically determined distal myopathy. ANN NEUROL 2019;85:899-906.Peer reviewe

Dominant Distal Myopathy 3 (MPD3) Caused by a Deletion in the HNRNPA1 Gene

Background and Objectives To determine the genetic cause of the disease in the previously reported family with adult-onset autosomal dominant distal myopathy (myopathy, distal, 3; MPD3). Methods Continued clinical evaluation including muscle MRI and muscle pathology. A linkage analysis with single nucleotide polymorphism arrays and genome sequencing were used to identify the genetic defect, which was verified by Sanger sequencing. RNA sequencing was used to investigate the transcriptional effects of the identified genetic defect. Results Small hand muscles (intrinsic, thenar, and hypothenar) were first involved with spread to the lower legs and later proximal muscles. Dystrophic changes with rimmed vacuoles and cytoplasmic inclusions were observed in muscle biopsies at advanced stage. A single nucleotide polymorphism array confirmed the previous microsatellite-based linkage to 8p22-q11 and 12q13-q22. Genome sequencing of three affected family members combined with structural variant calling revealed a small heterozygous deletion of 160 base pairs spanning the second last exon 10 of the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) gene, which is in the linked region on chromosome 12. Segregation of the mutation with the disease was confirmed by Sanger sequencing. RNA sequencing showed that the mutant allele produces a shorter mutant mRNA transcript compared with the wild-type allele Immunofluorescence studies on muscle biopsies revealed small p62 and larger TDP-43 inclusions. Discussion A small exon 10 deletion in the gene HNRNPA1 was identified as the cause of MPD3 in this family. The new HNRNPA1-related phenotype, upper limb presenting distal myopathy, was thus confirmed, and the family displays the complexities of gene identification.Peer reviewe

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis

Objective: Inclusion body myositis (IBM) has an unclear molecular etiology exhibiting both characteristic inflammatory T-cell activity and rimmed-vacuolar degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM.Methods: We performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n = 24), tibial muscular dystrophy (n = 6), and histopathologically normal group (n = 9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs).Results: We observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca2+-induced apoptotic pathways of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows increased expression and altered splicing usage in IBM muscles.Interpretation: Our analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles.</p

Mutations in the J domain of DNAJB6 cause dominant distal myopathy

Eight patients from five families with undiagnosed dominant distal myopathy underwent clinical, neurophysiological and muscle biopsy examinations. Molecular genetic studies were performed using targeted sequencing of all known myopathy genes followed by segregation of the identified mutations in the affected families using Sanger sequencing. Two novel mutations in DNAJB6 J domain, c.149C>T (p.A50V) and c.161A>C (p.E54A), were identified as the cause of disease. The muscle involvement with p.A50V was distal calf-predominant, and the p.E54A was more proximo-distal. Histological findings were similar to those previously reported in DNAJB6 myopathy. In line with reported pathogenic mutations in the glycine/phenylalanine (G/F) domain of DNAJB6, both the novel mutations showed reduced anti-aggregation capacity by filter trap assay and TDP-43 disaggregation assays. Modeling of the protein showed close proximity of the mutated residues with the G/F domain. Myopathy-causing mutations in DNAJB6 are not only located in the G/F domain, but also in the J domain. The identified mutations in the J domain cause dominant distal and proximo-distal myopathy, confirming that mutations in DNAJB6 should be considered in distal myopathy cases.Peer reviewe