Myotonic Dystrophy and Myotonic Dystrophy Protein Kinase

Progr. Histochem. Cytochem. Vol. 35 . No 3(2000) . pp. 187-251 http://www.urbanfischer.de/j ou rn a ls/p rag histcyt

Myotonic Dystrophy and Myotonic Dystrophy Protein Kinase Hideho Ueda . Shinichi Ohno . Takayoshi Kobayashi

With 20 Figures and 1 Table

URBAN & FISCHER 0079-6336/00/35/3-187 $ 15.00/0

HIDEHO UEDA, M. D., Ph. D. SHINICHI OHNO, M.D., Ph.D. Department of Anatomy, Yamanashi Medical University 1110 Shimokato, Tamaho, Yamanashi 409-3898 Qapan) TAKAYOSHI KOBAYASHI, M. D., Ph. D. Department of Neurology, Nakano General Hospital 4-59-16 Chuo, Nakano-ku, Tokyo 164-8607 Qapan)

Acknowledgements

This work was supported in part by grants from the Ministry of Health and Welfare to T. KOBAYASHI and by a grant-in-aid for scientific research 07670702 from the Ministry of Education, Science, Sport and Culture to T. KOBAYASHI.

~ Correspondence to: Dr. TAKAYOSHI KOBAYASHI, Department of Neurology, Nakano General Hospital, 4-59-16 Chuo, Nakano-ku, Tokyo 164-8607, Japan, Fax: 81-3-73-3381-4799

Contents 1 1.1 1.2 2 2.1 2.2 2.2.1 2.2.2 2.3

2.4 3 3.1 3.1.1 3.1.2 3.1.3

3.1.4 3.1.5

3.2 3.2.1 3.2.2

3.3 3.4 4 4.1 4.1.1

4.1.2 4.1.2.1 4.1.2.2 4.1.2.3 4.2 5

5.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical features and genetic loci of myotonic dystrophy .. . . . . Decreased DMPK expression due to trinucleotide repeat expansion Molecular genetics of myotonic dystrophy . . . . . . . . . . . . . . Triplet repeat instability during replication (change of chromosome structure) . . . . . . . . . . . . Loss of DMPK function Mouse model Dominantly inherited loss-of-function Possible dysfunction of DMPK and neighboring genes due to the change of chromatin structure containing extending triplet repeats in 3'UTR re-

.193 . . . . 193 197 199

~oo

200 204 206 207 207 208 208 211 213 214 214 214 217 218 218 219

A role for RNA metabolism in DM1 phenotype Localization of DMPK .. .. . . . Skeletal muscle . . . . . . . . . . . . . . . . . . . DMPK molecules in skeletal muscle . . . . . . . Predominant DMPK expression in type I muscle fibers DMPK localization in the terminal cisternae of sarcoplasmic reticulum DMPK localization in the neuromuscular junction DMPK localization in the muscle spindle . . . . . . . . . . Cardiac muscle. . . . . . . . . . . . . . . . . . . . . . . . . DMPK localization in intercalated discs and Purkinje fibers DMPK localization in corbular and junctional SRs Central nervous system . . . . . . . . Other tissues . . . . . . . . . . . . . . . . . . . . . Developmental localization of DMPK . . . . . . . Developmental localization of DMPK in skeletal muscle Developmental expression of DMPK gene in muscle cells in vivo and in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developmental expression of DMPK gene in human muscle cells by innervation in vitro . . .. . ... . . . . . . . . . . . . . . .. Developmental expression of DMPK and AChR subunit mRNAs in cultured human muscle cells . . .. . . . . . . . . . . . . . . . . . .. . . Developmental localization of DMPK in cultured human muscle cells Localization of DMPK in the neuromuscular junction in vitro . Developmental aspects of DMPK in CNS in vivo . . ... . ... . . Functions of DMPK . . . . . . . . . . . . . . . . . . . . . . . . . . ... DMPK protein belongs to a novel subfamily of serine-threonine protein kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .

199 202 202 202

. 219 219 220 221 224 225 227 227

190 . Contents

5.2 5.3 5.4 5.5 5.6 6 6.1 6.1.1 6.1.2 6.2 7

Phosphorylation of DMPK . . . . . . . . . . . . . . . . . . . . . . . " Modulation for skeletal muscle voltage-gated sodium channels . . . " Effects on Ca2 + metabolism by DMPK and relationship with other SRI ER proteins . . . . . . . . . . . . . . . . . . . . . . . A member of a subfamily of tumor suppressor genes Interaction with other proteins . . . . . . . . . Pathological aspects of DMPK . . . . . . . . . Pathological changes of adult type DM muscle Alterations of DMPK mRNA and its proteins Morphological changes of DM skeletal muscle Pathological aspects of congenital DM muscle Conclusions. . . . . . . . . . . . . . . . . . .

228 229 230 230 230 230 233 238 240

References

241

.

227 228

Abbreviations AChR a-BGT CAG cDM CNS COT-l CTG DHPR DM DMAHP DMPK

acetylcholine receptor a - bungarotoxin

cytosine-adenine-guanine congenital myotonic dystrophy central nervous system colonial temperature-sensitive-l cytosine-thymidine-guanine dihydropyridine receptor myotonic dystrophy DM locus-associated homeobox protein myotonic dystrophy protein kinase ER endoplasmic reticulum myosin heavy chain MHC MKBP DMPK-binding protein postnatal P PROMM proximal myotonic myopathy PDM proximal myotonic dystrophy RT-PCR reverse transcription-polymerase chain reaction ryanodine receptor RyR sHSP small heat shock protein UTR untranslated region warts wts

Abstract Myotonic dystrophy protein kinase (DMPK) was designated as a gene responsible for myotonic dystrophy (DM) on chromosome 19, because the gene product has extensive homology to protein kinase catalytic domains. DM is the most common disease with multisystem disorders among muscular dystrophies. The genetic basis of DM is now known to include mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'-untranslated region (UTR) of DMPK. Full-length DMPK was detected and various isoforms of DMPK have been reported in skeletal and cardiac muscles, central nervous tissues, etc. DMPK is localized predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in sekeletal muscle. In cardiac muscle it is localized in intercalated dises and Purkinje fibers. Electron microscopically it is detected in the terminal cisternae of SR in skeletal muscle and the junctional and corbular SR in cardia muscle. In central nervous system, it is located in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rough endoplasmic retriculum. The functional role of DMPK is not fully understood, however it may play an important role in Ca2+ homeostasis and signal transduction system. Diseased amount of DMPK may play an important role in the degeneration of skeletal muscle in adult type DM. However, other molecular pathogenetical mechanisms such as dysfunction of surrounding genes by structural change of the chromosome by long trinucleotide repeats, and the trans-gain of function of CUG-binding proteins might be responsible to induce multisystemic disorders of DM such as myotonia, endocrine dysfunction, etc. Key words: Ca2+ homeostasis - cardiac muscle - double immunofluorescent labeling electron microscopy - immunocytochemistry - isoforms of DMPK -location in central nervous system - multi systemic disorders - muscular dystrophies - myotonic dystrophy myotonic dystrophy protein kinase (DMPK) - signal transduction system - skeletal muscle.

1 Introduction 1.1 Clinical features and genetic loci of myotonic dystrophy Myotonic dystrophy (also known as dystrophia myotonica [OM], myotonia atrophica or Steinert disease) is an autosomal dominant inherited disorder with an incidence of about 1 in 8500 adults (HARPER, 1989). OM patients show multi systemic disorders including skeletal muscle (myotonia and distal muscular atrophy), cardiac muscle (cardiac conduction defect and cardiomyopathy), smooth muscle (gall bladder dyskinesia), eye lens (cataracts), the endocrine system (glucose tolerance deficiency, gonadal atrophy, premature balding), the immune system (hypogammaglobulinemia), and the central and peripheral nervous system (hypersomnia, apathy, inertia, dementia, personality difficulties, and peripheral neuropathy) (HARPER and RUDEL, 1994). The congenital form of OM (cOM) is more severe and characterized by general hypotonia and respiratory distress at birth. cOM also ~etards motor and mental development. In the last ten years, extensive genetic linkage analyses have been performed to determine the OM gene locus on chromosome 19q13.3 (BRUNNER et al. 1989; JOHNSON et al. 1990; HARLEY et al. 1991). The OM gene was first reported to be 14 kb and to encode 2.3 kb of mRNA with 15 exons and a protein, myotonic dystrophy protein kinase (OMPK), composed of 624 amino acids (BROOK et al. 1992; SHAW et al. 1993; MAHADEVAN et al. 1993). To date, two other human OMPK genes with slight differences of the deduced amino acid sequences in the N- or C-terminus in the same region have been reported by Fu et al. (1992) (639 amino acids) and SASAGAWA et al. (1994) (625 amino acids) (Fig. 1). The OM gene has a mutational expansion of a repetitive trinucleotide sequence (cytosine-thymidine-guanine: CTG)n located in a 3' untranslated region (3' -UTR) of the OMPK gene (ASLANIDIS et al. 1992; BROOK et al. 1992; BUXON et al. 1992; Fu et al. 1992; MAHADEVAN et al. 1992). The CTG repeat is highly polymorphic in the general population. The normal OM gene contains 5-35 (-37) CTG repeats in the 3'-UTR, and within this range the alleles are stably transmitted. When the repeat length exceeds 50 CTGs, the allele becomes unstable and expresses as disease. Patients with alleles ranging between 35 and 49 repeats have usually been found through their symptomatic offspring who have expansions in the 50-repeat range, so the alleles ranging between 35 and 49 repeats are considered "premutation" alleles. In the symptomatic cases, there is a significant inverse correlation between the age of onset and the number of repeats. The disease severity also correlates with the number of repeats. Roughly, mildly affected patients have 50-150 repeats, classical (adult type) OM patients have 100-1000 repeats and congenital OM (cOM) cases have more than 2,000 repeats. In OM families, an ealier onset of the disease as well as an increase in the severity of the clinical symptoms with transmission to successive generations, accompanied by an increase in the number of CTG repeats (genetic anticipation: ASHIZAWA et al. 1992a, b; HARLEY et al. 1992; BROOK et al.

194 . H .Ueda et al. 1 :HSAEVRLR · RLOOLVLOPGFLGLEp· L· L·DLLLGVHOE· . . . . LGASELAOO' KYVAOFLO\llAEPIVVRLKEVRLORODFEILKVIGRGAFSEVAVVKHKOT 93 1: HSAEVRLR · RLOOLVLOPGFLGLEp · L·L· DLLLGVHOE· .•.. LGASELAOD· KYVADFLQWAEPIVVRLKEVRLQRDDFEI LKVIGRGAFSEVAVVKHKQT 93 1 :HGGHFWPPEPYTVFMWGSPWEADSPRVKLRGREKGRQTEGGAFPL VSSALSGOPRFFSPTTPPAEP IVVRLKEVRLQRDDFEI LKVI GRGAFSEVAVVKMKQT 103

*

* ** *

****************************************

94 :GQVYAMKIMNKWDMLKRGEVSCFREERDVL VNGDRRWITOLHFAFODENYL YL VMEYYVGGDLL TLLSKFGERIPAEMARFYLAEIVMAIDSVHRLGYVHRDI 196 94: GOVYAMKIMNKWDMLKRGEVSCFREERDVL VNGDRRWITOLHFAFQOENYL YL VMEYYVGGDLL TLLSKFGERIPAEMARFYLAEIVMAIDSVHRLGYVHRDI 196 104: GOVYAMKIMNK\IIDMLKRGEVSCFREERDVL VNGDRR\IIITOLHFAFQDENYL YL VMEYYVGGDLL TLLSKFGERIPAEMARFYLAEIVMAIDSVHRLGYVHRDI 206

*******************************************************************************************************

197: KPDNI LLDRCGH I RLADFGSCLKL RADGTVRSL VAVGTPDYLSPE I LQAVGGGPGTGSYGPECD\II\IIALGVFAYEMFYGOTPFYA DSTAETYGKIVHYKEHLSL 299 197 : KPDNI LLDRCGH I RLADFGSCLKL RADGTVRSL VAVGTPDYL SPE I LQAVGGGPGTGSYGPECD\II\IIALGVF AYEMFYGQTPFY ADSTAETYGK IVHYKEHL SL 299 207: KPDNI LLDRCGH I RLADFGSCLKLRADGTVRSL VAVGTPDYLSPE I LOAVGGGPGTGSYGPECD\II\IIALGVF AYEMFYGQTPFYADSTAETYGK IVHYKEHLSL 309

