Sequestration of MBNL1 in tissues of patients with myotonic dystrophy type 2

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Neuromuscular Disorders 22 (2012) 604–616 www.elsevier.com/locate/nmd

Sequestration of MBNL1 in tissues of patients with myotonic dystrophy type 2 Z. Luka´sˇ a,⇑,1, M. Falk b,⇑,1, J. Feit a, O. Soucˇek a, I. Falkova´ a,b, L. Sˇtefancˇ´ıkova´ b, E. Janousˇova´ c, L. Fajkusova´ d, J. Zaora´lkova´ a, R. Hraba´lkova´ a a Institute of Pathology, Brno Faculty Hospital, Brno, Czech Republic Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic c Institute of Biostatistics and Analyses, Faculty of Medicine and Faculty of Science, Masaryk University, Brno, Czech Republic d Centre of Molecular Biology and Gene Therapy, Brno Faculty Hospital, Brno, Czech Republic b

Received 9 January 2012; received in revised form 2 March 2012; accepted 6 March 2012

Abstract The pathogenesis of myotonic dystrophy type 2 includes the sequestration of MBNL proteins by expanded CCUG transcripts, which leads to an abnormal splicing of their target pre-mRNAs. We have found CCUGexp RNA transcripts of the ZNF9 gene associated with the formation of ribonuclear foci in human skeletal muscle and some non-muscle tissues present in muscle biopsies and skin excisions from myotonic dystrophy type 2 patients. Using RNA-FISH and immunofluorescence-FISH methods in combination with a high-resolution confocal microscopy, we demonstrate a different frequency of nuclei containing the CCUGexp foci, a different expression pattern of MBNL1 protein and a different sequestration of MBNL1 by CCUGexp repeats in skeletal muscle, vascular smooth muscle and endothelia, Schwann cells, adipocytes, and ectodermal derivatives. The level of CCUGexp transcription in epidermal and hair sheath cells is lower compared with that in other tissues examined. We suppose that non-muscle tissues of myotonic dystrophy type 2 patients might be affected by a similar molecular mechanism as the skeletal muscle, as suggested by our observation of an aberrant insulin receptor splicing in myotonic dystrophy type 2 adipocytes. Ó 2012 Elsevier B.V. All rights reserved. Keywords: Myotonic dystrophy; Transcription of ZNF9; CCUG repeat expansion; Sequestration of MBNL1 protein; Ribonuclear foci; Insulin receptor alternative splicing

1. Introduction Both myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2) share a similar pathogenetic

⇑ Corresponding authors. Addresses: Department of Pathology, Brno Faculty Hospital, Jihlavska´ 20, CZ-625 00 Brno, Czech Republic. Tel.: +420 532233837 (Z. Luka´sˇ); Laboratory of Chromatin Function, Damage and Repair, Dept. of Molecular Cytology and Cytometry, Institute of Biophysics, Academy of Sciences of CR, Kralovopolska 135, 61265 Brno, Czech Republic. Tel.: +420 541517165; mobile: +420 728 084060 (M. Falk). E-mail addresses: [email protected], [email protected] (Z. Luka´sˇ), [email protected] (M. Falk). 1 The first and second author contributed equally.

0960-8966/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nmd.2012.03.004

pattern beginning with an expansion of CTG and CCTG nucleotide repeats due to mutation in the genes encoding dystrophia myotonica protein kinase (DMPK) and zincfinger protein 9 (ZNF9), respectively. Taneja et al. [1] were the first to describe and illustrate intranuclear foci containing CTG repeat expansions in skeletal muscle and fibroblasts of DM1 patients; the most of transcripts with expanded repeats remain retained in the nuclei of cells. Consequently, DMPK protein levels are reduced [2]. Miller et al. [3] demonstrated recruitment of RNA-binding proteins to the CUGexp foci in DM1 patients. Liquori et al. [4] showed that the second form of DM2 is also caused by an expansion, concretely of a CCTG repeat in the first intron of the ZNF9 gene. Consequently, co-localization

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of MBNL1 protein with DMPK expanded repeat transcripts in DM1 and DM2 cells was confirmed in vivo by Fardaei et al. [5]. The current model of the disease process in DM includes interaction of CUGexp or CCUGexp mRNAs with CUGbinding proteins, which leads to their sequestration at the volume of ribonuclear foci and, in turn, to an abnormal regulation of transcription and alternative splicing of their target pre-mRNAs. There are two groups of CUG-binding proteins: muscleblind proteins bind pre-mRNA through an evolutionary conserved tandem CCCCH zinc-finger domain. Human muscleblind homologues MBNL1, MBNL2, and MBNL3 promote inclusion or exclusion of specific exons on different pre-mRNAs by antagonizing the activity of the second group of binding proteins – CUG-BP and ETR 3-like factors (CELF proteins). The MBNL1 protein from the MBNL family promotes transition of splicing from foetal to adult exons (reviewed in Pascual et al. [6]), while CUGbinding protein (CUG-BP1) helps to retain foetal exons. CUG-BP1 binds to single-stranded RNA and does not localize to nuclear foci either in DM1 or DM2 [7–10]. Adult mouse skeletal muscle overexpressing CUG-BP1 reproduces molecular and physiological defects of DM1 tissue; the results of the study [10] suggest that CUG-BP1 also plays a role in the developmental regulation of abnormal splicing in DM1. On the other hand, in DM2, MBNL1 is sequestered on expanded CCUG repeat RNA which accumulates in nuclear foci like CUG repeat RNA, but CUG-BP1 is not overexpressed, suggesting that DM2 is primarily a disease of MBNL1 depletion [10]. This multistep model, common to both DM1 and DM2 [11,12], is well explored in skeletal muscle, but not in other cell types or tissues. MBNL proteins have no general effect on alternative splicing but rather regulate splicing of only specific targets [8]. Consequently, an inappropriate splicing of selected transcripts leads to the characteristic phenotype of DM1 and DM2 patients. For instance, the loss of MBNL1 results in an altered splicing of ClC-1 mRNA, expression of non-functional ClC-1 protein, and subsequently to the disruption of chloride conductance in muscle membrane [13,14]; this is manifested as myotonia, the characteristic symptom of myotonic dystrophies. Another core feature of DM1 observed in skeletal muscles is insulin resistance due to an altered splicing of the insulin receptor mRNA [15]. The list of the genes and exons known to be affected by spliceopathy in DM1 skeletal muscle, brain and heart is increasing [9]; similar results could be reasonably expected also in DM2, since gene expression analysis indicated no essential differences between processing of the ClC1 and insulin receptor pre-mRNAs in the muscle tissue of both DM1 and DM2 patients [12,16]. The splicing pattern of the insulin receptor, directly related to the DM2 phenotype, was described to be altered in in vitro differentiated DM2 myoblasts [17]. From the opposite point of view, DM1/2 presents a potential target for small molecule

