PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/85871 Please be advised that this information was generated on 2021-09-29 and may be subject to change. ISOFORMS IN MUSCLE AND BRAIN CELLS localization and function • ralph j.a. oude ophuis • 2011 9 789088 912344 > ISBN 978-90-8891234,-4 DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION Voor het bijwonen van de openbare verdediging van het proefschrift van RALPH J.A. OUDE OPHUIS DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION op vrijdag 1 april 2011 om 13:00u precies in de Aula van de Radboud Universiteit Nijmegen aan de Comeniuslaan 2 te Nijmegen Na afloop van de verdediging is er een receptie ter plaatse PARANIMFEN Susan Mulders [email protected] Rinske van de Vorstenbosch r.vandevorstenbosch(§) ncmls.ru.nl DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION ISBN-13 978-90-8891234-4 ISBN-10 90-8891-234-3 Printed by Proefsohriftmaken.nl || Printyourthesis.com Published by Uitgeverij BOXPress, Oisterwijk DMPK ISOFORMS IN MUSCLE AND BRAIN CELLS LOCALIZATION AND FUNCTION Een wetenschappelijke proeve op het gebied van de Medische Wetenschappen Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. mr. S.C.J.J. Kortmann, volgens besluit van het college van decanen in het openbaar te verdedigen op vrijdag 1 april 2011 om 13:00 uur precies door Raphaël Johannes Antonius Oude Ophuis geboren op 24 oktober 1978 te Sint-Oedenrode Promotor Prof. dr. B. Wieringa Copromotores Dr. D.G. Wansink Dr. J.A.M. Fransen Manuscriptcommissie Prof. dr. H.G. Brunner Prof. dr. J.A.M. Smeitink Dr. F.J.M. van Kuppeveld The studies presented in this thesis were performed at the Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, The Netherlands. Financial support was obtained from the Prinses Beatrix Fonds, the Stichting Spieren voor Spieren, the Muscular Dystrophy Association (MDA) and the Association Française contre les Myopathies (AFM). TABLE OF CONTENTS Chapter 1 9 General introduction • 1 Myotonic Dystophy type 1 11 • 2 DM1 molecular pathogenesis 12 • 3 DMPK protein 16 • 4 DMPK splice isoforms 18 • 5 Tail-anchored proteins 22 • 6 DMPK substrates and function 25 • 7 Aim and outline of this thesis 28 Chapter 2 31 DMPK protein isoforms are differentially expressed in myogenic and neu­ ral cell lineages Chapter 3 49 Topology and specificity of membrane insertion of tail-anchored DMPK isoforms Chapter 4 77 A tail-anchored myotonic dystrophy protein kinase isoform induces peri­ nuclear clustering of mitochondria, autophagy and apoptosis Chapter 5 97 Myotonic dystrophy protein kinase isoforms A and C exert differential control over Ca2+ mobilization in myotubes Chapter 6 121 Summarizing Discussion References 132 Nederlandse samenvatting 146 Abbreviations 150 Dankwoord 152 Curriculum vitae 156 Publications 157 i CHAPTER 1 GENERAL INTRODUCTION GENERAL INTRODUCTION Í • 1. Myotonic Dystophy type 1 (DM1) DM is the most common genetic determined muscular dystrophy in adults at a prevalence of 1:8000 (101) and was first described by Steinert (266), and Batten and Gibb (10) about one century ago. We now know that this multisystemic neuromuscular disorder can be divided into two subtypes with different prevalence: DM type 1 (DM1, also known as Steinert dis­ ease; MIM 160900) and DM type 2 (DM2, also known as proxim al myo­ tonic myopathy, MIM 602668). DM symptoms vary in severity and mostly manifest themselves as muscle related. Prominent symptoms include myotonia, distal and proxim al muscle weakness and atrophy, gastroin­ testinal transit problems, and cardiac aberrations like conduction de­ fects and arrhythmias. Cardiac problems are generally considered the leading cause of death in DM. Furthermore, the central nervous system can be affected, and cataract and endocrine problems can be involved, illus trating the multisystemic character of the disease (summarized in Fig. 1A). This plethora of symptoms together with long asymptomatic Figure 1 • DM1 molecular A Muscular pathogenesis: symptoms and myotonia +++ locus. distal muscle weakness +++ (A) Characteristic symptoms proximal muscle weakness ++ of DM1. The frequency of muscle atrophy ++ facial muscles ++ symptoms is indicated by a hip girdle muscles + scale from + to +++. Typical finger flexors +++ skeletal muscle features are neck muscles +++ illu s tra te d in the draw ing foot dorsiflexors ++ quadriceps +++ darkest shading indicates cardiac arrythmia ++ most severely affected skel­ gastrointestinal problems + etal m uscle areas. (B) DM1 uterus + locus organization. The (CTG)n repeat tra ct is Non-muscular cataract ++ located in a gene-dense re ­ hypogonadism ++ gion on human chromosome insulin insensitivity + 19q13 in exon 15 of the hypersomnia ++ DMPK gene, which overlaps mental retardation + with the promoter region of congenital DM + the SIX5 gene. Rectangles indicate exons (grey, DMWD; + present; ++ frequent; +++ usual black, DMPK; white, SIX5). The straight line represents B locus (19q13) N orm al 5 < (CTG)n < 35 introns and intergenic se­ DM1 quences. Arrows denote start P re m u ta tio n 35 < (CTG)n < 49 l L (CTG)n ^ and direction of transcription. M ild 50 < (CTG)n < 150 Disease classification anc C la ssic 100 < (CTG)n < 1000 corresponding number of -faotifr] i in //—tf-E—i-o o (CTG)n repeats is indicated DMWD DMPK SIX5 C o n g e n ita l (CTG)n > 1000 to the right. 