*******************************************************************************************************

300 : PL VDEGVPEEARDF IQRLLCPPETRLGRGGAGDFRTHPFFFGLD\IIDGLRDSVPPFTPDFEGATDTCNFDL VEDGL TAM · ... . ETl SD I REGAPLGVHLPFVG 397 300 : PL VDEGVPEEARDF IQRLLCPPETRLGRGGAGDFRTHPFFFGLD\IIDGLRDSVPPFTPDFEGATDTCNFDLVEDGL TAM · .. .. ETlSDIREGAPLGVHLPFVG 397 310 : PL VDEGVPEEARDFIQRLLCPPETRLGRGGAGDFRTHPFFFGLD\IIDGLRDSVPPFTPDFEGATDTCNFDLVEDGL TAMVSGGGETlSDIREGAPLGVHLPFVG 412

******************************************************************************

**** ** *** *** *** *****

398: YSYSCMAL RDSEVPGPTPMEL EAEQL LEPHVQAPSLEPSVSPQDET AEVAVPAA VP AAEAEAEVTLRELQEPLEEEVL TRQSL SREMEAI RTDNQNFASQLRE 500 398: YSYSCMAL RDSEVPGPTPMEVEAEQL LEPHVQAPSLEPSVSPQDETAEVAVPAAVPAAEAEAEVTLRELQEALEEEVL TRQSLSREMEAI RTDNQNFASQL RE 500 413: YSYSCMALRDSEVPGPTPMEVEAEQLLEPHVQAPSLEPSVSPQDETAEVAVPAAVPAAEAEAEVTLRELQEALEEEVL TROSLSREMEAIRTDNQNFASQLRE 515

******************** ************************************************** *******************************

501 : AEARNRDLEAHVRQLQERMEL LQAEGATAVTGVPSPRATDPPSHMAPRP\IILWASA· R\II\IIGQAPCTAATCCSLPGSLGLAYRRRFPCS· CSPLFCL VPPPWAAL 601 501 : AEARNRDLEAHVROLQERMEL LOAEGATAVTGVPSPRA TDPPSHLDGPPAVAVGQCPL VGPGP' MHRRHL LLPAR· . VP · RPGLSEAL SL LL FA VVLSRAAAL 599 516 : AEARNRDLEAHVROLOERMEL LQAEGATAVTGVPSPRA TDPPSHLDGPPAVAVGQCPL VGPGP . MHRRHLLLPAR · .vp · RPGLSEALSLLLFA VVLSRAAAL 614

********************************************

602 : G\II\IIPTPANSPQSGAA· OEPPALPEP 600 : GCIGL VAHAGQL TAVWRRPGAARAP 615 :GCIGLVAHAGQL TAVWRRPGAARAP

* * * *

* *

**

** * ***

625 624 639

Fig. 1. Comparison of amino acid sequences of DMPK. Amino acid sequences of human DMPKs (upper line: SASAGAWA et al. 1992; middle line: BROOK et al. 1992; lower line: Fu et al. 1992) are aligned to maximize their sequence homology. The conserved amino acid residues among all sequences are represented by asterisks.

1992; HUNTER et al. 1992; SHELBOURNE et al. 1992; TSILFIDIS et al. 1992; LAVEDAN et al. 1993) (Fig. 2). MARTORELL et al. (1996) reported clinical and molecular studies of three unrelated homozygous DM patients. In the first family, the homozygous patient showed the classical form ofDM with DM alleles of very different expansion sizes (60 and 1000 repeats). In the second family, a homozygous patient with mild symptoms carried a minimally expanded allele (64 repeats) and a "normal" allele (38 repeats) that increased in size when transmitted. The third homozygous case, who has two minimally expanded alleles (51 and 120 repeats), has had only late onset bilateral cataracts. This study showed that intergenerational enlargement occurs during transmission to the next generation. The pattern of intergenerational repeat-size instability is dependent on the sex of the transmitting parent: large average intergenerational increases, leading to very large CTG expansions, were more frequently found on transmission from females than from males (LAVEDAN

Myotonic dystrophy/ Myotonic dystrophy protein kinase · 195

et al. 1993). Thus, cDM transmission is predominantly maternal, and that is in contrast to cytosin-adenine-guanine (CAG)-repeat disease in which expansion size predominantly increases through paternal transmission (ASHlZAWA and ZOGHBI, 1997). The repeat length within the tissues or organs of individual patients has also been shown to increase with age, indicating ongoing mitotic instability in DM and reflected the progressive clinical symptoms (MARTORELL et al. 1995). CTG repeat expansion also occurs during somatic proliferation of DM fibroblasts in vitro (WOHRLE et al. 1995, and Fig. 3). No mutations have been found within the coding region of the DMPK gene in DM patients so far. Recently THORNTON et al. (1994) reported an autosomal dominant disorder similar to DM without CTG trinucleotide expansion at the DM locus. RICKER et al. (1994, 1995) named this disease proximal myotonic myopathy (PROMM) because these patients had predominantly proximal muscle weakness without any atrophy as opposed to the distal muscle atrophy and weakness seen in DM. Based on comparison of the phenotypes of DM and PROMM, PROMM is thought to be a more benign disorder. There are almost no obvious mental changes in PROMM patients, and death is extremely rare. Anticipation appears to be present, but to a milder, degree and a severe congenital form of PROMM is very rare if it even occurs (RICKER, 1999). However, recently clinical symptoms of brain disease, including mental changes with hypersomnia, Parkinsonian features, stroke-like episode and seizures (HUND et al. 1997) have been reported in PROMM. DAY et al. (1999) reported another multisystemic myotonic disorder, which was very similar to DM with distal muscle weakness but lacking CTG repeat expansion. They called this disorder myotonic dystrophy type 2 (DM2). Furthermore, MEOLA et al. (1996) and UDD et al. (1997) found a variant of PROMM with dystrophic features and named it proximal myotonic dystrophy (PDM). Recently RANUM et al. (1998) assigned the DM2 locus to chromosome 3q. RICKER et al. (1999b) also found that the majority of German PROMM families showed linkage to the DM2 locus, to which PDM was also mapped. It remains unknown whether DM2, PROMM and PDM represent different phenotypic expressions of the same responsible gene or they are different allelic disorders. The phenotypic resemblance among DM, DM2, PROMM and PDM complicates the diagnosis of these disorders. The possibility that these disorders are caused by mutations in different genes closely linked to the chromosome 3q region cannot be excluded (THORNTON and ASHlZAWA, 1999). The DM1 and DM2 disease loci in some typical PROMM families (RICKER et al. 1999b) and other families with multisystemic myotonic disorders have been excluded. Because of the genetic and phenotypic heterogeneity of these disorders, it is necessary to establish a new nomenclature foreseeing the future discovery of new disease loci and phenotypic variability. At the Second International Myotonic Dystrophy Consortium (IDMC) conference held in 1999, the consensus for a new nomenclature was reached so that all multisystemic myotonic disorders including DM, DM2, PROMM and PDM are collectively called myotonic dystrophies. These diseases will be consecutively named DMn followed by a number, such

196 . H. Veda et al.

I.

II.

M

F

feo R I

10.0 kb

~. 8

kb

e.e l . 4kt_

B

i<.b

J " kb __

Eco R I

fLO kb

4. 5

8. 6 kb 3. 4 ktt-t

loll

3 . " kb

III. 1 234 5

T1

U kb··"

T2

1 234 5

- 3 4 kh

Myotonic dystrophy/Myotonic dystrophy protein kinase . 197

as DM1, DM2, DM3, based on their locuses and regardless of the clinical phenotype. Therefore, patients in DM families with the expanded CTG repeats in chromosome 19 are now designated DM1 (ASHIZAWA, 2000).

1.2 Decreased DMPK expression due to trinucleotide repeat expansion In DM1 patients, drastic changes in different somatic cells of a single patient have also been observed (mitotic or somatic instability) (ANVRET et al. 1993; JANSEN et al. 1994; MARTORELL et al. 1996). Small pool PCR also demonstrated that the smear composed of multiple, coalesced fragments can be resolved into discrete ones derived from single cells (MONCKTON et al. 1995). Studies of length of (CTG)n triplet repeats in DM patient tumors showed that the longest expansion of the repeats was observed, compared with non-tumor tissues of the same organs, muscles and leukocytes (somatic instability: JINNAI et al. 1999). Our recent studies using long-term DM fibroblast cultures showed that DMPK protein decreased with the expansion of trinucleotide repeats during somatic proliferation (Fig. 3). Other groups have also shown that CTG expansion resulted in the reduction of DMPK mRNA and protein levels, although one study reported that a DM patient had increased expression of the DMPK gene (SABOURIN et al. 1993). In cultured lymphoid cells derived from a homozygous DM patient, both DMPK mRNA and 72kD full-length DMPK protein levels were significantly reduced (CASKEY et al. 1996). On the basis of these observations, we conclude that the CTG expansion negatively affects DMPK expression. Fig.2. Molecular genetic analysis of a DM family with monozygotic twins. I. A DM pedigree with monozygotic twins. The parents have no clinical symptoms of DM. The twins (Tl and T2) recognized grip myotonia at the ages of 24 and 20, respectively. They have mild distal muscle atrophy and weakness of the upper limbs. Their younger brother (B) recognized grip myotonia at the age of 25. He has very slight distal weakness of the upper limbs. II. Southern blot analyses of the father (F), mother (M) and younger brother (B). DNA was extracted from peripheral blood samples. Genomic DNA was digested by Eco RI and BgII, and Southern blot analyses were performed using a 32P-Iabeled DMPK gene probe. Control (each lane 1) has 9.8 kb and 8.6 kb bands with Eco RI digestion and a 3.4 kb band with BgII digestion. The father (F: lane 2) has 10.0 kb and 9.8 kb bands with Eco RI digestion and 3.7 kb and 3.4 kb bands with BgII digestions, respectively. He has about 100 trinucleotide repeat expansion. The mother (M: lane 2) has no trinucleotide repeat expansion. The brother (B: lane 2) has 11.0 kb and 8.6 kb bands with Eco RI digestion and 4.5 kb and 3.4 kb bands with BgII digestions. He has about 400 trinucleotide repeat expansion. III. Southern blot analyses of the twins (Tl and T2). DNA was extracted from peripheral blood samples. Genomic DNA was digested by Eco RI (lanes 2 and 3) and Bam HI (lanes 4 and 5) and Southern blot analyses were performed using a 32P-Iabeled DMPK gene probe. Lanes 3 and 5 in T1 and T2 are samples of the patients Tl and T2. Lane 1 shows size markers digested by AHindIII. Lane 2, 4 are controls. T1 and T2 have about 700-1400 and 500-700 trinucleotide repeat expansions, respectively. (From KOBAYASHI et aI., unpubI.)

98 . H .Veda e t la.

A

OM

control 123456

.... B

...

L~ :~2L'

2021

membrane fraction

soluble fraction Pr

Pr PI Pr Pt

t'X" ...

- 66

- ,29

NC

Pr PI

-

- 48.5

OM

PI

kDa NC

66

Myotonic dystrophy/Myotonic dystrophy protein kinase' 199

In the following chapters, we describe the molecular genetics of OMl, localization of OMPK in skeletal and cardiac muscles, the central nervous system and other tissues, developmental localization of OMPK, functions of OMPK and pathological aspects of OMPK in adult OMI and cOM.

2 Molecular genetics of myotonic dystrophy To date, molecular cytopathological mechanisms of DM1 have been proposed by several researchers (WIERUNGA, 1994; HARRIS et al. 1996; CASKEY et al. 1996; KORADEMIRNICS et al. 1998; SINGER, 1998). Fig. 4 shows the chromosome localization of the OMI gene, the gene structure and expected function of the responsible OM1 gene, and the possible molecular pathophysiological mechanisms of OM1.