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therapy; since the pathogenesis of these disorders involves sequestration of splicing factors such as MBNL1 and CUG-BP1, caused by an RNA gain-of-function mutation, an agent that antagonizes this process could potentially be used therapeutically. Indeed, Pentamidine was proved to reverse the splicing defects associated with myotonic dystrophy [18]. The structural organization and composition of the foci is still incompletely known. Recently, the spatial relations of the components of the splicing apparatus and the MBNL1-containing ribonuclear foci in cell nuclei have been explored using immunohistochemical and electronmicroscopic methods. The RNA-containing nuclear foci are round electron dense spaces which contain RNPs and proved to be immunopositive for MBNL1. These nuclear domains are surrounded by perichromatin fibrils (PFs), a morphological equivalent of hnRNP (heterogeneous nuclear ribonucleoproteins – complexes of RNA and protein present in the cell nucleus during gene transcription and subsequent post-transcriptional modification of the newly synthesized pre-mRNA), and by perichromatin granules (PG), small nuclear RNPs (snRNP). These snRNP as well as hnRNP co-localize in the nuclear foci with MBNL1 protein. The 3rd nuclear RNA-containing structure, interchromatin granules (IG, nuclear speckles) do not co-localize – unlike IG in DM1 – with the PF and PG in DM2 [19,20]. The sequestration of splicing factors involved in early phases of pre-mRNA processing supports the hypothesis of general alteration in the maturation of several mRNAs, which could lead to pathological dysfunction in dystrophic patients. To better understand the role of the influence of CUG/ CCUG repeat expression on the development of DM1/ DM2 pathology, it is necessary to establish the identity of the tissues and cell types in which the expanded mRNAs and RNA-binding proteins are expressed. DMPK mRNA was found to be expressed in a range of adult mouse tissues that show pathology in DM1, but also in the intestinal epithelium, liver and cartilage, which had not been reported to show abnormalities in DMPK/ mice. DMPK RNA was not found in the ovary, pancreas and kidney [21]. To date, there are still no comprehensive data about the expression of DMPK or ZNF9 genes in human tissues. DMPK transcripts were detected in foetal eyes and in adult conjunctival and corneal epithelia, uvea, cellular layers of the retina, optic nerve, and in the sclera [22]. There are only several reports about CUGexp-containing foci in human non-muscle tissues: Mankodi et al. [23] illustrated ribonuclear intranuclear foci present in a smooth muscle cell of an intramuscular arteriole of a patient with DM1. Sequestration of muscleblind proteins in the nuclear foci of mutant CUGexp RNA and deregulated alternative splicing in cortical and subcortical neurons was reported by Jiang et al. [24]. Nuclear CUGexp RNA foci were also described in DM1 cardiomyocytes [25], subsynaptic nuclei of muscle fibres and in motor neurons of DM1 neuromuscular junction [26]. In addition, expression of mutant

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DMPK mRNA was shown to be accumulated in foci in the gallbladder smooth muscle cells of DM1 patients [27]. Finally, Bonifazi et al. [28] demonstrated by RNA-FISH analysis the presence of CUGexp RNA foci in the nuclei of amniotic cells of a DM1 foetus. The ZNF9 gene is ubiquitously expressed but the level of the ZNF9 post-transcriptional modification of the newly synthesized RNA (pre-mRNA) or ZNF9 protein in human tissues is not known. To date, the expression of CCUGexp RNA or colocalization of CCUGexp RNA foci with MBNL1 protein only in muscle tissue and cell cultures has been reported [5,23,29,30]. In this report, we examine the presence of CCUGexp RNA transcripts of the ZNF9 gene associated with the formation of ribonuclear foci in human muscle and non-muscle tissues present in muscle biopsies and skin excisions from patients with DM2. We also study the expression of MBNL1 protein, quantify the frequency of nuclei containing the CCUGexp and MBNL1 foci, and determine the rate of co-localization, i.e. possible sequestration of the MBNL1 within CCUGexp repeats in the tissues examined. Based on our analysis of the insulin receptor splicing in adipocytes taken from a DM2 patient, we show that the colocalization of the expanded repeats with the MBNL1 protein has functional consequences and may disturb some MBNL1 functions, such as splicing activity. Therefore, we speculate that the co-localization of the expanded repeats with the MBNL1 protein may result in alteration of some MBNL1 functions and, consequently, non-muscle tissues included in the DM2 phenotype may theoretically be affected by the same or a similar molecular mechanism as the skeletal muscle. 2. Materials and methods 2.1. Tissue samples Diagnostic muscle biopsies and skin excisions of 12 patients with DM2 (9 muscle biopsies, 4 skin excisions) and 9 non-DM controls (6 muscle biopsies, 3 skin excisions) were collected after obtaining the written informed consent. The numbers of nuclei in tissues examined of DM2 patients and non-DM controls are listed in Table 1. The samples were deep-frozen and the histopathological and histochemical examination [31] as well as fluorescence in situ hybridization (FISH) and immunohistochemical (IHC) or immunofluorescence (IFL) methods were applied on frozen sections. The tissue cells in the biopsies were identified by the general texture of the section and, in some cases, by the immunohistochemical reactivity of the cell types (neurofilament proteins, smooth muscle actin, S100 protein, CD34). The diagnosis of DM2 was confirmed by mutation analysis of blood samples, using the “Tetraplet-primed PCR” method modified from Triplet-primed PCR [32,33]. 2.2. Immunofluorescence Immunofluorescence (IFL) was performed according to Holt et al. [34]. Briefly, fresh frozen sections (6 lm thick)

were placed on SuperfrostÒ Plus microscope glasses (Menzel-Gla¨ser), dried, fixed by acetone–methanol (1:1) at room temperature (RT), washed in PBS at RT, and incubated for 1 h at 37 °C with the primary mouse antibody MB1a [19] diluted 1:3 in diluting buffer as described below. Alternatively, namely for the high-resolution confocal microscopy (see the particular paragraph), the slides were spatially fixed in 4% paraformaldehyde in PBS for 5 min at RT, and incubated with the MB1a primary antibody as in the case of the acetone–methanol fixation. Subsequently, the sections were washed in PBS at RT and incubated with the secondary antibody labelled with Alexa FluorÒ 488 (Alexa FluorÒ 488 Goat Anti-Mouse SFX Kit, Molecular Probese, Invitrogen) for 1 h at 37 °C. Following incubation, the slides were washed in PBS at RT and mounted in Vectashield Mounting Medium (Vector Laboratories, USA). Nuclear chromatin was counterstained with 0.075 lg/ml diamino-2-phenylindole (DAPI). The image acquisition and evaluation of the results were performed on a Leica DMRXA2 fluorescence microscope. 2.3. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) of frozen muscle sections for CUG repeats was performed according to Holt et al. [34]. Frozen sections (6 lm) were placed onto SuperFrost Plus slides (Menzel-Gla¨ser), air-dried at room temperature (RT) for 30 min, fixed in modified Carnoy’s fixative (73% ethanol; 25% acetic acid; 2% formalin (Sigma)) for 30 min at 4 °C, and washed in PBS at RT followed by PBS at RT. The sections were incubated in prehybridization buffer (30% formamide (Sigma) in 2 SSC (Eppendorf)) at RT, and thereafter hybridized with the probe (1 ng/ll) at 37 °C in hybridization buffer (30% formamide, 2 SSC, 0.02% BSA (Invitrogen), 66 ng/ll yeast tRNA (Invitrogen), 2 mM vanadyl complex (Sigma)). Probe for DM1 was 50 CAG CAG CAG CAG CAG CAG CAG-30 20 –O-MeRNA50 Fl, for DM2: 50 -CAG GCA GGC AGG CAG GCA GG-30 20 –O-Me-RNA50 Fl (both Generi Biotech, Czech Republic). The sections were washed in a post-hybridization wash (30% formamide; 2 SSC) for 30 min at 45 °C followed by 1 SSC washing for 30 min at RT. Finally, the sections were mounted by DAPI (Vectashield Mounting Medium, Vecor Laboratories, USA). The slides were observed in a Leica DMRXA2 fluorescence microscope. 2.4. Combined immunofluorescence and in situ hybridization For combined immunofluorescence and in situ hybridization (IFL-FISH), the same antibodies, dilutions and procedures as for the immunohistochemistry were used. The sections were dried, fixed by acetone–methanol (1:1) at RT, and incubated with the primary mouse antibody for 1 h at 37 °C. The secondary antibody was labelled with Alexa FluorÒ 488 (Alexa FluorÒ 488 Goat Anti-Mouse SFX Kit, Molecular Probese, Invitrogen).