1 • General Introduction periods and a variable age of onset make DM a difficult to diagnose dis­ order (94). Contrary to DM2, DM1 can also occur as a severe congenital form characterized by high neonatal mortality, hypotonia, mental retar­ dation and respiratory distress (101). In the early nineties of the twentieth century the mutation underly­ ing DM1 was discovered by several groups on chromosome 19q13 in the 3'-UTR of the DMPK gene (Fig. 1B) (26, 86, 175). Several years later, a (CCTG)n mutation in intron 1 of the zinc finger protein 9 (ZNF9) mapping to chromosome 3q21 was identified as the cause of DM2 (167, 225). For DM1, it has been found that severity of symptoms and length of the repeat are correlated (Fig. 1B). An unaffected person carries a short repeat composed of 5-35 CTG triplets, while mildly affected patients carry 35-150 CTG units in their affected allele. Patients with the classical DM1 phenotype have repeats of 100-1000 CTGs and the most severely affected individuals carry a repeat of over 1000 CTGs (101, 116). Once the (CTG)n repeat crosses a threshold of 35 CTG triplets, it becomes un­ stable, resulting in expansion in somatic tissues (187) and in successive generations. Repeat expansion causes anticipation, meaning that severity of disease increases between generations and symptoms appear earlier in life (31, 102, 187). In order to better understand the multisystemic character of DM1 it is of paramount importance to gain extensive knowledge about DMPK mRNA and protein functions and expression patterns. • 2. DM1 molecular pathogenesis DM1 is now classified as an unstable mini- or m icrosatellite or DNA expansion disorder, a heterogeneous family of disorders to which also Huntington's disease, FRAXA, various types of spinocerebellar ataxia (94) and several other neurodegenerative disorders belong. Upon discovery that the mutation leading to DM1 is localized in the 3'-UTR of the DMPK gene, it was realized that special mechanisms of pathogenesis must be operational because formation of abnormal protein structure cannot be involved. Hence, several hypotheses were postulated to explain the mul- tisystemic nature of DM1 pathology. • 2.1. RNA pathogenesis The most prevalent and best supported explanation for the molecular pathology of DM1 is an RNA-based toxic gain-of-function mechanism, in which newly formed transcription products containing large (CUG)n repeats are trapped in the nucleus, where they aggregate with nuclear proteins to form ribonuclear protein complexes of abnormal nature (75, 111, 129, 181). The end result is global m isregulation of gene expres­ sion (43, 65, 225). Just lately, it was speculated that soluble transcripts containing long CUG-repats are also able to bind transcription and splice factors, in this way contributing to gene misregulation (132). Length of the repeat is correlated with binding of nuclear proteins, nuclear reten­ tion and formation of nuclear aggregates (54, 58). Analysis of ribonuclear foci in muscle and brain tissue from DM1 patients revealed presence of splice factors of the muscleblind family (MBNL 1, 2 and 3) within the 1 1 • General Introduction 12 ribonucleoprotein (RNP) aggregates. It is therefore commonly believed that the amount of MBNL available for proper regulation of splicing is reduced. Although not found in nuclear foci but believed to bind soluble, expanded transcripts, other RNA processing factors able to bind (CUG) n repeats are upregulated or are behaving abnormally, like CUG-binding protein 1 (CUG-BP1) a member of the CUG-BP and ETR3-like (CELF) fam ilies of factors (132, 278) or HnRNP-H (139). Furthermore, several other proteins not involved in splicing such as protein kinase R (276), transcription factor SP1 (specific protein 1), signal transducers and ac­ tivators of transcription (STAT1 and 3), the retinoic acid receptor gamma subunit (58, 65) and Nkx2.5 (306) may also be involved in DM1 via an RNA-mediated mechanism. In DM1 tissues, multiple splice abnormalities have been described (58), including in muscle: splicing of the mRNAs for chloride channel 1 (ClC-1 ) (40, 180), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (143), cardiac troponin T (110, 219), insulin receptor (IR) (248), and the ryanodine receptor (143). In brain, processing of the mRNAs for Tau (164, 253), and amyloid precursor protein (129) is misregulated.