2.1 Triplet repeat instability during replication (change of chromosome structure) Progressive changes of the number of triplet nucleotides (expansion-deletion mechanisms) have been reported for the CTG (CAG), CGG (GCC) and GAA (Crr)

Fig. 3. Southern (A) and Western (B) blot analyses of DMPK gene and protein in DM and control fibroblasts in long-term culture. A: Fibroblasts from muscle biopsies of 4 DM patients and 3 control cases were cultured for more than 13 weeks. Genomic DNAs were extracted from fibloblasts at 7 weeks and 13 weeks of culture. Each 20 I-tg of genomic DNA was digested by Bam HI. Southern blot analyses were performed using a 32P-Iabeled DMPK gene probe, and the changes of (CTG)n repeats during somatic proliferation in culture were compared. DM, lane 1: case 1 (7 weeks of culture), 1.3 kb and 10.5 kb; lane 2: case 1 (13 weeks of culture), 1.3 kb and 10.5 kb; lane 3: case 2 (7 weeks of culture) 1.3 kb and 5.7-8.5 kb, lane 4: case 2 (13 weeks of culture) 1.3 kb and 8.5-11.5 kb; lane 5: case 2 (21 weeks of culture) 1.3 kb and 9.0-11.5 kb; lane 6: case 3 (7 weeks of culture) 1.3 kb and 13 kb, lane 7: case 3 (13 weeks of culture) 1.3 kb and 13 kb; lane 8: case 4 (7 weeks of culture) 1.3 kb and 13.5 kb, lane 9: case 4 (13 weeks of culture) 1.3 kb and 13.5 kb. Controls. lane 1: control 1 (7 weeks of culture), lane 2: control 1 (13 weeks of culture), lane 3: control 2 (7 weeks of culture), lane 4: control 2 (13 weeks of culture), lane 5: control 3 (7 weeks of culture), lane 6: control 3 (13 weeks of culture), all 1.3 kb. In case 2, the DNA fragment elongates from 7.1 kb (midportion) at 7 weeks of culture (lane 3) to 10 kb (midportion at 13 weeks of culture (lane 4) and 10.25 kb (midpoint) at 21 weeks of culture (lane 5). The 3 other cases and 3 control cases have no change of (CTG)n repeats at 7 and 13 weeks of culture. B: Western blot analyses were performed on the membrane (left panel) and soluble fractions (right panel) of homogenates of DM case 2 cultured fibroblasts using DMPK antibody against the Cterminus. 70 kDa (arrow in left panel) and 55 kDa DMPK proteins (arrow in right panel) decrease in 13 weeks of culture (Pt) in comparison with those in 7 weeks of culture (Pr), whereas the normal control has no change of D MPK protein in 7 and 13 weeks of culture. Each lane includes equal amounts of myosin heavy chain protein. (From SHIMOKAWA et a!., unpuh!.)

200 . H. Ueda et al.

--

B

H

""""', u

.

U ll. t

[

SerfThr·Kinase Consensus

o

~

Change of chromosome structure by ItTG)n triplet repeat (CUG)n triplet repeat RNR-bindlng proteins

DMPK~l ~p ~

gene

,.::;:.;:;;;'::: .;:',,:'.;:;;",,:',"'. ,,;';:1:' ,;.>: .:.

tI

;"f'

'i'~':""

RbnDrmal regulatlDn 01 OMPk mRNIl Redu ced ll M FlHI' mAN/ (nuclear retentlDn Df lPutant transcripts) ODminant-negatlDe RNR mutatiDn Reduced

OM~K

proteIn

Ilelltie ncy of Na,/Ic:' -flTPase

• RbnDrmal homeostasis of [Cali

Apoptosls and/or necrosis

~ Degeneration of lPusde

I}

5 Cal .- flTPase

Maturation arrest J~t ~Llff.c h' fJf 3 p l~ r'; :-o f F f ~'.'i.!r~ '~('

~:,g'nne~u:;/ ,· , .lfo1

I .m.
Fig. 4. Chromosome localization (A) of the DMI gene, the gene structure (B), expected function of the responsible DM1 gene (C), and molecular pathophysiological mechanisms of DMI (D). A. The DM gene is localized in 13.3 of chromosome 19q. B. Schematic structural organization of the DMI region showing the proximity of the 59 and DMAHP genes to DMPK. Localization of the unstable (CTG)n expansion within 3'-UTR of DMPK and the Alu insertion within DMPK are shown (modified from H ARRIS et aI., 1996). C. Expected function and structure of DMPK genes. D. Hypothetical molecular pathophysiological mechanisms of DMl.

Myotonic dystrophy/Myotonic dystrophy protein kinase' 201

sequences (KANG et al. 1995a, b; WELLS, 1996; WELLS et al. 1998; TISHKOFF et al. 1997). Intensive studies have focused on the triplet repeat instability. Recent studies of replication of repeat sequences in yeast suggest that a replication division is formed and that the lagging structue is appropriately single-stranded; however, repeating triplets autohybridize and form 'hairpin' structures (TISHKOFF et al. 1997; GORDENIN et al. 1997, KUNKEL et al. 1997), and DNA polymerase could misread the hairpin structure (Okazaki fragments would not be bound to the hairpin structure). The same hairpin formation would playa quite important role in triplet expansions in human diseases. It is reported that new alternative DNA secondary structures that map within the repeat tracts during reannealing of complementary strands, containing an equal number of repeats, form into linear duplex DNA molecules. The formation of these alternative DNA structures (slipped-strand DNA structures, which are termed S-DNA), increases with the triplet repeat length (PEARSON and SIND EN, 1996). These S-DNA structures can be formed between complementary strands, and the structural complexity of S-DNAs formed increases in proportion to the increasing repeat number. Electron microscopy has demonstrated that S-DNA structures are composed of multiple loops or hairpins (PEARSON et al. 1998). When the Okazaki fragments are synthesized on the lagging strand, the fragments containing the triplet repeats would form hairpin structures with themselves or loop back on the template strands. This would trigger the replication of the already replicated triplets from the template strand. Endonuclease would not be able to repair this duplication. After DNA ligation, a corresponding repeat expansion would be added to the template strand, and finally the triplet repeat expansion would occur in that region. Expanded CTG triplet repeats have been shown to form the strongest natural nucleosome positioning elements (WANG and GRIFFITH, 1995). Recently the presence of 75-130 CTG repeats was shown to cause an increase in nucleosome assembly in Xenopus borealis (WANG and GRIFFITH, 1995). In both in vivo (WANG and GRIFFITH, 1995) and in vitro (WANG et al. 1994) studies, the nucleosome stability was dependent on the number of repeats. The efficiency of the nucleosome formation would increase with the size of the repeat. Further evidence for the change in chromatin structure due to the triplet repeat expansion came from studies in muscle nuclei of unrelated DM patients with the large expansion of CTG repeats. OTTEN and TAPSCOTT (1995) showed that an inaccessible and DNAse I-resistant site was detected just adjacent to the triplet repeat. These data suggest that the structure of expanded triplet repeats changes the local chromatin structure, which would affect the expression of DMPK and other neighbourmg genes.

202 . H. Veda et al.

2.2 Loss of DMPK function 2.2.1 Mouse model

REDDY et al. (1996) bred DMPK knock-out mice. Heterozygous mice showed no symptoms characteristic of DM phenotypes, suggesting that the dominantly inherited human disease is not simply due to loss-of-function of the gene (haploinsufficiency). However, homozygous mice developed a late-onset progressive myopathy (completely loss-of-function). JANSEN et al. (1996) also reported knocking out the endogenoeous mouse DM gene and made simultaneously producing overexpression of the normal human DM gene. These knocked-out/overexpressed DMPK mice did not develop any characteristic features of human DM phenotype. However, mice with> 20 copies of the human DM gene showed cardiac myopathy and reduced life span. Recently, GOURDON et al. (1997) bred transgenic mice using the complete 45 kb human genomic locus including a defective DM gene (55 CTG repeats) and two surrounding genes, DMR-N9 and DMPAHP. In their study only about 7% of descendants showed any increase in the number of CTG repeats. Both germinal and somatic instability were noted, but there was no parental bias. In addition, no DM phenotype was seen in their mice.

2.2.2 Dominantly inherited loss-of-function

DM patients are heterozygous for the expansion mutation, with one normal and one mutant DMPK gene. If the expansion mutation destroys the ability of the mutant gene to produce functional DMPK, theoretically the DMPK protein would be reduced more than 50 %. According to the haploinsufficiency model, sufficient protein is not produced by the normal allele to prevent biochemical abnormalities (50 % is not enough). If the most severe disease is due to having 50 % of the normal DMPK level (0 % from the mutant allele and 50 % from the normal allele) and adult onset is due to having 80 % of the normal protein level (for example 30 % from the mutant allele and 50 % from the normal allele), then DM1 disorder would be explained by the haploinsufficiency model. However, the simple haploinsufficiency model is not able to explain the more than 50 % decrease of DMPK protein in adult DM skeletal muscle (HOFMANN-RADVANYI et ai. 1993a; NOVELLI et al. 1993; UEDA et ai. 1999). In DM1, the loss of protein production from the mutant allele could be less than 50 %, because the expanded CTG repeats in the 3'-UTR would prevent poly (A) tailing or interfere with mRNA and/or protein synthesis G. WANG et al. 1995). Regarding the molecular pathophysiological basis of cDM, dysfunction of other genes would be more important in producing hypotonia and respiratory distress, because DMPK is relatively well preserved in skeletal muscle even in the severe cases of cDM, who were born as floppy infants and died within one year with more than 1600 CTG repeats in leukocytes and skeletal muscle in our studies (TANAKA et aI., unpub1.).

Myotonic dystrophy/Myotonic dystrophy protein kinase· 203

2.3 Possible dysfunction of DMPK and neighboring genes due to the change of chromatin structure containing extending triplet repeats in 3'UTR region Most studies have shown a negative effect of the (CTG)n and (CGG)n repeats on gene transcription and translation (Fu et al. 1993; CARANGO et al. 1993; PARSONS et al. 1998). Y. WANG et al. (1995) reported that the precise analysis of poly (AY RNA from adult DM patients showed dramatic decreases of both mutant and normal DMPK RNAs. Therefore both normal and expanded DMPK genes are transcribed in DM skeletal muscle, but the mutant RNA containing the abnormal expansion has a dominant effect on RNA metabolism by preventing the accumulation of poly (AY RNA (dominantnegative RNA mutation). Aggregates of a trinucleotide repeat in the nucleus in DM muscle cells have been observed in in situ hybridization (TANEJA et al. 1995; DAVIS et al. 1997), and altered processing of DMPK transcription (KRAHE et al. 1995) has been also reported. If these abnormal transcriptions manifest at the RNA level in DM muscle, the expression of DMPK protein might decrease to less than 50 % of that in normal muscle. The expanded CTG repeats that interfere with the local chromatin configuration might affect the expression of both DMPK and neighboring genes. The putative downstream gene, which encodes a homeodomain protein, has been named DM locusassociated homeobox protein (DMAHP). DMAHP has a conserved lysine residue that is a feature of a small subset of homeodomain proteins related to the sine oculis gene of Drosophila. Homeodomain proteins are known to be transcription factors, which regulate gene expression by binding to DNA. Recently, it was reported that the gradient morphogen bicoid (bcd), a homeodomain protein, which binds DNA and transcriptionally activates target genes at different threshold concentrations. It also binds to RNA and acts as a translational repressor to generate an opposing gradient of the homeodomain protein caudal (cad) (DUBNAU and STRUHL 1996). DMAHP mRNA is expressed in many tissues of normal and DM patients, including skeletal muscle, fibroblasts, lymphocytes, heart and brain. The enhancer element and the first exon of DMAHP are in close proximity to exon 15 of the DMPK gene. The DMAHP gene may be susceptible to regional chromatin changes (OTTEN and TAPSCOTT 1995; BOUCHER et al. 1995; KLESERT et al. 1997), and the expanded CTG repeats could affect transcription from the DMAHP homeobox gene. Recent studies show that DMAHP transcript levels in fibroblasts and myoblasts from DM patients are significantly lower than in normal control cells (KLESERT et al. 1997; THORNTON et al. 1997). Quantification of the mRNA transcribed from the mutant DMAHP allele showed that the mRNA of DMAHP was reduced to 10-30 % of normal (KLESERT et al. 1997), or its transcription was sometimes completely inactivated (THORNTON et al. 1997). THORNTON et al. (1997) also showed that DMAHP expression in myoblasts, muscle and myocardium was reduced by the DM mutation and that the magnitude of this effect depended on the extent of the CTG repeat expansion. It is quite interesting that the DMAHP sequence is very similar to a murine transcription factor that regulates the expression of the Na+IK+ -ATPase a1-subunit

204 . H. Ueda et al.

in dev~loping skeletal muscle (KAWAKAMI et al. 1996). Dysregulation of DMAHP may be responsible for the immaturity of skeletal muscle in cDM and abnormal innervation in adult onset of DMl. Another neighboring gene, gene 59, which is upstream of the DMPK gene, is also a candidate gene for influence by the CTG repeat expansion GANSEN et al. 1992; SHAW et al. 1993). The mouse homologue of this gene, DMR-N9, is located 1.1 kb upstream of the 3' end of DMPK (Fig. 4B) GANSEN et al. 1992; JANSEN et al. 1995). This 7 kb gene contains five exons and encodes a 650 amino acid protein. Two regions within this gene show significant homology to WD repeats, which are highly conserved amino acid sequences found in a family of proteins involved in signal transduction. The actual function of DMR-N9 is still unknown. However, it is strongly expressed in brain and testis. Recently the human homologue to DMR-V9 (59 gene) has been identified and shown to be expressed in brain, testis, heart, liver, kidney and spleen but not detected in skeletal muscle GANSEN et al. 1995). If repeat expansion does alter local chromatin structure, then this could affect 59 gene. The altered transcription from gene 59 may account for some of the clinical features observed in DM1 patients: mental retardation and reduced fertility. The presence of a large CpG island at the 3' end of the DMPK gene indicates that the (CTG)n repeat expansion could disrupt the function of the island, affecting expression of other associated genes. Myotonia is one of the main clinical features of DM. It is of interest that DMPKknockout mice have progressive degenerative changes in skeletal muscle but no myotonia, suggesting that myotonia is not related to the lack of DMPK protein. DM muscle has a low resting membrane potential (KOBAYASHI et al. 1990) and remains an apamin receptor (RENAUD et al. 1986). The existance of a decreased resting membrane potential and the presence of an apamin-sensitive Ca2+-activated K+ channel can create repetitive action potentials (myotonia). The remaining apamin receptors, decreased resting membrane potential, decreased Na+/K+-ATPase and SR Ca2 +-ATPase activities (BENDERS et al. 1993), and polysynapse (KOBAYASHI et al. 1990a) may be a maturation-related disturbance of the DM muscle membrane. These phenomena are probably responsible for the abnormal regulation of these neighboring genes of DMPK.