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Table 1 The numbers of cell nuclei examined in DM2 patients and non-DM controls for the particular tissue. Diagnosis Patient P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12

DM2 DM2 DM2 DM2 DM2 DM2 DM2 DM2 DM2 DM2 DM2 DM2

R Control C1 C2 C3 C4 C5 C6 C7 C8 C9 R

Muscular dystrophy Neural atrophy Muscle, normal Skin, prurigo Neural atrophy Varices cruris Myogenic disorder Myositis Erythema nodosum

Skeletal muscle 228

Vascular media

Endothelium

Adipocytes

Schwann cells

50 51 410 259 23 13 23 3 41 26 33 27

84

12

104

307 83 30 36 18 22 36 18 28 16

280 30

234 72

19

51

1559

959

678

351

461

20 56 10

50

27

7

13

25

31

14 17

443 97 88 118 157 173 255

Epidermis

Hair sheath

Sweat gland

606

30

85

979 275 160

491 184 293

228 112 48

2020

998

473

72 6

46

16

40

9

76

6 4

10

37 133

46 12 133

21 17 19

18

115

56

10

18 118

90

122

78

212

138

128

P: patients with DM2; C: non-DM controls. Notes: skin excisions were examined outside the lesion.

Following the incubation, the slides were washed, fixed in 4% paraformaldehyde PBS at RT, and prehybridized in 30% formamide in 2 SSC. Next, the tissue sections were hybridized for 16 h at 37 °C with 1 ng/ll of a 50 -end cy3-labelled RNA probe, containing either CUG or CCUG repeats (OPC-purified 20 -O-Me-RNA, 50 -20mers, Generi Biotech), in a hybridization buffer consisting of 30% formamide, 2 SSC, 0.02% BSA, 66 lg/ml yeast rRNA, and 2 mM vanadyl complex. Afterwards, the slides were washed in 1 SSC for 30 min (RT) and mounted in Vectashield Mounting Medium (Vector Laboratories, USA). Nuclear chromatin was counterstained with DAPI. Both RNA foci and MBNL1 protein were visualized on a Leica DM RXA2 fluorescence microscope, alternatively using a high-resolution confocal regime as described below. 2.5. Image acquisition and confocal microscopy Image acquisition and confocal microscopy were performed on an automated Leica DM RXA fluorescence microscope in a wide-field or confocal, high-resolution regime. This configuration enabled us to inspect cell nuclei in 3D-space, which allowed us to discriminate between true reaction signals, lipofuscin and autofluorescence, as well as to confirm mutual co-localization of MBNL1 and CCUGexp foci in all the three planes (Fig. 1). For confocal image acquisition, the microscope was equipped with a CSU10a Nipkow disc (Yokogawa, Japan) for confocal imaging, a

CoolSnap HQ CCD-camera (Photometrix, Tucson, AZ, USA), an Ar/Kr-laser (Innova 70C, Coherent, Palo Alto, CA, USA), and an oil immersion Plan Fluotar objective (100/NA1.3) [35]. Automated exposure, image quality control, and other procedures were performed using FISH 2.0 software [36]. The exposure time and dynamic range of the camera in the red, green and blue channels were adjusted to the same values for all slides to obtain quantitatively comparable images. Up to forty serial optical slices (according to the thickness of tissue sections) were captured at 0.2 lm intervals along the z-axis at a constant temperature of 26 °C. 2.6. Image analysis The count of positive nuclei was determined in each tissue type as the rate between the number of signal-bearing nuclei and the number of all evaluable nuclei (PN/RN) present in a high-power field (HPF). For each tissue type the values were calculated from at least 10 HPF or 100 nuclei (Table 1). The tissue cells in the biopsies were identified by the general texture of the section, in some cases by the immunohistochemical reactivity of the cell types (neurofilament proteins, smooth muscle actin, S100 protein, CD34). The numbers of CCUGexp and MBNL1 foci per nucleus were determined by visual inspection of maximal images composed of individual confocal slices. Co-localization of MBNL1 and CCUGexp foci was determined in confocal regime as a >50% overlay (Fig. 1) of the particular signals at original

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Fig. 1. Co-localization of CCUGexp RNA and MBNL1 protein foci as determined by the IFL-FISH method in combination with a high-resolution confocal microscopy. The main panel shows three nuclei of DM2 skeletal muscle (TOPRO-3, artificially blue), two of them with ribonuclear CCUGexp (red) and MBNL1 foci (green); the maximal image in the X–Y coordinate plane, composed from 20 optical confocal slices with a z-step of 2 lm, is displayed. Right panels demonstrate, at single confocal slices, spatial preservation of nuclei after their fixation with paraformaldehyde (see Section 2) and co-localization of CCUGexp and MBNL1 foci in all the three planes (X–Y, X–Z, and Y–Z) (the middle nucleus from the main maximal image is displayed). The bottom panel shows images in separated red and green channels (red: CCUGexp foci, green: MBNL1 protein).

images (without deconvolution). The size of the foci was determined in pixels (1 pix = 165  165 nm), after manual demarcation of the signal area on the maximal image. 2.7. Statistical analysis of the data A comparison of the percentages of CCUGexp and MBNL1 positive nuclei in different tissues was performed. Positive nuclei were further examined to analyse the number and size of CCUGexp and MBNL1 foci. The percentage of CCUGexp and MBNL1 positive nuclei in each tissue was computed as a mean of the percentages of positive nuclei in each microscopic field weighted by the number of nuclei in the microscopic field. Generalized linear mixed models were used for the analysis of the percentage of positive nuclei, the number and size of foci in different tissues to account for the correlation of microscopic fields

from one patient. Differences in the size of CCUGexp foci according to the categories of the size of MBNL1 foci were tested using a Kruskal–Wallis ANOVA test. A level of statistical significance a = 0.05 was used. The analyses were performed in the statistical software PASW Statistics 19 and software R. The results of comparison of the size of CCUGexp and MBNL1 foci were visualized using box plots prepared in the software STATISTICA for Windows 9.1. 2.8. Semiquantitative PCR for insulin receptor The expression analysis of the insulin receptor isoform A and B (IR-A, IR-B) mRNAs, respectively, was performed in skeletal muscle and adipose tissue, according to Savkur et al. [15], with small adaptations. A High Pure RNA Isolation Kit (Roche, #11828665001) was employed for isolation of

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total RNA from the homogenized skeletal muscle tissue, an RNeasy Lipid Tissue Mini Kit (Qiagen, #704804) for isolation from the adipose tissue. Altogether 200 ng of RNA and the Transcriptor First-Strand cDNA Synthesis Kit (Roche) were used for reverse transcription with the primer 50 TTGGGGAAAGCTGCCACCGT-30 . By using a Hot Start Master Mix Kit (Qiagen), PCR analysis was accomplished from 5 ll of cDNA in a total volume of 50 ll, with a primer pair (50 -CCAAAGACAGACTCTCAGAT-30 and 50 -AACATCGCCAAGGGACCTGC-30 ) [15] flanking the exons 10 and 12. Thirty cycles consisting of 95 °C/30 s, 60 °C/30 s, and 72 °C/30 s were followed by a final extension at 72 °C for 10 min. Ten ll of PCR products was separated on 10% PAGE gel (acrylamide:bis-acrylamide 29:1) in TBE buffer. The size of PCR products was 131 bp (IR isoform A) and 167 bp (IR isoform B).

