Knockout mice and myocyte cell models to study the pathogenesesis and phenotype rescue of muscular plectinopathies
Final Report Abstract
The most common disease caused by plectin deficiency, epidermolysis bullosa (EB)-MD, is characterized by muscular dystrophy and severe skin blistering. EB-MD patients and plectin-deficient mice display massive desmin aggregation, the hallmark of myofibrillary myopathies (MFMs). Our previous studies had shown that the 4 major plectin isoforms expressed in muscle are crucial for the integrity of myofibers by specifically targeting and anchoring desmin IF networks to Z-disks, costameres, mitochondria, and the nuclear/SR membrane system. Having established myocyte cell culture systems mimicking the pathological hallmarks of MFMs in phase I of our FOR1228 project and taking advantage of our ample collection of conditional and muscle isoform-specific plectin knockout mouse lines, our major objectives for project phase II were: (i) To gain a better understanding of the pathogenesis underlying plectinopathies (and MFMs in general) by unraveling the mechanisms of plectin isoformmediated IF-docking at distinct cellular locations, including the neuromuscular synapse, ii) to develop and assess treatment concepts for the removal of protein aggregates from MFM myofibers, including phenotype rescue attempts through gene transfer; and iii) to analyze the biomechanical properties and mechanotranduction potential of plectin-deficient myocytes and myofibers in comparison to wild-type and other cell types. Our studies to objective (i) revealed that plectin isoform 1 (P1) by binding and thereby recruiting desmin IFs to myonuclei affects the nuclear morphology and gene expression pattern, and thus plays an important role in mechanotransduction. Equally important roles were revealed for P1d and P1b in affecting structural and functional features of sarcomeric Z-disks and mitochondria, respectively, while P1f could be shown to be of vital importance for neurosynapse integrity and to affect muscle cell metabolism, i.e. glucose uptake. The outcome of objective (ii)-based studies was equally satisfying, as 4-PBA, a chemical chaperon, was found to lead to the removal of protein aggregates, ex vivo in MFM myotube cultures as well as in vivo in mouse muscles, combined with improved muscle performance. This study provided the basis for ongoing initiatives for clinical test trials with MFM patients. Under this objective we also prepared and tested cDNA expression plasmids for plectin “mini” versions, setting up the stage for gene therapeutic approaches with MFM mice and eventually human patients. In the framework of objective (iii), we could show that dysfunctional plectin severely affects the stiffness, adhesion strength, and cytoskeletal dynamics of myoblasts with partially divergent implications for other cell types. In a series of complementary studies, the dominant role of plectin in mechanotransduction, as revealed in our studies with muscle cells, could be confirmed and extended to other cell systems, including vascular endothelial cells, fibroblasts, keratinocytes, and different cancer cell systems. In all, our results significantly advanced our understanding of the molecular mechanisms underlying plectinopathies and other MFMs, and opened a way for a possible treatment of related diseases.
Publications
- Linking cytoarchitecture to metabolism: Sarcolemma-associated plectin affects glucose uptake by destabilizing microtubule networks in mdx myofibers. Skeletal Muscle 2013; 3: 14
Raith M, Valencia RG, Fischer I, Orthofer M, Penninger J, Spuler S, Rezniczek AG, Wiche G
(See online at https://doi.org/10.1186/2044-5040-3-14) - The many faces of plectin and plectinopathies: Pathology and mechanisms. Acta Neuropathol 2013; 125:77-93
Winter L, Wiche G
(See online at https://doi.org/10.1007/s00401-012-1026-0) - Aciculin interacts with filamin C and Xin and is essential for myofibril assembly and maintenance. J Cell Sci 2014;127: 3578-3592
Molt S, Bührdel JB, Yakovlev S, Schein P, Orfanos Z, Kirfel G, Winter L, Wiche G, van der Ven PFM, Rottbauer W, Just S, Belkin AM, Fürst DO
(See online at https://doi.org/10.1242/jcs.152157) - Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle. J Clin Invest 2014; 124: 1144-1157
Winter L, Staszewska I, Mihailovska E, Fischer I, Goldmann WH, Schröder R, Wiche G
(See online at https://doi.org/10.1172/JCI71919) - Mechanosensing through focal adhesion-anchored intermediate filaments. FASEB J 2014; 28: 715-729
Gregor M, Osmanagic-Myers S, Burgstaller G, Wolfram M, Fischer I, Walko G, Resch GP, Jörgl A, Herrmann H, Wiche G
(See online at https://doi.org/10.1096/fj.13-231829) - Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn-plectin 1f complexes. Mol Biol Cell 2014; 25: 4130-4149
Mihailovska E, Raith M, Valencia RG, Fischer I, Al Banchaabouchi M, Herbst R, Wiche G
(See online at https://doi.org/10.1091/mbc.E14-06-1174) - Networking and anchoring through plectin: a key to IF functionality and mechanotransduction. Curr Opin Cell Biol 2015; 32: 21-29
Wiche G, Osmanagic-Myers S, Castañón MJ
(See online at https://doi.org/10.1016/j.ceb.2014.10.002) - Plectin isoform 1-dependent nuclear docking of desmin networks affects myonuclear architecture and expression of mechanotransducers. Hum Mol Genet 2015; 24: 7373-7389
Staszewska I, Fischer I, Wiche G
(See online at https://doi.org/10.1093/hmg/ddv438) - Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle. Hum Mol Genet 2015; 24: 4530-4544
Winter L, Kuznetsov AV, Grimm M, Zeöld A, Fischer I, Wiche G
(See online at https://doi.org/10.1093/hmg/ddv184) - Plectin reinforces vascular integrity by mediating vimentinactin network crosstalk. J Cell Sci 2015; 128.4138-4150
Osmanagic-Myers S, Rus S, Wolfram M, Brunner D, Goldmann WH, Bonakdar N, Fischer I, Reipert S, Zuzuarregui A, Walko G, Wiche G
(See online at https://doi.org/10.1242/jcs.172056)