2.4 A role for RNA metabolism in DM1 phenotype It is known that RNA-protein interactions playa crucial role in correct processing and trafficking of RNA molecules within cells (ST JOHNSTON, 1995). A number of studies strongly suggest that abnormal RNA-protein interactions playa crucial role in the molecular pathogenesis of DM1. One model of the molecular pathogenesis of DMl hypothesizes that RNAs from the expanded allele create a gain-of-function mutation by the inappropriate binding of proteins to the CUG repeats. Recently it was reported that DMPK mRNA in DM patient fibroblasts and muscle cells accumulated abnormally in

Myotonic dystrophy/Myotonic dystrophy protein kinase' 205

focuses within the nucleus (five to hundreds of copies), while the normal cells maintained the transcripts around cytoplasmic perinuclear regions in the cytoplasm (TANEJA et al. 1995; DAVIS et al. 1997; HAMSHERE et al. 1997). The focuses of DMPK RNA contained non-poly (At RNA and the pathological repeat (TANEJA et al. 1995). Formation of nuclear focuses is a new mechanism for preventing transient export and affecting a loss of gene function. Recently, TIMCHENKO et al. (1996a, b) isolated a CUG-binding protein from HeLa cells that binds to synthetic (CUG)s RNA oligonucleotides. Detailed analysis of this protein revealed two novel isoforms of a heterogenous nuclear ribonucleoprotein (hnRNP) of the type called hNab50, which were called CUG-BP1 (49 kDa) and CUG-BP2 (51 kDa). hnRNP proteins are typically present within the nucleus, where they bind to poly (At RNA and are involved in nuclear RNA processing, and their expressions are essential for cell growth (ANDERSON et al. 1993; WILSON et al. 1994). CUG-BP1 and CUG-BP2 are able to preferentially bind the 3' -UTR of poly (At DMPK mRNA, suggesting that these hnRNPs may be transcript-specific or interact with only a subset of mRNAs. In normallymphoblasts, CUG-BP1 is found predominantly in the cytoplasmic fraction and CUG-BP2 predominantly in the nucelar fraction. However, in lymphoblast cell lines from DM1 patients the majority of CUG-BP2 shifts its localization from the cytoplasmic fraction to the nuclear one. Shifted CUG-BP2 may be involved both in splicing of mRNAs containing (CUG)n repeats and in their transport (trans-dominant RNA disorder by CUG binding proteins: CASKEY et al. 1996; PHILIPS et al. 1998). So far the only protein other than the CUG-BPs that has been shown to bind RNA triplet repeats is the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis and its close relative Bacillus pumilus (BABITZKE, 1997). Using an in vitro photocrosslinking assay, the CUG-BP/hNab50 present in Hela cell nuclear extract was shown to bind to RNAs that include the 3' -untranslated region of DMPK mRNA, but not to actin mRNA (CASKEY et al. 1996). The 3' -UTR expanded CTR repeats are transcribed, but not processed appropriately, and the transcripts accumulate in the nucleus. In the nucleus the expanded RNA molecules bind excessive numbers of transport proteins to sequester them from their normal localization in the cytoplasm. Abnormal RNA-protein complexes are not able to leave the nucleus. This would prevent translation of a number of mRNA species, including normal DMPK mRNA from the normal allele, leading to loss of DMPK, insulin receptors and other proteins. Abnormal levels of insulin receptor mRNA were observed in muscles from adult DM1 and cDM (MORRONE et al. 1997), which could explain the increased insulin resistance seen in many DM1 patients. Recently ROBERTS et al. (1997) reported that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 (CUGBP/hNab50), were altered in homozygous DM patients and that CUG-BP/hNab50 was a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patients and DMPK knock-out mice, show that DMPK regulates both phosphorylation and intracellular localization

206 . H. Veda et al.

of the CUG-BP/hNab50 protein. Decreased levels of DMPK in DM homozygous patients and DMPK knock-out mice lead to the elevation of the nuclear concentration of the hypophosphorylated form of CUG-BP/hNab50. DMPK-mediated phosphorylation of CUG-BPlhNab50 results in dramatic reduction of the CUG-bp2, hypophosphorylated isoform. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter the phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BPlhNab50. The sequestration of CUG-BPI hNab50 and decreased levels of DMPK affecting processing and transport of specific subclasses of mRNAs may be, in part, molecular pathophysiological mechanisms of DM. The excessive binding to the extended CTG repeats would create a shortage of transport proteins required for cytoplasmic localization of many RNAs. This could account for the discrepancy seen between normal total RNA and decreased to poly (At RNA levels and would explain the trans-dominant effect of the mutant gene. This model could also explain the multi systemic nature of the disease and the dramatic clinical variability. Various cells and tissues would be affected in different manners, depending on which subset of RNAs was involved in each cell type.

3 Localization of DMPK It is important to know the exact localization of DMPK in normal tissues in order to understand the physiological functions of DMPK. The predicted molecular weight of the translational product of the longest DMPK mRNA coding region is 71,100 daltons. It has been reported that there are at least two DMPK isoforms: -70 kDa full-length and - 50 kDa truncated forms (TIMCHENKO et al. 1995; SHIMOKAWA et al. 1997) in human skeletal muscle. Furthermore, multiple splicing forms of DMPK mRNA have been also described (Fu et al. 1993; CARANGO et al. 1993; HOFMANN-RADVANYI et al. 1993a; HOFMANN-RADVANYI and JUNIEN, 1993b; NOVELLI et al. 1993). So far polyclonal and monoclonal antibodies raised against a variety of epitopes in the DMPK sequence by different investigators have recognized a range of polypeptides species from 42 kDa to 84-kDa (MAHADEVAN et al. 1993; van der VEN et al. 1993; DUNNE et al. 1994; TIMCHENKO et al. 1995; BUSH et al. 1996). Different sizes of immunoreactive polypeptide appear to be specific to certain tissues such as 70 and 55 kDa DMPK in skeletal muscle (SHIMOKAWA et al. 1997) and 84-85 kDa DMPK in heart (BUSH et al. 1996). These different sizes of DMPK immunoreactive proteins are probably products of splice variants (JANSEN et al. 1992) or differently initiated translations (TIMCHENKO et al. 1995), variably phosphorylated or posttranslationally modified forms, or products of a very closely related gene. In this chapter, we describe the localization of DMPK in skeletal and cardiac muscles, the central nervous system, the lens, and various other tissues.

Myotonic dystrophy/Myotonic dystrophy protein kinase . 207

3.1 Skeletal muscle 3.1.1 DMPK molecules in skeletal muscle

On the basis of the eDNA sequence, human DMPK protein was previously reported to have two amino acid sequence variations in the C-terminus (Fu et al. 1992; BROOK et al. 1992; SASAGAWA et al. 1994). Using two specific antibodies against the C-terminus or mid-portion of human DMPK, a full-length protein of 70 kDa is detected in the membrane-rich fraction and a truncated protein of 55 kDa in the soluble fraction of human skeletal muscle (Fig.5). In our recent study an DMPK antibody against the Nterminus recognizes the 70 kDa protein, but not the 55 kDa one. These data suggest that the 55 kDa protein is an N-terminus truncated form of DMPK protein. WHITING et al. (1995) and MAEDA et al. (1995) reported protein species of 71 and 80 kDa and -74 and - 82 kDa in whole tissue extracts from human skeletal and cardiac muscles, respectively. Previous studies have reported the molecular mass of DMPK as 42-62 kDa (Fu et al.

23123

.-

KOa

-94

-43

-30 M

c

Fig.S. Western blot analysis of DMPK in human skeletal muscle homogenates. Full-length DMPK is detected with two different anti-DMPK antibodies that recognize the midportion of DMPK (M: left three lanes) and C-terminus of DMPK (C: right three lanes) as an immunolabeled 70-kDa band in human skeletal muscle homogenate (each lane 3). Each lane 1: negative staining of DMPK in COS-l cells; each lane 2: DMPK expresses in cDNA transfected COS-l cells; and each lane 3: human skeletal muscle homogenate.

208 . H. Ueda et al.

1993; VAN DER VEN et aL 1993; SALAVATORI et aL 1994; KOGA et aL 1994; BREWSTER et aL 1993). These DMPK proetins in skeletal muscle in previous reports may represent either truncated forms or degradation products of full-length DMPK. 3.1.2 Predominant DMPK expression in type I muscle fibers

Myosin and actin are major muscle structure proteins. Skeletal muscle is divided into two types: type I and II, based on their contraction patterns. Type I muscle contains slow myosin heavy chain (MHC), and type II contains fast MHC. Low-power views of immunofluorescent double labeling with DMPK and slow or fast MHCs show that DMPK is exclusively colocalized in type I muscle fibers (KOBAYASHI et aL 1997; Fig. 6). The intensity of DMPK labeling of type I muscle fibers is variable (strong, moderate and weak in Fig. 6). It is well known that ATP-dependent calcium pumps in sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) are responsible for the maintenance of low free Ca2+ concentration in cytoplasm. The ATP pumps are encoded by a family of structurally related enzymes and termed the sarcoplasmic or endoplasmic reticulum calcium ATPases (SERCA). The SERCA I gene is expressed in type II (fast) skeletal muscle. SERCA II gene is subject to tissue-dependent processing that is responsible for the generation of SERCA lIa in type I skeletal, cardiac and smooth muscles and the SERCA lIb, which is expressed in all muscle types. DMPK is exclusively colocalized with SERCA IIa ATPase (Fig. 7). DUNNE et aL (1996) reported that a monoclonal antibody, which recognized the 64 kDa isoform of DMPK, detected DMPK protein specifically in type I muscle fibers. VAN DER VEN et aL (1993) and SALVATORI et aL (1994) also reported type I-dominant immunostaining using different antibodies against DMPK. It is well known that type I muscle atrophy is one of the significant finding of DM muscle pathology. DMPK might have different physiological roles among subclasses of type I muscle fibers, because type I muscle fibers with equal concentrations of slow MHC and SERCA II ATPase have different concentrations of DMPK. Furthermore, excitation-contraction coupling (EC-coupling) proteins such as ryanodine receptors (RyRs) and dihydropyridine receptors (DHPRs) are predominantly localized in type II muscle fibers (TANAKA et aI., unpubL). Thus DMPK is unigue among EC-coupling-related proteins because it is predominantly localized in type I muscle fibers. 3.1.3 DMPK localization in the terminal cisternae of sarcoplasmic reticulum

High-power views of immunofluorescent double labeling of DMPK and slow MHC in a longitudinal section of human skeletal muscle show that a binding pattern with a doublet of DMPK exists between a cross-striated pattern of slow MHC (Fig. 6). This finding indicates that DMPK is localized in I bands. In adult muscle fibers, DMPK is

Myotonic dystrophy/ Myotonic dystrophy protein kinase' 209

Fig. 6. Double immunofluorescent labeling ofDMPK (a, d, g) and slow (b, h) and fast (e) MHCs in adult skeletal muscle. c, f and i: Simultaneous double-immunofluorescent photomicrographs of DMPK/siow (c and i) and fast MHCs (f). Low power views (a-f) show that DMPK staining is completely negative in fast muscle fibers (d-f), and DMPK exclusively expressed in slow muscle fibers (a-c). The intensity ofDMPK in slow muscle fibers is strong (arrows), moderate (arrowheads) and weak (double arrowheads). In high-power views of a longitudinal section of human skeletal muscle, DMPK is detected (g) between cross-striations of slow MHC (h). No colocalization of DMPK and slow MHC is observed (i). Bars: 100 !!min (a-f) and 10 !!min (g-i). (From KOBAYASHI eta1.1997)

210 . H. Veda et al.