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3. Results 3.1. Confirmation of DM2 diagnosis of the samples examined The diagnosis of DM2 was confirmed by mutation analysis of blood samples, using the “Tetraplet-primed PCR” method. In all DM2 cases, an expanded allele [(CCTG)n > 50–100] of the ZNF9 gene was detected (Suppl. Table 1), while it was absent in non-DM controls. 3.2. Histopathological features of muscle biopsies in DM2 patients Our histopathological findings correspond to the data previously published (reviewed in [37,38]). In two cases, however,

Fig. 2. Demonstration of CCUGexp RNA and MBNL1 protein foci in skeletal muscle and non-muscle tissues of DM2 patients. CCUGexp RNA foci (red) and MBNL1 protein foci (green) were simultaneously detected by IFL-FISH method in (panel A): skeletal muscles, vascular media and endothelium, (panel B): adipocytes, Schwann cells and epidermis of DM2 patients. The foci were not observed in non-DM control tissues (panels A and B). The main images: wide-field views on the tissue; inserts: selected nuclei (identified by the red arrow at wide-field images), multiplied from the original image as indicated. For selected nuclei (panel B), green channel of MBNL1 and red channel of CCUGexp are displayed separately to demonstrate their colocalization; Chromatin was counterstained with DAPI (artificially blue); maximal microscopic images displayed were composed from 10 confocal slices 0.2 lm wide. Abbreviations: I, fascicle of interstitial cells with intranuclear foci (presumably capillary endothelium and nerve fibres) between muscle fibres (SM); L, lipofuscin; M, media; E, endothelium.

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an inflammatory infiltration of the endomysium by T cells, and in one case a picture indistinguishable from the congenital fibre type disproportion, were found [39].

3.4. Frequency of nuclei containing MBNL 1 protein and comparison with the frequency of CCUGexp – positive nuclei in DM2 tissues

3.3. The presence of nuclear foci containing ZNF9 transcripts in human tissues

As follows from the IFL staining, MBNL1 protein was expressed in a high frequency of nuclei in all tissues studied (Figs. 2 and 3). This is a further evidence that human MBNL1 is not muscle-specific [34]. A comparison of the frequency of MBNL-positive with CCUGexp-positive nuclei (Figs. 2 and 3) indicates that a number of MBNL1-positive nuclei do not contain CCUGexp ZNF9 transcript. These results hold both for the tissues fixed with acetone–methanol or formaldehyde, although slightly different values of MBNL1-positive nuclei were obtained by these two methods (Figs. 3A and B, respectively). The frequency of MBNL1-positive nuclei in other examined tissues varied between 79.9% and 91.9% for the acetone–methanol fixation (Fig. 3A and Suppl. Table 3) or between 86.7% and 99.3% for the formaldehyde (Fig. 3B and Suppl. Table 4); the differences observed between these two methods can be attributed to the fact that cross-linking of tissue polypeptide chains by formaldehyde generally contributes to a higher retention of proteins in tissue sections (Fig. 4). Anyway, in both cases this provides evidence of a high level of expression of the MBNL1 protein in all (i.e. also non-muscle) tissues studied. Only skeletal muscles and the epidermis, with the highest and lowest percentage of MBNL1 positive nuclei after formaldehyde fixation, respectively, provided statistically significant differences from other tissues (Suppl. Table 4). In non-DM controls, the proportion of the cells expressing MBNL1 protein in all tissues examined was very similar compared to that in patients with DM2. Table 2 shows that the differences in the percentage of MBNL1 positive

We have first analyzed the retention of expanded CCUG mRNA (further termed CCUGexp) in the foci in cross-striated muscle fibres (skeletal muscle also served as a positive control tissue) obtained from genetically confirmed DM2 patients and non-DM controls. By employing the simple RNA-FISH or combined IFL-FISH method, the CCUGexp foci were directly visualized in situ in 66.4% nuclei in the skeletal muscle of DM2 patients (Figs. 1–3). In other tissues, the proportion of the nuclei that contained CCUGexp foci differed: in sweat gland, adipocytes, Schwann cells, endothelial cells, and vascular smooth muscle the values fluctuated from 57.9% to 76.8% (Fig. 3) (for the significance of these differences see Suppl. Table 2). In epidermal and follicular keratinocytes the number of positive nuclei was markedly lower, 30.8% and 47.6% respectively (Figs. 2 and 3 and Suppl. Table 2). These results thus provide unequivocal evidence that the transcription of the expanded repeats of the ZNF9 gene takes place at a level ascertainable by the in situ hybridization method also in non-muscle tissues which may be involved in DM2 pathology [40]; hence, these tissues may theoretically be also affected by the same molecular pathological mechanism as skeletal muscles; however, the transcription levels of the ZNF9 gene in the tissues studied differ (see later). As expected, the foci were completely absent from the nuclei of all non-DM2 control muscle fibres and other tissues analyzed.

CCUGexp

40

ed ia

nd Va

sc ul a

rm

gl a

cle m us

ea t Sw

te s cy

ce ll s n

el et al

ul a sc Va

Ad ip o

gl a

cle

ea t Sw

n

el et al Sk

hw an Sc

m us

ee t sh

rm is

ai r H

do th e

cy

id e Ep

En

Ad ip o

nd

0

rm ed ia

0

ce ll s

10

l

20

10

te s

20

Tissue

30.8

30

Sk

30.8

30

57.9

47.6

50

ee t

40

62.1

59.0

sh

50

66.4

60

hw an

47.6

68.1

70

Sc

57.9

76.8

80

rm is

62.1

59.0 60

96.8 89.2

86 2 86.2

ai r

66.4

99.3

97.5

95.7

93.4 86.7

90

H

70

100

l

76.8

68.1

B

id e

79.9

91.5

do th e

83.3

91.9

89.2

87 5 87.5

86 5 86.5

Ep

80

90

86 4 86.4

En

100

% positive nuclei

A % positive nuclei

MBNL1

Tissue

Fig. 3. The percentage of CCUGexp and MBNL1 positive nuclei compared for skeletal muscle and other soft tissues of DM2 patients. Tissues for examination of MBNL1 foci were fixed either using acetone–methanol (panel A) or formalin (panel B). Grey bars: nuclei positive for MBNL1 protein, black bars: nuclei containing CCUGexp foci. See Suppl. Table 2 for a significance of differences.

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6), while the nucleoplasmic signals (clouds) of MBNL1 protein outside the foci or in nuclei devoid of the CCUGexp foci were less distinct or more diffuse (Fig. 6), particularly in the sections fixed by formaldehyde (Fig. 4). The same diffuse “granular” pattern of intranuclear MBNL1 signals was recorded in all tissues from non-DM persons (Fig. 2 and 6). An analysis of the number of CCUGexp foci per nucleus (Table 3, for statistics see Suppl. Table 5) demonstrates that two and more foci appear more often in soft tissues with a higher frequency of the CCUGexp signal-containing nuclei (adipocytes, endothelia, vascular smooth muscle, Schwann cells and skeletal muscle) (Fig. 3) than in ectodermal derivatives (epidermis, hair follicle and sweat glands) (Fig. 3). The size of sequestered MBNL1 protein signals (MBNL1 foci) was proportional to the size of the CCUGexp foci in every tissue studied (Fig. 5). 3.6. Aberrant splicing of the insulin receptor pre-mRNA in adipocytes from DM2 patients

Fig. 4. Immunodetection of MBNL1 protein in soft tissues of DM2 patients compared for formalin (left panels) and acetone–methanol (right panels) fixed tissues. The main images: wide-field views on the tissue (vascular endothelium and media, Schwann cells); inserts: selected nuclei (identified by the red arrow at wide-field images) multiplied from the original image as indicated, showing the signal and nuclear distribution of MBNL1 in detail. Green: MBNL1, blue (artificially): chromatin counterstaining (DAPI). M, media; E, endothelium.

nuclei between the patients and the controls are not statistically significant in any tissue.2 Taken together, the majority of cells from both DM2 patients and non-DM controls expressed the MBNL1 protein in all the tissues examined. In DM2 cells, MBNL1 was more or less sequestered by CCUGexp ribonuclear foci, depending on the tissue type. Markedly, the proportion of the expressed and sequestered protein was relatively low in epidermal and hair follicle cells. 3.5. The expression pattern of MBNL1 protein and CCUGexp in DM2 tissues In an attempt to quantify and compare MBNL1 sequestration in CCUGexp foci we have analyzed the expression pattern, the frequency per nucleus (Table 3 and Suppl. Tables. 5–7), and the size of these foci (Fig. 5) in human tissues. MBNL1 was usually partly co-localized with CCUGexp in intensely fluorescent, sharp demarcated foci (Figs. 1, 2 and 2 This result is surprising since, in the light of recent reports proving a reduction of the rate of protein translation in patients with myotonic dystrophy 2 [41–43], rather a lower level of MBNL1 protein expression could be expected in DM2 patients.

Finally, we have analyzed whether the sequestration of MBNL1 protein by CCUGexp foci observed in non-muscle tissues could be of some pathological relevance. To address this question, we have followed, by semiquantitative PCR, the expression of the IR-A and IR-B splicing isoforms of the insulin receptor (IR) mRNAs in adipose tissue (adipocytes are very active in glucose uptake) taken from nonDM controls and DM2 patients (Fig. 7). The results were compared with the expression of IR isoforms in the skeletal muscle, where the shift from non-DM persons to DM2 patients is well known [15]. It is evident from Fig. 7 that IR-B dominates over the IR-A splicing variant both in the skeletal muscle (Fig. 7, Lines C2–C4) and adipocytes (Fig. 7, Line C1) of non-DM controls. On the other hand, in DM2 patients IR-A predominates in the skeletal muscle (Fig. 7, Lines A–E) and, importantly, it is markedly increased also in the adipose tissue (while IR-B is significantly reduced) (Fig. 7, Line F). 4. Discussion The expression of MBNL1 in vascular smooth muscle cells and endothelium, Schwann cells and differentiated fat cells has not yet been reported, and, consequently, a possible sequestration of the MBNL1 protein in these tissue types has not been specified in DM2 patients. In this report, we demonstrate that expression of MBNL1 protein and co-localization and possible sequestration with CCUGexp takes place also in non-muscle tissues enumerated above. The results show that nucleoplasmic MBNL1 is present in DM2 myonuclei, as well as in the nuclei of other tissues studied. However, the results pose several questions. Question one: What is the cause of the observed disproportion in the rate of MBNL1 protein sequestration among the tissues studied? The level of MBNL1 seems to be quite high in

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Table 2 Comparison of the percentage of MBNL1 positive nuclei in skeletal muscles and soft tissues of DM2 patients and non-DM controls, fixed either with acetone–methanol (left panel) or formalin (right panel). Acetone–methanol fixation

Adipocytes Endothelium Epidermis Hair sheet Schwann cells Skeletal muscle Sweat gland Vascular media

Formalin fixation

% Positive nuclei in tissue of patients/% positive nuclei in tissue of controls

p-Value

% Positive nuclei in tissue of patients/% positive nuclei in tissue of controls

p-Value

83.3%/83.9% 86.4%/93.2% 79.9%/73.6% 86.5%/75.0% 87.5%/83.6% 89.2%/80.6% 91.9%/75.0% 91.5%/89.3%

0.955 0.606 0.340 0.465 0.979 0.471 0.454 0.740

86.7%/83.3% 93.4%/100.0% 86.2%/83.0% 95.7%/91.8% 97.5%/92.2% 99.3%/97.0% 89.2%/99.0% 96.8%/96.4%

0.832 0.335 0.996 0.192 0.279 0.561 0.115 0.928

Table 3 The summary of the number of CCUGexp foci per nucleus in DM2 tissues. Tissue

Number of CCUGexp foci per nucleus 1 focus

2 and more foci

Total

Adipocytes Endothelium Epidermis Hair follicle Media Schwann cells Skeletal muscle Sweat gland

1 (6.7%) 3 (9.7%) 70 (85.4%) 62 (82.7%) 12 (33.3%) 12 (30.8%) 18 (33.3%) 52 (72.2%)

14 28 12 13 24 27 36 20

15 31 82 75 36 39 54 72

(93.3%) (90.3%) (14.6%) (17.3%) (66.7%) (69.2%) (66.8%) (27.8%)

(100.0%) (100.0%) (100.0%) (100.0%) (100.0%) (100.0%) (100.0%) (100.0%)

A

400

B

700

Siz ze of CCUGexp foci (in pixe els)

350

Siz ze of CCUGexp foci (in pixe els)

all the cell types studied, despite some variation of the percentage of MBNL1 positive nuclei in the tissues. Consequently, the level of MBNL1 sequestration mostly depends on the expression level of the ZNF9 gene (i.e. CCUGexp repeats), which differs in individual tissues. The proportion of CCUGexp-negative cells in the skeletal muscle, other soft tissues, and in sweat glands is lower than that in epidermal and hair follicle/sheath cells (Fig. 3). For comparison, we have found only one report on the rate of expression of nuclear foci in DM2 skeletal muscle – the foci were recorded in >70% of the nuclei with a rate of co-localization >90% [30]. The disproportion is therefore difficult to explain: one possibility is a different proliferation rate.