Fig. 7. Double immunofluorescent labeling of DMPK (a, d) and SERCA II ATPase (b, e) on adult skeletal muscle. c and f: Simultaneous double immunofluorescent pictures of DMPK/SERCA II ATPase. Low power views (a-c) show that DMPK is colocalized in SERCA II ATPase-positive muscle fibers. The intensity of DMPK is strong (arrows), moderate (arrowheads) and weak (double arrowheads) in SERCA II ATPase-positive muscle fibers (a-c) similar to slow muscle fibers in Fig. 6. In high-power views, a binding pattern of DMPK (d) is detected between a cross-striated pattern of SERCA II ATPase (e), and no clear colocalization of DMPK and SERCA II ATPase is observed (f). Bars: 100 [-tm in (a-c) and 10 [-tm in (d-f). (From KOBAYASHI et al. 1997)

colocalized with myosin in the border of both bands. SERCA ATPases are known to be localized in longitudinal SR, which exists in I bands. A banding pattern of DMPK also exists between SERCA II ATPase, and no colocalization is detected (Fig. 7). Immunoelectron microscopy clearly demonstrates that DMPK is localized in the terminal cisternae of the SR, but not in the longitudinal SR (Fig. 8). At higher magnification, diffusion of immunoreactive products into the lumen of the terminal cisternae suggest that DMPK is localized at the junctional face of the terminal cisternae. SALAVATORI et al. (1994) reported that DMPK is a membrane-bound protein, especially localized in the SR heavy fractions, although their DMPK molecular mass was 55 kDa. More recently SALVATORI et al. (1997) reported that DMPK is localized both in the surface membranes and within the skeletal muscle fibers at the A-I band boundary. In addition, Western blot analysis from purified SR fractions showed that triads and the

Myotonic dystrophy/ Myotonic dystrophy protein kinase' 211

terminal cisternae of SR are immunoreactive for 85 and 54 kDa DMPK proteins. Furthermore, when a full-length DMPK eDNA is transfected into a cultured rat L6 cell line, about 95 % of DMPK is recovered from the light microsomal fraction (SAITOH et al. 1996). Recently, a native full-length DMPK was purified from rat skeletal muscle SR (KOIKE et al. 1998). These biochemical studies confirm that DMPK is a SR protein, which is specifically localized in the terminal cisternae of SR in skeletal muscle. 3.1.4 Localization of DMPK in the neuromuscular junction

Fig. 9 shows that DMPK is localized in an a-bungarotoxin (a-BGT) binding site in adult human tissue. Several previous studies reported that DMPK was localized predominantly in neuromuscular junctions and myotendinous junctions in adult tissues (VAN DER VEN et al. 1993; MAEDA et al. 1995; TACHI et al. 1995; WHITING et al. 1995; DUNNE et al. 1996). However, PHAM et al. (1998) reported that co-localization of DMPK

Fig. 8. Localization of DMPK in the terminal cisternae of the sarcoplasmic reticulum in human skeletal muscle. Confocal images show a striated pattern at a low magnification (a). The highpower field demonstrates that a striation consists of a doublet (a, inset). Immunoelectron micrograph shows that DMPK is localized in the terminal cisternae of the sarcoplasmic reticulum (arrows in b). Asterisks indicate terminal cisternae. M, mitochondria; T, transverse tubule; Z, Z line. Bar in (a): 10 flm; bar in inset, 3 flm; bar in (b): 200 nm.

212 . H. Veda et al.

Fig. 9. Double immunofluorescent labeling of DMPK (a) and a-BGT (b) on adult skeletal muscle by confocal laser scanning microscopy. In adult skeletal muscle, a cross-striated pattern of DMPK is observed in a longitudinal section (a), and the varicose pattern of DMPK (arrow in (a)) is colocalized with the exact same pattern of a a-BGT binding site (arrow in (b)). Bars: 10 ~lm. (From KOBAYASHI et al. 1997)

with acetylcholine receptors at neuromuscular junctions was not observed with their panel of monoclonal antibodies raised against the catalytic and coil domains of DMPK, which could detect a major 55 kDa and minor 72-80 kDa proteins on Western blots of human skeletal muscle. Abnormalities of the neuromuscular junction were previously reported in biopsied materials from extrafusal muscle fibers of DM patients (Me DERMOT, 1961; ENGEL and BROOKE, 1966; COERS et al. 1973). Existence of apamin receptors in DM muscle (RENAUD et al. 1986) may cause repetitive discharge of DM muscle (appearance of myotonia), and the remaining apamin receptors in DM muscle might be one of the phenomenon of immature DM skeletal muscle due to defective innervation. Investigation of the exact ultrastructural localization of DMPK in neuromuscular junctions is important to understand the functional role of DMPK in synaptic signal transmission and maturation of muscle fibers.

Myotonic dystrophy/ Myotonic dystrophy protein kinase · 213

3.1.5 DMPK localization in the muscle spindle

DMPK is also localized in the intrafusal muscle fibers of muscle spindles (Fig. 10). Other studies using different antibodies to DMPK reported that DMPK was detected in muscle spindles (VAN DER VEN et al. 1993; TACH! et al. 1995). Abnormalities of the intrafusal fibers in DM have been reported (DANIEL and STRICH, 1964; SWASH, 1972). The hypotonia, that frequently occurs in cDM patients, might be due to the dysfunction of muscle spindles.

Fig. 10. Localization of DMPK in a human skeletal muscle spindle. Semiserial cross-sections of biopsied DM skeletal muscle are stained with HE (hematoxylin-eosin) (a), histochemical staining of myofibrillary ATPase (pre-incubation pH 4.2) (b), immunohistochemical stainings of slow (c) and fast (d) MHCs and DMPK (e). DMPK is localized in intrafusal muscles of a muscle spindle (arrow). Bars: 100 !-1m.

214 . H. U eda et al.

3.2 Cardiac muscle Sudden death is the most serious complication in DM patients, and it is often caused by either complete atrioventricular blocks or ventricular arrhythmias (MOTTA et al. 1979; HIROMASA et al. 1988; PHILLIPS and HARPER, 1997). Our immunoblot data show that 70 kDa and 55 kDA protein bands of DMPK are detected in human cardiac muscle (VEDA et al. 1998). So far variable molecular weights for DMPK, including 62 kDa (KOGA et al. 1994),70 kDa (SASAGAWA et al. 1994),74 kDa (WHITING et al. 1995), two 80 and 71 kDa isoforms (MAEDA et al. 1995),85 and 54 kDa (SALVATORI et al. 1997), and a 72-80 kDa doublet (PHAM et al. 1998), have been reported. 3.2.1 DMPK localization in intercalated discs and Purkinje fibers

On the cellular level, DMPK in cardiac muscle has been demonstrated to locate at intercalated discs and Purkinje fibers in the heart by the light microscopy (MAEDA et al. 1995; SALVATORI et al. 1997; VEDA et al. 1998; PHAM et al. 1998). Confocal images of DMPK show a striated banding pattern in common cardiac working myofibers and impulse-conducting myofibers similar to that in skeletal muscles (Figs. llA and B). DMPK immunoreactivity is strongly recognized around intercalated discs, while that of both SERCA II and ryanodine receptor is not strong there. The intercalated disc is a unique structure in cardiac muscle cells, containing numerous fasciae adherens, desmosomes and gap junctions. The gap junctions are composed of connexins, and their channel pore facilitates the permeability of ions and other small molecules. The presence of DMPK on both sides of the intercalated disc suggests that it may play an important role in signal transduction and may be related to the cardiac conduction abnormalities in DM patients. 3.2.2 DMPK localization in corbular and junctional SRs

Cardiac myofibers are classified as two types: common cardiac myofibers and impulse-conduction myofibers. The only significant structural difference between the two in mammals is the absence of a T-tubule system in the conduction myofibers (SOMMER and JOHNSON, 1968). The cardiac Tor SR system is different from that of skeletal muscle. The T-tubules are located around a Z band and often run axially, the so-called transverse-axial tubular system (TATS), and are generally larger than those of skeletal muscle. Moreover, cardiac SR has been classified into three domains: network SR, junctional SR and corbular SR in terms of their morphological features. The network SR comprises anastomosing networks above the Z or M line, called Z or M retes, which share SERCA and phospholamban in uniform distribution (J ORGENSEN et al. 1982; J ORGENSEN and JONES, 1987). The junctional SR, located nearly above the Z band is sorted into the interior junctional SR, which encircles T-tubules partially or completely, and the

Myotonic dystrophy/Myotonic dystrophy protein kinase . 215

z

z

'. ,

c Fig. 11. Localization of DMPK in human cardiac muscle. Confocal images of common cardiac working fibers (a) and impulse-conducting fibers (b) show a banding pattern similar to human skeletal muscle. Thick labeling bands (arrows in a and b) are observed along intercalated discs. No significant differences are recognized between common cardiac working and impulse-conducting fibers. Immunogold labeling shows that DMPK is detected in the corbular sarcoplasmic reticulum in human cardiac muscle (arrowheads in c). Z, Z line. Bars: in (a and b): 10 [.lm; bars: in (c): 200 nm.

peripheral junctional SR, which often couples to the sarcolemma (FORBES and VAN NIEL, 1988; OGATA and YAMASAKI, 1990). The corbular SR is abundant in all cardiac myofibers, especially in impulse-conduction and atrial myofibers. The corbular SR, containing electron-dense materials and independent of the T-tubular system, is characteristic of cardiac muscle myofibers. The primary structural difference between junctional SR and corbular SR is that the junctional SR is physiologically connected to either a Ttubule or sarcolemma via 'feet' structures, whereas the corbular SR is not. Thus, it is suggested that Ca2+ release from the junctional SR might be triggered by signals depending on the physical contact with sarcolemma via feet structures, while corbular SR might be triggered to release Ca2+ by a diffusible agent ORGENSEN et al. 1985, 1993). Ultrastructurally, DMPK is expressed mainly in corbular and junctional SR (Fig. 11 b) but not in network SR. Moreover, DMPK is associated with junctional SR closely opposed to T-tubules in interior regions (UEDA et al. 1998). Around the intercalated disc most immunoreactive products of DMPK are recognized at various distances from the

a

216 . H. Ved a e t al.

A

B Fig. 12. Drawings representing DMPK localization (red) in skeletal (A) and cardiac (B) muscles. Arrows indicate corbular SR and arrowheads indicate junctional SRi M, M-line; Mt, mitochondria; SR, sarcoplasmic reticulum; T, transverse tubule; Z, Z line.

Myotonic dystrophy/Myotonic dystrophy protein kinase· 217

intercalated disc, but some are localized in its neighbourhood. At a higher magnification, DMPK are recognized away from the fascia adherens. Electrophysiological studies have demonstrated that the His-Purkinje system is damaged in DM patients, and this impulse-conduction disturbance becomes progressively worse with age (PRYSTOWSKY et al. 1979). It has been also demonstrated that an increased CTG repeat length is correlated with the severity of conduction abnormalities (MELACINI et al. 1995), although the relationship between DMPK expression and clinical phenotype is still not clear. Fig. 12 depicts DMPK localization in skeletal and cardiac muscles.

3.3 Central nervous system Cognitive disturbances, mental retardation, sleep disorder, behavioral changes, and difficult personalities can be recognized in adult-type DM patients (BUNGENER et al. 1998; DELAPORTE et al. 1998). Moderate to severe mental retardation and severe muscle hypotonia are frequently observed in cDM patients. Thus it is important to know the exact localization of DMPK in the central nervous system. Recently, BALASUBRAMANYAM et al. (1998) characterized the localization and developmental expression of DMPK protein in rat brain and spinal cord using a monospecific rabbit antiserum produced against bacterially expressed DMPK. Their DMPK antibody, which recognizes an epitope in the a-helical "coiled-coil" region of DMPK, detected a 53 kDa band in protein extracts from adult rat spinal cord and cerebellum, and a 51 kDa band in cerebral cortex extracts. WHITING et al. (1995) reported a similar molecular weight protein in rat brain extract. VAN DER VEN et al. (1993) reported that DMPK was detected in mouse cerebellar Purkinje cells and in cells of Ammon's horn and stated that "most neurons" were stained by their DMPK antibody. Furthermore WHITING et al. (1995) described the tissue localization of a "41-50 kDa" DMPK isoform in adult rat brain using a polyclonal antibody raised against the portion of the DMPK polypeptide from amino acid 471-629 "coiledcoil" and transmembrane regions. They reported that DMPK was detected in the hippocampus, cerebellum and cerebral cortex of adult rat. Recently BALASUBRAMANYAM et al. (1998) reported that DMPK was detected in adult brain except in the basal ganglia, the thalamus, and the amygdala. In the spinal cord, motor neurons of the ventromedial region are the most DMPK-immunoreactive. In the large neurons of the spinal cord, the immunoreactivity appears exclusively in the cytoplasm. Staining in dendrites is usually confined to the proximal portion. In the midbrain and hindbrain, there is prominent immunostaining in neurons of the nuclei of cranial nerves III and IV and in the mesencephalic nucleus. The staining in the cells of the mesencephalic trigeminal nucleus is particularly intense. DMPK is not observed in any thalamic nuclei or in the inferior or superior colliculi. Staining is also absent in the cen-

218 . H. Veda et al.

tral gray matter and in the deep mesencephalic nuclei. In the cerebellum, there is light but specific immunostaining in the Purkinje cell layer. There is no apparent staining in the molecular layer, the granule layer or the white matter of the cerebellar cortex. However, there is prominent staining in all of the cerebellar nuclei. In the cerebral neocortex, neurons throughout all cortical areas and in all layers of the neocortex are very lightly immunoreactive to DMPK, but populations of neurons located in layers II and V are stained prominently. In the hippocampus and entorhinal cortex, there is very little specific staining in the hippocampal formation in the adult. Electron microscopically, DMPK is detected within the somata in aggregates of rough ER in all DMPK- positive neurons. Proximal and distal dendrites have immunostaining distributed irregularly throughout the cytoplasm. In the proximal dendrites, DMPK is mainly detected in the vicinity of microtubules. DMPK is not observed in Golgi, mitochondria or lysosomes.