600

300 250 200 150 100 50 0

<=50 50-100 >100 Size of MBNL1 foci (in pixels)

A normal consequence of mitosis in eukaryotes is transitory repression of the transcription [44]. Epidermal (and hair follicle) cells are characterized by a relatively high proliferation rate compared to that in a differentiated skeletal muscle (permanently in the G0 phase) or in other tissues examined (entering the cell cycle only on activation signal). Alternatively, the regulation of the ZNF9 (CCUGexp) expression in ectodermal tissues may be differently regulated. Question two: Is the sequestration of the MBNL1 protein into the ribonuclear foci sufficient enough to deplete its activity from the nucleoplasm? Nucleoplasmic MBNL1 is only partially sequestered into the ribonuclear foci of expanded repeats in the skeletal muscle (reviewed in [34]). Our results (Fig. 3) demonstrate that the MBNL1 protein remains unsequestered in about 23–33% of the MBNL1containing myonuclei and yet the DM2 pathological phenotype develops. We also demonstrate aberrant splicing of the insulin receptor in the human adipose tissue despite only a partial sequestration of MBNL1. Question three: Are there any specific target genes or pre-mRNAs for MBNL1 in other tissues examined? Do these targets correspond to those in the skeletal muscle (or heart and brain) in patients with DM2? How about the tissues examined in this study? Previous studies suggested that insulin response defects might play a key role in the metabolic manifestations of DM1 potentially leading to type 2 diabetes [45–47]. The

p<0 001 p<0.001

500

Median

400

25%-75%

300

Min-max

200 100 0

<=50 50-100 >100 Size of MBNL1 foci (in pixels)

Fig. 5. Correlation between the sizes of the CCUGexp and co-localizing MBNL1 foci. Results are displayed separately for (A) ectodermal DM2 tissues (epidermis, hair sheath, sweat gland) and (B) soft DM2 tissues (skeletal muscle, vascular media and endothelium, adipocytes and Schwann cells).

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Fig. 6. Nuclear distribution of MBNL1 protein in skeletal muscle and epidermis of non-DM controls and DM2 patients. The level of MBNL1 sequestration by CCUGexp foci in DM2 skeletal muscle and epidermis is visually compared in the main images (MBNL1: green, CCUGexp: red, nuclear chromatin: TOPRO-3, artificially blue). Quantification of MBNL1 sequestration by CCUGexp foci is provided as histograms showing distributions of pixels according to their intensity in the green channel (from 0 [black] to 255 [green]). Histograms were made separately for CCUGexp foci and extrafocal nuclear area. The maximal images, composed from 20 optical confocal slices with a z-step of 0.2 lm, were used for demarcation of the foci area and signal quantification in the Adobe Photoshop CS5 software.

Fig. 7. An aberrant splicing of the insulin receptor in DM2 skeletal muscle and adipose tissue. Insulin receptor splicing is compared for skeletal muscles and adipose tissue of non-DM controls and DM2 patients, as the expression levels of insulin receptor splicing variants IR-A and IR-B mRNAs. The ratios of IR-A to IR-B were determined by semiquantitative PCR. Figure shows IR-A (131 kb) and IR-B (167 kb) PCR amplicons separated on 10% PAGE gel (acrylamide:bis-acrylamide 29:1) in TBE buffer. Lines A–E: skeletal muscle of DM2 patients P3, P9, P7, P6, and P1; Line F: adipocytes of a DM2 patient P3 (the same patient as in the Line A); Line C1: adipocytes from the DM1/DM2 negative control (taken from surgery); Lines C2–C4: DM1/DM2 negative skeletal muscles; Line C: non-cDNA control (non-template PCR control). Size marker: 1 kb DNA ladder.

muscles of DM1 patients contain lower than normal levels of insulin receptor RNA and protein [48] and display an abnormal regulation of insulin receptor mRNA splicing, resulting in a predominant expression of the lower-signalling non-muscle IR-A isoform associated with a weaker metabolic response in skeletal muscle tissue and cultured skeletal muscle cells from DM1 [14,15]. Importantly, MBNL1 was found to be the primary determinant of CUGexp foci formation and aberrant insulin receptor splicing in the myoblasts of patients with myotonic dystrophy [49].

Insulin receptor splicing in insulin-responsive tissues (skeletal muscle, liver and adipose tissue) was also studied in transgenic mice carrying the human genomic DM1 region with expanded (>350 CTG) repeats [50]. The mice displayed a tissue-specific and age-dependent abnormal regulation of insulin receptor mRNA splicing in all the tissues studied. Splicing abnormalities of insulin receptors prior to the development of muscle pathology were also reported in DM2 muscle [16]. Recently, Sen et al. established that MBNL1 binds specifically to the IR RNA [51] and overexpression or knockdown of MBNL1 altered the levels of exon 11 inclusion. The present finding of nuclear CCUGexp foci in fat cells and sequestration of MBNL1 protein in these foci in DM2 patients therefore suggest a role of the MBNL1 protein in abnormal splicing of the insulin receptor. Our demonstration of the shift in the proportion of IR-B/IR-A in human DM2 adipocytes is in accordance with this idea. The peripheral nerve expresses predominantly the high-affinity insulin receptor lacking exon 11. The insulin receptor was localized to paranodal terminal Schwann cell loops and microvilli and to the paranodal axolemma, while the mRNA was found in Schwann cells, endothelial cells, and pericytes [52]. If the splicing of the receptor in Schwann cells is controlled by MBNL1, its sequestration may contribute to an increased insulin resistance of DM2 patients. Studies carried out to verify the involvement of the peripheral nervous system (PNS) concerned only DM1 patients [53–57]. There are also only few reports that concern the presence of RNA foci or aberrant alternative splicing controlled by MBNL1 in vascular smooth muscle cells. Co-localization of ribonuclear inclusions and MBNL1 foci was reported in the gallbladder smooth muscle of a DM1 patient [25]. However, we do not know whether the

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sequestration of MBNL1 in the vascular smooth muscle demonstrated in this report is related to any specific target gene or exon. Thus, what could be the possible molecular pathways that lead to an altered smooth muscle function or contractility in DM1/DM2 patients? Contrary to the cell types discussed above, there are no indications on the MBNL1dependent alternative splicing of insulin receptors in vascular smooth muscles. Other possible candidates for an altered alternative splicing controlled by MBNL1, described for DM1, could be represented by a-actinin [58] and the ryanodine receptor [59]. Human endothelial cells possess insulin receptor mRNA and specific receptor proteins whose physicochemical properties are similar to those of insulin receptors in other tissues [60–62]. If the splicing of the IR were regulated by MBNL1 protein, similarly to the skeletal muscle, then its disturbance could contribute to the insulin resistance in human tissues. To date, there is no literature on MBNL-regulated splicing of insulin receptor mRNA in epidermal or adnexal cells. In fact, these tissues provided unique results concerning the MBNL1 protein distribution in the nucleus and its co-localization with CUG/CCUG expanded repeat foci, showing a low rate of sequestration of the protein in the cells while intranuclear MBNL1 signals were present in the majority of epidermal and adnexal cells. Nevertheless, age-dependent hyperhidrosis was reported in 20–30% of DM2 patients and early-onset male frontal bolding was observed in approximately 20% of German and 50% of American men aged 21–34 years [63]. Mueller et al. [64] hypothesize that an association between myotonic dystrophy and pilomatricomas (as well as other tumours reported in DM patients) involves up-regulation of beta-catenin, possibly via the actions of CUGBP and MBNL1 proteins.

5. Conclusions The present results show that the MBNL1 protein is expressed and more or less sequestered into the CCUGexp nuclear foci also in analyzed non-muscle tissues of DM2 patients. Since the process of sequestration of MBNL1 in skeletal muscles is involved in the pathogenesis of DM1/ DM2, the sequestration of the protein in soft and other tissues may represent a potential source of pathology and contribute to the pathological phenotype in DM1/DM2 patients. The degree of ZNF9 RNA expression and MBNL1 colocalization differs among individual tissues. The level of MBNL1 protein expression has been found to be relatively high in all tissues. On the other hand, the level of the CCUGexp transcript differs. In the skeletal muscle, soft tissues and sweat glands, more than 50% of the cells express the CCUGexp transcript of ZNF9 protein, while a lower percentage of epidermal and hair sheath cells express this

transcript. Therefore, it seems that different tissues show different levels of MBNL1 sequestration in DM1/2 patients. However, Ho et al. [65] showed that formation of RNA foci and disruption of MBNL1-regulated splicing are separable events. Recent reports also suggest that the DM2 phenotype may not depend solely on the expression of toxic mutant CCUGexp transcripts, but may also be due to alterations of ZNF9 expression [41–43]. Chen et al. [66] demonstrated in animal models that haploinsufficiency of ZNF9 contributes to the DM2 phenotype. Therefore, other factors contributing to the DM1/DM2 pathogenesis must also be taken into consideration. Acknowledgements This work was supported by the grants NS/9877-4/2008 of the Ministry of Health of CR, LD12039 of the Ministry of Education, IAA500040802 of the Grant Agency of the AS CR and the P302/10/1022 and P302/12/G157 grants of the Grant Agency of CR. Monoclonal antibody MB1a was supplied by Professor Glenn Morris, MDA Monoclonal antibody resource, Wolfson CIND, RJAH Orthopaedic Hospital, Oswestry, UK. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.nmd.2012.03.004. References [1] Taneja KL, McCurrach M, Schalling M, Housman D, Singer RH. Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J Cell Biol 1995;128:995–1002. [2] Davis BM, McCurrach ME, Taneja KL, Singer RH, Housman DE. Expansion of a CUG trinucleotide repeat in the 30 untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc Natl Acad Sci USA 1997;94:7388–93. [3] Miller JW, Urbinati CR, Teng-Umnuay P, et al. Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy. EMBO J 2000;19:4439–48. [4] Liquori CL, Ricker K, Moseley ML, et al. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 2001;293:864–7. [5] Fardaei M, Rogers MT, Thorpe HM, et al. Three proteins MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expandedrepeat transcripts in DM1 and DM2 cells. Hum Mol Genet 2002;11:805–14. [6] Pascual M, Vicente M, Monferrer L, Artero R. The muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing. Differentiation 2006;74:65–80. [7] Timchenko NA, Welm AL, Lu X, Timchenko LT. CUG repeat binding protein (CUGBP1) interacts with the 50 region of C/EBPbeta mRNA and regulates translation of C/EBPbeta isoforms. NAR 1999;27:4517–25. [8] Ho TH, Charlet-B N, Poulos MG, Singh G, Swanson MS, Cooper TA. Muscleblind proteins regulate alternative splicing. EMBO J 2004;23:3103–12.

Z. Luka´sˇ et al. / Neuromuscular Disorders 22 (2012) 604–616 [9] Osborne RJ, Thornton CA. RNA-dominant diseases. Hum Mol Genet 2006;15:R162–9. [10] Lin X, Miller JW, Mankodi A, et al. Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. Hum Mol Genet 2006;15:2087–97. [11] Lee JE, Cooper TA. Pathogenic mechanisms of myotonic dystrophy. Biochem Soc Trans 2009;37:1281–6. [12] Botta A, Vallo L, Rinaldi F, et al. Gene expression analysis in myotonic dystrophy: indications for a common molecular pathogenic pathway in DM1 and DM2. Gene Expr 2007;13:339–51. [13] Charlet-B N, Savkur RS, Singh G, Philips AV, Grice EA, Cooper TA. Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol Cell 2002;10(1):45–53. [14] Osborne RJ, Lin X, Welle S, et al. Transcriptional and posttranscriptional impact of toxic RNA im myotonic dystrophy. Hum Mol Genet 2009;18:1471–81. [15] Savkur RS, Phillips AV, Cooper TA. Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet 2001;29:40–7. [16] Savkur RS, Philips AV, Cooper TA, et al. Insulin receptor splicing alteration in myotonic dystrophy type 2. Am J Hum Genet 2004;74:1309–13. [17] Cardani R, Baldassa S, Botta A, et al. Ribonuclear inclusions and MBNL1 nuclear sequesteration do not affect myovlast differentiation but alter gene splicing in myotonic dystrophy type 2. Neuromuscul Disord 2009;19:335–43. [18] Warf MB, Nakamori M, Matthys CM, et al. Pentamidine reverses the splicing defects associated with myotonic dystrophy. Proc Natl Acad Sci USA 2009;106:18551–6. [19] Holt I, Mittal S, Furling D, Butler-Browne GS, Brook JD, Morris GE. Effective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles. Genes Cells 2007;12:1035–48. [20] Perdoni K, Malatesta M, Cardani R, et al. RNA/MBNL1-comtaiting foci in myoblast nuclei from patients affected by myotonic dystrophy type 2: an immunocytochemical study. Eur J Histochem 2009;53:151–8. [21] Sarkar PS, Han J, Reddy S. In situ hybridization analysis of Dmpk mRNA in adult mouse tissues. Neuromuscul Disord 2004;14:497–506. [22] Winchester CL, Ferrieer RK, Sermoni A, Clark BJ, Johnson KJ. Characterization of the expression of DMPK and SIX5 in the human eye and implications for pathogenesis in myotonic dystrophy. Hum Mol Genet 1998;8:481–92. [23] Mankodi A, Urbinati CR, Yuan QP, et al. Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum Mol Genet 2001;10:2165–70. [24] Jiang H, Mankodi A, Swanson MS, et al. Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum Mol Genet 2004;13:3079–88. [25] Mankodi A, Lin X, Blaxall BC, Swanson MS, Thhornton CA. Nuclear RNA foci in the heart in myotonic dystrophy. Circ Res 2005;97:1152–5. [26] Wheeler TM, Krym MC, Thornton CA. Ribonuclear foci at the neuromuscular junction in myotonic dystrophy type 1. Neuromuscul Disord 2007;17:242–7. [27] Cardani R, Mancinelli E, Saino G, Bonavina L, Meola G. A putative role of ribonuclear inclusions and MBNL1 in the impairment of gallbladder smooth muscle contractility with cholelithiasis in myotonic dystrophy type 1. Neuromuscul Disord 2008;18:634–40. [28] Bonifazi E, Gullotta F, Vallo L, et al. Use of RNA fluorescence in situ hybridization in the prenatal molecular diagnosis of myotonic dystrophy type 1. Clin Chem 2006;52:319–22. [29] Mankodi A, Teng-Umnuay P, Krym M, Henderson D, Swanson M, Thornton CA. Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann Neurol 2003;54:760–8. [30] Lucchiari S, Pagliarani S, Corti S, et al. Colocalization of ribonuclear inclusions with muscle blind like-proteins in a family with myotonic dystrophy type 2 associated with a short CCTG expansion. J Neurol Sci 2008;275:159–63.