3.4 Other tissues

Cataracts are a common complication of DM. ABE et ai. (1999) examined the expression of the mutated allele of the DMPK gene in the lens epithelium of patients with DM or senile cataracts and also studied lens epithelial cell changes in DM patients. Lens epithelial cell densities are extremely reduced in DM patients but DMPK mRNA is detected in the lens epithelium. However, no information on DMPK protein expression in lens has been reported so far. DMPK is also detected in pancreas (Fu et ai. 1993). Various muscle cell lines (508 cells, C2 cells, C3H), non-muscle cells (10T1/2 cells) and dividing myoblasts have been reported to have certain levels of DMPK transcript (LAURENT et ai. 1997). Human normal and DM fibroblasts express DMPK protein (Fig. 3, SHIMOKAWA et aI., unpubI.). DMPK is localized in the cytoplasm of these cells, but the precise subcellular localization is not examined yet.

4 Developmental localization of DMPK So far there have been a few reports concerning the developmental expression of the DMPK gene and protein during muscle development in vivo and in vitro (LAURENT et ai. 1997; KAMEDA et ai. 1998) and in rat brain in vivo (BALASUBRAMANYAM et ai. 1998).

Myotonic dystrophy/Myotonic dystrophy protein kinase' 219

4.1 Developmental localization of DMPK in skeletal muscle 4.1.1 Developmental expression of DMPK gene in muscle cells in vivo and in vitro

Using RNA in situ hybridization, JANSEN et al. (1996) have shown that DMPK transcripts are absent from limb buds of 10. 5-day-old mouse embryos, but present in all skeletal muscle at 18. 5 days. LAURENT et al. (1997) also studied the DMPK mRNA level in mouse limb tissues during development and cultured muscle cells at different developmental stages in comparison with the mRNA levels of the well documented basic helix-loop-helix myogenic factors (Myf5, Myogenin, MyoD and MRF4). In in vivo experiments, DMPK transcripts are detectable at 10. 5 day post-coitum, increase until delivery and remain stable after birth in parallel with the muscle regulatory factor MRF4 mRNA. In vitro DMPK mRNA expression tentatively decreases at 6-8 days in culture and begins to increase from 9-10 days with the suppression of MyoD mRNA, when the muscle contraction starts. However, this upregulation of DMPK does not depend on transfected muscle-specific basic helix-loop-helix factors. Thus, DMPK mRNA expression occurs mainly as a results of muscle differntiation.

4.1.2 Developmental expression of DMPK gene in human muscle cells by innervation in vitro

Muscle fibers mature and function by precise stimulation from motor neurons (innervation) in vivo. To examine the successive developmental processes of DMPK localization in human muscle, we co-cultured human muscle cells in a monolayer with explants of fetal rat spinal cord with attached dorsal root ganglia (KOBAYASHI et al. 1987, 1995). This co-culture system is a very suitable model for investigating the developmental expression of DMPK at several stages of human muscle development including mononucleated cells (myoblasts), aneurally cultured non-contracting human muscle cells and mature innervated muscle fibers with cross-striations. In this co-culture system, innervated continuously contracting human muscle fibers become more mature than aneural muscle cells morphologically (ASKANAS et al. 1987; FURUYA et al. 1991; KAMEDA et al. 1993; KOBAYASHI et al. 1987, 1995), biochemically (MARTINUZZI et al. 1986, 1987, 1988), and electrophysiologically (KOBAYASHI et al. 1990b; MIYAZAKI et al. 1990; SAITO et al. 1990; MICHIKAWA et al. 1991). In the following sections we describe the developmental expression and localization of DMPK mRNA and protein in muscle cells and neuromuscular junctions using this co-culture system.

220 . H. Ueda et al.

4.1.2.1 Developmental expression of DMPK and AChR subunit mRNAs in cultured human muscle cells From our reverse transcription-polymerase chain reaction (RT-PCR) studies of DMPK and AChR subunit transcripts assayed chronologically in aneural cultures, short- and long-term co-cultures, DMPK mRNA is expressed in all aneural and innervated stages (Fig. 13). There are no differences in DMPK mRNA expression between contracting and non-contracting cultures in either short- or long-term co-culture. Interestingly, short-term co-cultures express less DMPK mRNA than aneural cultures, while long-term co-cultures show more expression of DMPK mRNA compared with aneural

R

1 2 3 4 5

------..............

--

B

DMPK 63PDH AchR c AchR'i

-~--

G3PDH

Fig. 13. Developmental changes of DMPK (A) and AChR subunit transcripts (B) in aneural and innervated cultures. G3PDH is used as a positive control. Agarose gel showing the ethidium bromide stained products of RT-PCR. Lane 1, aneural culture (41 days of culture); lane 2, early innervation culture without muscle contractions from 20 days of muscle culture alone and 21 days of co-culture with spinal cord; lane 3, early innervation culture undergoing continuous muscle contractions from 27 days of muscle culture alone and 31 days of co-culture with spinal cord; lane 4, long-term innervation culture without muscle contractions from 17 days of muscle culture alone and 59 days of co-culture with spinal cord; lane 5, long-term innervation culture undergoing continuous muscle contraction from 17 days of muscle culture alone and 59 days of co-culture with spinal cord. DMPK mRNA expresses in aneural and innervated cultures. Whereas aneural cultures have strong expression of y-subunit mRNA and no expression of E-subunit mRNA, weak but clear expression of E-subunit mRNA is detected in early co-culture without muscle contractions (line 2). Strong expression of y-subunit mRNA and decreased expression of E-subunit mRNA appear in short-term co-culture with continuous muscle contractions at about 17 days (line 3). In long-term co-culture, this reciprocal expression of y-subunit mRNA and E-subunit mRNA is more enhanced (line 5) than in short-term co-culture with continuous muscle contractions, whereas only weak expression of E-subunit mRNA is detected in long-term co-culture without muscle contractions (line 4).

Myotonic dystrophy/Myotonic dystrophy protein kinase . 221

cultures or short-term co-cultures. This temporal decrease of DMPK expression in short-term culture may be the reason that DMPK is expressed diffusely through the cytoplasm of all aneural muscle cells; however, after cells are innervated and start to contract, DMPK becomes restricted to the terminal cisternae of SR in the muscle fibers, that will become the adult type of slow muscle fibers (KAMEDA et al. 1993). 4.1.2.2 Developmental localization of DMPK in cultured human muscle cells

Immunoblot of DMPK detects an approximately 65 kDa DMPK in aneurally cultured muscles. From our developmental studies in vitro, mononucleated cells are not DMPK-positive in very immature aneural cultures and only a few myotubes with several nuclei have both DMPK and myosin. In immature aneural culture, DMPK is located at the center of the myotubes, while myosin is located at the periphery and sometimes encircle (Fig. 14). In more mature aneural culture, DMPK is partially positive in a crossstriated pattern in some myotubes. Innervated contracting muscle fibers have both DMPK and myosin, but the staining intensity of DMPK is weak compared with that in aneural cultures. In short-term coculture, myosin is observed in a cross-striated pattern in almost all continuouscontracting muscle fibers, but DMPK expression appears faint. Therefore, the arrangement of structural proteins such as myosin and actin precedes that of the DMPK. In long-term co-culture with continuous muscle contractions, DMPK shows a clear crossstriated pattern with doublets between the myosin positive bands (Fig. 15). Less than 10% of contracting cross-striated muscle fibers have a clear cross-striated pattern of DMPK. In this co-culture system, aneural muscle cells have mainly fetal type MHCs (the percentage of adult slow-positive muscle cells is about 0.9 %), and after innervation, 8.3 % of innervated muscle fibers become adult slow-MHC positive, which probably corresponds to adult type I muscle (KAMEDA et al. 1993). In adult human muscle, DMPK is detected in type I (slow-MHC positive) muscle (VAN DER YEN et al. 1993; DUNNE et al. 1996; KOBAYASHI et al. 1997). In our co-culture system, less than 10 % of innervated muscle fibers have cross-striated bands of DMPK after innervation. These muscle fibers probably correspond to adult type I muscle fibers. The transient decrease of DMPK mRNA in short-term co-culture might be related to the dramatic change in DMPK from the fetal type in aneural muscle cells to the adult type in cultured innervated muscle fibers, in which differentiation from fetal type to adult slow and fast types occurs. Ultrastructural localization of DMPK differs greatly between aneural and innervated human muscle cells. In immature aneual myotubes, immunoproducts against DMPK are observed as linear or dense deposits on some thin membrane sacs in the cytoplasm. DMPK-positive membranous organelles are seen among mitochondrial clusters. The formation of the Z line is not clear. In some portions of these membranous structures, DMPK immunoproducts is observed as narrow duct-like canals. In these areas, association with myofibrils is rare. After innervation, the myofilaments are regularly arranged

222 . H. Veda et al.

Fig. 14. Double immunofluorescent labeling of DMPK (red) and myosin (green) in aneurally cultured human muscle cells. DMPK is diffusely observed in an aneurally cultured spindle-shape myotube and myosin is detected in the periphery of the myotube (a). In a relatively mature myotube, a partially cross-striated pattern of myosin is detected. In this myotube DMPK and myosin are not co-localized (b). The staining of DMPK is basically homogeneous, but in some areas it is finely pigmented in the cytoplasm. Nuclei are identified as negative DMPK staining (c). In a more mature myotube, a cross-striated patttern of myosin staining is observed. DMPK is centrally located, and a small part of the DMPK-positive area shows a striated pattern (arrow in d). Twentynine days of culture. Bar, 10 [lm. (From KAMEDA et al. 1998)

Fig. IS. Double immunofluorescent labeling of DMPK (a)/myosin (b) and F-actin (d)IDMPK (e) in cultured innervated human muscle fibers. Panels c and f show simultaneous double immunofluorescent pictures of DMPK/myosin and F-actin/DMPK. DMPK (a, e) and myosin (b) and F-actin (d) are in cross-striated labeling patterns. DMPK shows a doublet pattern [arrows in a and e are DMPK negative bands between DMPK-positive doublets (arrowheads in a and e)]. The simultaneous double fluorescent picture of DMPK/myosin (c) shows DMPK immunostaining in periodic patterns between myosin bands. In the borders of the myosin and DMPK bands, small colocalized areas of myosin and DMPK (yellow) are sometimes detected (arrows in c). Co-culture for 40 days. Bars: 10 [lm. (From SHIMOKAWA et al. 1997)

Myotonic dystrophy/Myotonic dystrophy protein kinase' 223

224 . H. Ueda et al.

at the sarcomere. Some membranous structures become apparent between the bundles of myofibrils. Immuno-positive membranous sacs are detected in some of these membranous structures, which are sometimes closed and frequently located in I bands. In some portions, the membranous structures are transversely oriented at the A-I border of the sarcomere. In long-term co-cultures, DMPK immunoreactivity is localized at the edge of membranous sacs located near the border of the A and I bands. In muscle fibers with well-arranged myofibrils, DMPK immunoreactivity appears as small round sacs near the A-I borders. It has been reported that T-systems in innervation cultures are formed 2 to 3 weeks after innervation and become relatively mature after 6 to 7 weeks of innervation (ASKANAS et al. 1987). Positive staining for DMPK in very similar structures in the terminal cisternae of the SR in continuously contracting muscle fibers is observed after more than 10 weeks of innervation. Therefore, the distinctive differences between the ultrastrsuturallocalizations of DMPK in aneural and innervated cultures are that DMPK-positive intramembranous structure appear more regularly in the I band region, and some small membrane organelles with positive DMPK become apparent in the triad region of innervated contracting muscle fibers in long-term co-cultures. It is believed that SR differentiates from ER. FLUCHER et al. (1993) reported that the association of the SR with myosin myofibrils was an early event in the periodic organization of the membrane system. Ultrastructural analysis suggests that the SR is derived from the ER at the organizing Z line. DMPK is expressed in early immature myotubes, suggesting that DMPK appears in the ER before the formation of the SR. Developmentally, DMPK is distributed diffusely in a partially granular pattern in the cytoplasm of aneural muscle cells and is less associated with myosin, while DMPK is organized at the I band and A-I junction in innervated contracting muscle fibers, so the organization of DMPK in the SR might be under neuronal control. In human muscle cultures, aneurally cultured myotubes rarely contract, but after innervation, a bundle of continuously contracting muscle fibers with miniature endoplate potentials and endoplate potentials appears (MICHIKAWA et al. 1991). The contraction of muscle fibers might be an important factor in the organization of DMPK in the SR. We previously observed that the distribution of dystrophin is influenced by muscle contractile activity (PARK-MATSUMOTO et al. 1991), so innervation affects the distribution of not only cytoskeletal proteins, but also the intramembranous proteins. In our developmental studies in vitro, DMPK appears on ER or SR during the very early stages of the organization of intracellular membranous structures and then localizes in the terminal cisternae of the SR.