615

[31] Dubowitz V, Sewry CA. Muscle biopsy. A practical approach. 3rd ed. London: Elsevier Saunders; 2007. [32] Warner JP, Barron LH, Goudie D, et al. A general method for the detection of large CAG repeat expansions by fluorescent PCR. J Med Genet 1996;33(12):1022–6. [33] Falk M, Vojtı´skova´ M, Luka´s Z, Kroupova´ I, Froster U. Simple procedure for automatic detection of unstable alleles in the myotonic dystrophy and Huntington’s disease loci. Genet Test 2006;10:85–97. [34] Holt I, Jacquemin V, Fardaei M, et al. Muscleblind-like proteins: similarities and differences in normal and myotonic dystrophy muscle. Am J Pathol 2009;174:216–27. [35] Kozubek M, Kozubek S, Lukasova E, et al. High-resolution cytometry of FISH dots in interphase cell nuclei. Cytometry 1999;36:279–93. [36] Kozubek M, Kozubek S, Lukasova E, et al. Combined confocal and wide-field high resolution cytometry of fluorescent in situ hybridization-stained cells. Cytometry 2001;45:1–12. [37] Vihola A, Bassez G, Meola G, et al. Histopathological differences of myotonic dystrophy type 1 (DM1) and PROMM/DM2. Neurology 2003;60:1854–7. [38] Day JW, Ranum LP. RNA pathogenesis of the myotonic dystrophies. Neuromuscul Disord 2005;15:5–16. [39] Luka´sˇ Z, Kroupova´ I, Bednarˇ´ık J, et al. The muscle biopsy in myotonic dystrophy in the era of molecular genetics (article in Czech). Cesk Slov Neurol N 2007;70:395–401. [40] Harper PS. Myotonic dystrophy. 3rd ed. London: W.B. Saunders; 2004. [41] Salisbury E, Schloser B, Schneider-Gold C, et al. Expression of RNA CCUG repeats dysregulates translation and degradation of proteins in myotonic dystrophy 2 patients. Am J Pathol 2009;175:748–62. [42] Huichalaf C, Schoser B, Schneider-Gold C, Jin B, Sarkar P, Timchenko LT. Reduction of the rate of protein translation in patients with myotonic dystrophy 2. J Neurosci 2009;29:9042–9. [43] Raheem O, Olufemi SE, Bachinski LL, et al. Mutant (CCTG)n expansion causes abnormal expression of zinc finger protein 9 (ZNF9) in myotonic dystrophy type 2. Am J Pathol 2010;177:3025–36. [44] Hartl P, Gottesfeld J, Forbes DJ. Mitotic repression of transcription in vitro. J Cell Biol 1993;120:613–24. [45] Frasca F, Pandini G, Scalia P, et al. Insulin receptor isoform A, a newly recognized, high-affinity insulin-like growth factor II receptor in fetal and cancer cells. Mol Cell Biol 1999;19:3278–88. [46] Sesti G, Marini MA, Tullio AN, et al. Altered expression of the two naturally occurring human insulin receptor variants in isolated adipocytes of non-insulin-dependent diabetes mellitus patients. Biochem Biophys Res Commun 1991;181:1419–24. [47] Moxley RT, Kingston WJ, Minaker KL, Corbett AJ, Rowe JW. Insulin resistance and regulation of serum amino acid levels in myotonic dystrophy. Clin Sci (Lond) 1986;71:429–36. [48] Morrone A, Pegoraro E, Angellini C, Zammarchi E, Marconi G, Hoffman EP. RNA metabolism in myotonic dystrophy: patient muscle shows decreases insulin receptor RNA and protein consistent with abnormal insulin resistance. J Clin Invest 1997;99:1691–8. [49] Dansithong W, Paul S, Comai L, Reddy S. MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1. J Biol Chem 2005;280:5773–80. [50] Guiraud-Dogan C, Huguet A, Gomes-Pereira M, et al. DM1 CTG expansions affect insulin receptor isoforms expression in various tissues of transgenic mice. Biochim Biophys Acta 2007;1772:1183–91. [51] Sen S, Talukdar I, Liu Y, Tam J, Reddy S, Webster NJG. Muscleblind-like 1 (Mbnl1) promote insulin receptor exon 11 inclusion via binding to a downstream evolutionarily conserved in tronic enhancer. J Biol Chem 2010;285(33):25426–37. [52] Sugimoto K, Marakawa Y, Zhang X, Xu G, Sima AA. Insulin receptor in rat peripheral nerve: its localization and alternatively spliced isoforms. Diabetes Metab Res Rev 2000;16(5):354–63. [53] Panayiotopoulos CP, Scarpalezos S. Dystrophia myotonica. Peripheral nerve involvement and pathogenetic implications. J Neurol Sci 1976;27: 1–16. [54] Borenstein S, Noe¨l P, Jacquy J, Flamentdurand J. Myotonic dystrophy with nerve hypertrophy. Report of a case with electro-

616

[55]

[56]

[57]

[58]

[59]

[60]

Z. Luka´sˇ et al. / Neuromuscular Disorders 22 (2012) 604–616 physiological and ultrastructural study of the sural nerve. J Neurol Sci 1977;34(1):87–99. Schro¨der JM, Himmelmann F. Fine structural evaluation of altered Schmidt–Laterman incisures in human sural nerve biopsie. Acta Neuropathol 1992;83:120–33. Kurt S, Karaer H, Kaplan Y, et al. Combination of myotonic dystrophy and hereditary motor and sensory neuropathy. J Neurol Sci 2010;288:197–9. Gantelet J, Kraftsik R, Delaloye S, Gourdon G, Kuntzer T, BarakatWalter I. The expansion of 300 CTG repeats in myotonic dystrophy transgenic mice does not induce sensory or motor neuropathy. Acta Neuropathol 2007;114:175–85. Vicente M, Monferrer L, Poulos MG, et al. Muscleblind isoforms are functionally distinct and regulate alpha-actinin splicing. Differentiation 2007;7:427–40. Kimura T, Nakamori M, Lueck JD, et al. Altered mRNA splicing of the skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in myotonic dystrophy type 1. Hum Mol Genet 2005;14:2189–200. Sugimoto K, Marakawa Y, Zhang X, Xu G, Sima AA. Insulin receptor in rat peripheral nerve: its localization and alternatively spliced isoforms. Diabetes Metab Res Rev 2000;16:354–63.

[61] Cerosimo E, DeFronzo RA. Insulin resistance and endothelial dysfunction: the road map to cardiovascular diseases. Diabetes Metab Res Rev 2006;22:423–36. [62] Finol H, Torres S, Rabucha A, Saenz H. Intramuscular capillary abnormalities in a case of myotonic dystrophy (Steinert’s disease). Acta Cient Venez 1992;43:284–9. [63] Day JW, Ricker K, Jacobsen JF, et al. Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum. Neurology 2003;60: 657–64. [64] Mueller CM, Hilbert JE, Martens W, Thornton CA, Moxley RT, Greene MH. Hypothesis: neoplasms in myotonic dystrophy. Cancer Causes Control 2009;20(10):2009–20. [65] Ho TH, Savkur RS, Poulos MG, Mancini MA, Swanson MS, Cooper TA. Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy. J Cell Sci 2005;118:2923–33. [66] Chen W, Wang Y, Abe Y, Cheney L, Udd B, Li YP. Haploinsufficiency for ZNF9 in ZNF9+/ mice is associated with multiorgan abnormalities resembling myotonic dystrophy. J Mol Biol 2007;368: 8–17.