4.1.2.3 Localization of DMPK in neuromuscular junction in vitro Motor neurons in the spinal cord not only induce muscle contractions, but also regulate the expression of many crucial proteins that are associated with synaptic transmission and mechanical activity (contractions). In particular, the nicotinic AChRs are especially sensitive to neurtrophic controls. AChRs are present predominantly at the

Myotonic dystrophy/Myoto nic dystrophy protein kinase' 225

neuromuscular junction in adult muscle, whereas AChRs are also found in the nonjunctional membranes in fetal muscle, as well as in denervated adult muscle (CHANGEUX et al. 1981; CONTI-TRONCONI and RAFTERY, 1982). Molecularly genetically different forms of the nicotinic AChR occur in mammalian skeletal muscle during development. A transient form of the AChR in fetal muscle consists of a2, ~, Yand 6 subunits, whereas in adult muscle the y subunit is replaced by the f-subunit (TAKAI et al. 1985; MISHINA et al. 1986). A switch from the y-subunit to f-subunit mRNA also occurs in rat diaphragms denervated by cutting phrenic nerve and reinnervated after mechanical crushing (WITZEMANN et al. 1987, 1991). We studied the expression of AChR subunit mRNAs with the same cultures used to examine the expression of DMPK mRNA in vitro (Fig. 13). High expression of ysubunit AChR mRNA was observed, and f-subunit AChR mRNA was not detected in aneural cultures. After innervation, f-subunit AChR mRNA appeared in both continuously contracting and non-contracting cultures. Interestingly, f-subunit mRNA increased and y-subunit mRNA decreased in longer-term co-culture containing well developed cross-striated muscle fibers with continuous contractions. However, in noncontracting co-cultures, weak expression of f-subunit AChR mRNA and relatively high expression of y-subunit AChR mRNA was sustained. This observation clearly shows that the initial appearance of f-subunit AChR mRNA is dependent on innervation, although further switching from y-subunit to f-subunit AChR mRNA occurs by muscle contractions. Immunofluorescent double labeling of DMPK and a-BGT show that a linear or varicose pattern of DMPK is colocalized with a cluster of AchR (a-BGT binding sites), where a nerve terminal is detected using phase-contrast microscopy (Fig. 16).

4.2 Developmental aspects of DMPK in CNS in vivo Studies of developing mouse embryos have indicated that DMPK mRNA is present symmetrically and segmentally within the neural crest, specifically in the myotomes, as early as day 10.5. All smooth and striated muscles express DMPK mRNA at substantiallevels by day 18.5, at which stage DMPK mRNA is seen in cardiomyocytes. The DMPK expression occurs later in the CNS than in other tissues GANSEN et al. 1996). BALASUBRAMANYAM et al. (1998) examined developmental changes of DMPK in rat brain from birth to 28 days postnatal (P28). No specific DMPK immunostaining has detected before P7 in any brain region, but the expression increased during the 2nd and yd postnatal weeks, peaking by P21. The intensity of the staining then decreased to adult levels by P28. These developmental changes of DMPK expression are most prominent in the spinal cord, but similar temporal changes are also observed to a lesser extent in all CNS areas. The newborn-rat spinal cord shows no DMPK immunoreaction. Positive immunostaining could be detected by the end of the 15t postnatal

226 . H. Ueda et al.

Fig. 16. Double immunofluorescent labeling of DMPK (a) and a-BGT (b) in innervated cultured human muscle cells. c: A simaltaneous image of double immunofluorescent pictures of DMPKI a-BGT and phase-contrast photomicrographs (d) by confocal laser scanning microscopy. The exact same co-localization of DMPK (a) and a-BGT binding site (b) is observed in the triple immunofluorescent (c) and phase contrast image (d), in which a nerve terminal is clearly detected (arrowheads). Co-culture was for 38 days. Bars: 10 ftm. week and by P21, DMPK is detected in all neuronal cell types, particulary in the large motoneurons of the ventral spinal cord. The pattern of staining appears to be entirely neuronal, even in the first postnatal weeks. DMPK is present in the cytoplasm and proximal dendrites throughout development, similar to adults. At P28, there is a striking change in the pattern and intensity of DMPK immunostaining. Staining intensity significantly diminishes, and the distribution of the immunostain is restricted to neurOns in the ventral horn region. The immunostaining characteristics of the P28 spinal cord persist into adulthood. The hippocampus and cerebral cortex show the same developmental changes. DMPK immunostaining becomes apparent at P7. There is a marked decline in the intensity of immunostaining at P28, and this pattern persists into adulthood. In both the hippocampus and the cerebral cortex, there is nO change in the location or type of cells that are immunostained across the developmental period. In the cerebellum, immunostaining appears betweeen P7 and P14 and does not seen decline after P21, in contrast to the hippocampus and cerebral cortex. Specific staining

Myotonic dystrophy/Myotonic dystrophy protein kinase· 227

occurrs primarily in the cell bodies of Purkinje neurons, but it is also present in some cells in the granule cell layer. From P14 onward, the staining intensity does not change as significantly as that in the spinal cord does, but it appears to be more restricted to Purkinje cells. At all ages, the intensity of staining is much less in the cerebellum than in other CNS regions. These developmental observation are consistent with recent data on the temporal sequence of DMPK mRNA expression in mice. In situ hybridization produces no signal in the brains of rat embryos and neonates, and the earliest detection of signal is at 14 days of age in the cerebellum and hippocampus GANSEN et al. 1996)

5 Functions of DMPK 5.1 DMPK protein belongs to a novel subfamily of serine-threonine protein kinases Sequence analysis reveals homology between DMPK and members of a subfamily of the cyclic AMP-dependent serine/threonine protein kinases (SHAW et al. 1993; DUNNE et al. 1994; TIMCHENKO et al. 1995). Members of this family are present in a diverse variety of organisms. The greatest amino acid sequence similarities of the catalytic domains of human DMPK have been found in genes such as WART (wts) in Drosophila melanogaster GUSTICE et al. 1995), colonial temperature-sensitive-1 (COT-1) of the fungus Neurospora crassa (YARD EN et al. 1992), DBF2 GOHNSTON et al. 1990) and DBF20 (TOYN et al. 1991) of Saccharomyces cerevisiae. Potential orthologues have also been identified in plants (WATSON et al. 1995) and PK428 (ZHAO et al. 1997). In all family members, serine/threonine kinase-specific and nucleotide-binding consensus sequences are present within the catalytic domain. The products of these genes share two distinguishing properties: amino acid inserts between residues 43 and 49 in the catalytic domain and a long non -conserved N -terminal domain.

5.2 Phosphorylation of DMPK Analysis of the kinase specificities of the full-length and truncated isoforms of DMPK indicates that they phosphorylate different amino acid residues. A 54 kDa protein is phosphorylated at tyrosine residues in human skeletal muscle (ETONGUE-MAYER et al. 1994), while a recombinant DMPK autophosphorylates a recombinant truncated DMPK with a specificity for Threonine and Serine residues (DUNNE et al. 1994). Another study reported that the full-length recombinant DMPK autophosphorylated a Serine residue and phosphorylated the synthetic peptide Gly-Arg-Gly-Leu-Ser-LeuSer-Arg, which contained a Serine residue in the phosphorylation site (TIMCHENKO et

228 . H .Veda et al.

al. 1995). ETONGUE-MAYER et al. (1998) also reported that a 54 kDa human protein kinase (most probably DMPK) displayed serine/threonine kinase activity in heart and tyrosine kinase activity in skeletal muscle. Recently, it was reported that an RNA CUGbinding protein (CUG-BP/hNab50) is a substrate for DMPK both in vivo and in vitro (ROBERTS et al. 1997).

5.3 Modulation for skeletal muscle voltage-gated sodium channels MOUNSEY et al. (1995) reported that coexpression of human DMPK with rat skeletal muscle Na+ channels in Xenopus oocytes reduced the amplitude of Na+ currents and accelerated current decay. This phenomenon is explained by the presence of a potential phosphorylation site in the inactivation mechanism of the channel. CHAHINE et al. (1998) also reported the effect of a recombinant mouse DMPK on the functional properties of human skeletal muscle and cardiac voltage-gated sodium channels in Xenopus oocytes. Coexpression of DMPK with skeletal voltage-gated sodium channels in oocytes resulted in a significantly lower peak sodium current amplitude as compared to that in cells expressing sodium channels alone. However, DMPK had no effect on the level of expressed sodium current in oocytes expressing cardiac ones, so the effect of DMPK on sodium channels is isoform- specific despite conservation of a putative phosphorylation site between the two isoforms.

5.4 Effects on Ca2+ metabolism by DMPK and relationship with other SRIER proteins Two reports have demonstrated a probable involvement of DMPK in the phosphorylation of voltage-dependent Ca 2+ release and Na+ channels (MOUNSEY et al. 1995; TIMCHENKO et al. 1995). In addition, recent studies reported that calcium homeostasis changed in DMPK-Iacking mice (REDDY et al. 1996) and in cultured mouse DMPKdeficient myotubes GANSEN et al. 1996). Regarding calcium homeostasis, JACOBS et a1. (1991) reported that cultured human OM muscle cells showed a significantly high [Ca2+]i in the presence of external Ca2+, and proposed that this phenonenon was due to an influx of Ca2+ through voltage-operated nifedipine-sensitive Ca 2+ channels: dihydropyridine receptors (DHPR). It has been reported that the ~ subunit of the DHPR-containing Serine residue is phosphorylated in vitro by full-length 72 kDa DMPK (TIMCHENKO et a1. 1995). DHPR is located in the T-tubules of adult skeletal muscle and cooperates with ryanodine receptors (RyRs) localized at the terminal cisternae of the SR (FRANZINI-ARMSTRONG and JORGENSEN, 1994). DAMIANI et a1. (1995) reported the defective expression of the slow/cardiac isoform of Ca2+-binding protein calsequestrin, together with an increased phosphorylation

Myotonic dystrophy/Myotonic dystrophy protein kinase' 229

activity of membrane-bound Ca2+-calmodulin-dependent protein kinase, in adult DM skeletal muscle. Another research group reported that their DMPK knock-out mice showed only inconsistent and minor size changes in head and neck muscle fibers at older ages GANSEN et al. 1996). However, calcium homeostasis in differentiated cultured myotubes from DMPK knock-out mice was altered (BENDERS et al. 1997). Taken this information together, we speculate that DMPK may play an important functional role in the modulation of EC coupling or an indirect role in the modulation of Na+ channels in the plasmalemma. Furthermore the fact that DMPK is localized in the ER of neurons leads us to think that abnormal phosphorylation might induce the central nervous system symptoms. It will be interesting to clarify the association between DMPK and other SR/ER molecular markers such as calsequestrin, Ca2+_Mg2+ ATPase, RyR, DHPR and traidin to understand the mechanisms of EC-coupling and signal transductions in muscle cells and neurons.

5.5 A member of a subfamily of tumor suppressor genes The catalytic domain of DMPK shares -45 % identity with the Drosophila warts (wts) gene, which encodes a protein that suppresses tumors (WATSON, 1995). Based on comparisons of functional domains and amino acid homology, DMPK appears to belong to a new protein family, some members of which are known to play crucial regulatory roles in cell differentiation, replication and signal transduction GUSTICE et al. 1995; LEUNG et al. 1995; ISHIZAKI et al. 1996; WATANABE et al. 1996). Homozygous wts-I - gene clones develop prominent outgrowths on the adult cell body, resulting from increased proliferation of the wts-I - cells and increased deposition of adult cuticle between epithelial cells GUSTICE et al. 1995). This indicates that wts has a tumor suppressor function, which may be representative of this subfamily of proteins. The yeast cell cycle proteins DBF2 and DBF20 (WATSON et al. 1994) have been shown to posses serine/threonine kinase activity as DMPK (DUNNE et al. 1994). DBF2 is required and its activity increases during anaphase and/or telophase of mitosis (TOYN et al. 1994), suggesting that DBF2 functions in cell cycle regulation. Cotl-I - mutants in Neurospora display local hyphal cellular hypertrophy, suggesting that the Cotl gene may have an important role in the regulation of hyphal elongation (YARDEN et al. 1992). DM patients often have pilomatrixoma, neural crest-derived tumors, parathyroid adenoma and small bowel carcinomas, which may reflect the DMPK gene function as a tumor suppressor. Recently, we saw a 44 year-old DM patient with expanded 1230-1430 CTG repeats who had parathyroid and thyroid adenomas and ovarian tumor (HIRASAKI et al. 1999, unpubl.). The inactivation of both alleles of DMPK might have contributed to her tumor development. A second 'hit' may be also required for tumorigenesis (KNUDSON, 1993). Determining the presence of a possible second mutation in tumorigenic tissues from DM patients would support this hypothesis.

230 . H. Ueda et al.

5.6 Interaction with other proteins Using the yeast two hybrid system, Fu (1996) identified a novel protein that specifically interacted with the DMPK. This protein interacts both in vivo and in vitro and exhibits a high degree of homology to an snRNP, D1, so this novel protein is probably a member of the signal transduction pathway, which is possibly responsible for the manifestation of the DM phenotype. Recently, SUZUKI et al. (1998) also identified a novel protein that bound and activated DMPK. This DMPK-binding protein, MKBP, is a novel member of the small heat shock protein (sHSP) family and localizes at the Z band and the neuromuscular junctions, where DMPK is concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. Importantly, the amount of MKBP protein, but not other sHSP proteins, is selectively upregulated in skeletal muscle from DM patients. These data suggest that an interaction between DMPK and MKBP is involved in the molecular pathogenesis of DM.

6 Pathological aspects of DMPK To understand the pathophysiological mechanism of DM skeletal muscle degeneration, here we show quantititative DMPK expression in DM skeletal muscles by Western blot analysis, and compared the localization of DMPK in DM skeletal muscles with well-known DM pathological changes (UEDA et al. 1999). DMPK alterations in severe cDM skeletal muscle will also be described briefly.

6.1 Pathological changes of adult type DM muscle 6.1.1 Alterations ofDMPK mRNA and proteins

Western blot analysis of biopsied skeletal muscle from DM patients showed that both 70 kDa and 55 kDa protein bands dramatically decrease (Fig. 17). A reduced level of DMPK mRNA in the skeletal muscle of adult-form DM patients has been reported to be associated with trinucleotide amplification (Fu et al. 1993; CARANGO et al. 1993; HOFMANN -RADVANYI et al. 1993 b; TIMCHENKO et al. 1995). Several studies reported that DMPK proteins decreased in DM skeletal muscle (VAN DEN VEN et al. 1993; KOGA et al. 1994; MAEDA et al. 1995), although some researchers reported no change in the amount of DMPK protein (BHAGWATI et al. 1996). WANG et al. (1995) reported that analysis of poly (At RNA from classical adult-onset DM patients showed dramatic decrease of both the mutant and normal DMPK RNA. Both normal and expanded DMPK genes are transcribed in DM skeletal muscle, but the abnormal expansion-containing RNA has a dominant effect on RNA metabolism by

Myotonic dystrophy/ Myoto nic dystrophy protein kinase' 231

R

2

3

4

567

kDa --97 --66

- - 48.5

- - 29

B

2

3 4

5

6 7

kDa --66 - - 48.5

--29

Fig. 17. Western blot analysis of human skeletal muscle biopsies of DM patients and controls. Biopsied samples of DM patients (lanes 1-4) and controls (lanes 5-7) were analyzed by immunoblotting with anti-DMPK antibody. The controls have both 70 kDa and 55 kDa proteins, but DM patients have both dramatically decreased 70 kDa proteins and decreased 55 kDa proteins. (A) membrane-rich fraction. (B) soluble fraction. Lane 1, muscle from DM case 1; lane 2, muscle from DM case 2; lane 3, muscle from DM case 3; lane 4, muscle from DM case 4; lane 5, muscle from a normal 22-year-old male; lane 6, muscle from a normal 30-year old male and lane 7, muscle from a normal 50-year-old female. Detailed information of DM cases is given in Table 1.

++

+

muscle myotonia, distal muscle atrophy, cataract, conduction block muscle myotonia, muscle atrophy & hypotonia, hatchet face, cataract, Af

13

15

48

59

F

4

++

+

+

+++

++

+++

+

+

+

Central nuclei & nuclear chains type 1 type 2

+

+

+

r

r

r

r

+

+

+

++

++

Sarcoplasmic ring fiber mass type 1 type 2 type 1 type 2

r: rare, + : sometimes, ++: often, +++: remarkable in one specimen. We examined more then two different specimens in each case. (From Ueda et ai., 1999).

++

M

muscle myotonia, distal muscle atrophy & hypotonia, hatchet face, cataract, diabetes mellitus

3

14

37

type I type II fiber fiber atrophy hypertrophy

Histological and histochemical features

M

muscle myotonia, distal muscle atrophy & hypotonia, hatchet face, frontal baldness, testicular atrophy

clinical feature

2

3

Duration of symptoms (years)

29

Age (years)

M

Case Sex No

Table 1. Clinical and histological and histochemical features of patients with myotonic dystrophy.

N

~

~

.,'"c-

c:

;:r:

N

'"

Myotonic dystrophy/ Myotonic dystrophy protein kinase' 233

preventing the accumulation of poly (At RNA (dominant-negative RNA mutation). Nuclear aggregation of DMPK trinucleotide repeat transcription (TANEJA et al. 1995) and altered processing of DMPK transcription (KRAHE et al. 1995) have also been reported. If these abnormal transcriptions manifest at the RNA level in DM muscle, the expression of DMPK protein might decrease to less than half of that of normal muscle. SASAGAWA et al. (1996) reported that CTG repeat expansion caused DMPK cDNA translation to decrease in COS-1 cells transfected with artificial (CTG) repeat expansion. Altogether, the dramatic DMPK decrease in DM muscle is possibly due to a dominantnegative mutation at the RNA level (TIMCHENKO et al. 1996a, b). 6.1.2 Morphological changes of DM skeletal muscles

Table 1 summarizes the histological and histochemical features of DM skeletal muscles. Our previous study demonstrated that DMPK in DM skeletal muscle dramatically decreased (SHIMOKAWA et al. 1997). Immunohistochemically, however, the expression of DMPK and its pathological changes vary greatly even in the same specimen. Fig. 18 shows that DMPK is exclusively colocalized in slow (type I) muscle fibers and not found in fast (type IIa, lIb, and lIc) ones. Type I muscle fibers that show almost the same intensity of slow MHC have weak, moderate and strong intensities of DMPK, which is similar to the controls (KOBAYASHI et al. 1997). Almost all angular atrophic fibers are DMPKpositive with weak or moderate intensity, and these atrophic fibers are detected from very slightly pathological to severely degenerated lesions. DMPK-negative (type II) muscle fibers are usually hypertrophic. Typical central nuclei and nuclear chains are mainly observed in DMPK-positive (type I) muscle fibers but are also found in some DMPKnegative (type II) muscle fibers. Typical sarcoplasmic masses are observed in DMPKpositive muscle fibers. Peripheral sarcoplasm like with a homogeneous appearance was strongly labeled with DMPK in comparison with the typical sarcoplasmic mass. Typical sarcoplasmic masses are rarely observed in DMPK-negative (type II) muscle fibers. Many hypertrophic ring fibers are DMPK-negative, but some DMPK-positive ring fibers are detected. Pyknotic nuclear clumps are DMPK-negative. In lesions with early DM pathological changes, large muscle fibers of different sizes have homogeneously distributed DMPK in the cytoplasm with various stainining intensities, but angular atrophic fibers have weak DMPK staining. Hypercontracted fibers show strong DMPK labeling in the peripheral sarcoplasm, but it is very weak in the central parts. With progressive muscle degeneration, the regular cross-striated pattern of DMPK disappears and irregular granular DMPK-positive substances appear (Fig. 19). In moderately to severely degenerated DM muscle fibers, the localization of DMPK becomes heterogeneous, and strongly positive DMPK-areas are often observed in the periphery of the sarcoplasm or just underneath the sarcolemma. DMPK-positive granules are variable in size, and DMPK-negative areas are often observed. In the more advanced stages, irregular DMPKpositive granules are distributed more sparsely in the sarcoplasm.

234· H . Veda et al.

Myotonic dystrophy/Myotonic dystrophy protein kinase' 235

Ultrastructurally, most muscle fibers show a focal dilatation of intramembranous structures and disorganization of Z bands. In addition, abnormal triads are often recognized as irregular dilatations and distortions of the T-tubules and extremely swollen SR. In more degenerated regions, myofilaments are irregularly organized, and the typical triad structure is not recognized. DMPK immunoreactivity is detected in the terminal cisternae of SR (Fig. 199), even in the swollen ones. In our studies, well-known DM pathological features such as type I muscle fiber atrophy, central nuclei, nuclear chains, and sarcoplasmic masses, with the exception of ring fiber formation, are observed predominantly in DMPK-positive (type I) muscle fibers. Accordingly, the disregulation of DMPK might be responsible for these pathological changes in DM skeletal muscle. A strong DMPK-positive region appears in the homogeneous area on the peripheral sarcoplasm before the formation of a typical sarcoplasmic mass, so the sarcoplasmic mass may be one of the degenerative products containing DMPK-positive materials. Immunoelectron microscopy demonstrated that DMPKpositively swollen SRs appear between well preserved myofibrils. These findings are consistent with earlier work of MUSSINI et al. (1970) and the recent study by REDDY et al. (1996). MUSSINI et al. (1970) carefully selected DM patients at the very early stages and found microvacuoles on I bands by light microscopy and considerable disorganization of the SR. Furthermore, DMPK-knockout mice developing late onset progressive myopathy show focal dilation of the SR, degenerating mitochondria, and focal loss of the Z band at the ultrastructural level (REDDY et al. 1996). Fig. 18. Histological, histochemical, and immunohistochemical photomicrographs of biopsied DM muscle in comparison with the localization of DMPK and the classical DM pathological changes. a, f, h, j, I, nand p: HE staining, b: myofibrillary ATPase staining after preincubation of pH 4.5, and immunohistochemical stainings of fast MHC (c), slow MHC (d and q) and DMPK (e, g, i, k, m, 0 and r). DMPK localizes in type I (slow MHC-positive) muscle fibers at different intensities but does not localize in type II (fast MHC-positive) muscle fibers. 1, type I muscle fiber; 2A and 2B, type IIA and lIB muscle fibers. Classical DM pathological changes stained with HE are compared with peroxidase immunostaining ofDMPK and slow MHC. Most muscle fibers with central nuclei (arrows in f) are DMPK-positive (arrows in g). Type II muscle fibers (DMPK-negative) are hypertrophic (g). Many muscle fibers with nuclear chains (arrows in h) are also DMPK-positive (arrows in i), but one muscle fiber ('f in h) is DMPK-negative C in i). A typical sarcoplasmic mass (arrow in j) has irregular staining ofDMPK (arrow in k). Dense peripheral staining ofDMPK (arrow in m) often appears in a homogeneous lesion of the peripheral sarcoplasm (arrow in I) in a D MPK -positive muscle fiber, which is assumed to be the very early formation of a 'sarcoplasmic mass'. Sarcoplasmic masses are usually observed in DMPK-positive fibers. A nuclear clump (arrowhead in I) is DMPKnegative (arrowhead in m). Typical ring fibers in a DMPK-negative muscle fiber (arrow in nand 0) and a DMPK-positive (r) type I (slow MHC-positive in q) fiber (arrow in p-r) are shown. Ring fibers are more common in type II muscle fibers than in type I muscle fibers. Photomicrographs of a-e, h, and i are taken from case 3, those of f, and g are taken from case 1, those of j-r are taken from case 2. Bar, 100 [lm in a-i and 50 [lm in j-r. Detailed information of DM cases is given in Table 1.

236 . H. Veda et al.

BENDERS et al. (1997) reported that their DMPK-knockout mice had no apparent abnormality light-microscopically, but their myotubes exhibited a higher resting intracellular Caz+([Caz+]J and smaller and slower Caz+ transients due to high extracellular potassium concentration. It has been also reported that cultured DM muscle cells showed significantly higher cytoplasmic Caz+ levels than normal muscle cells in the presence of extracellular Caz+, while almost no difference between them was recognized in a Ca2+free buffer GACOBS et al. 1991). If the intracellular Caz+ remains high, it subsequently activates various proteases that induce cell death. DAMIANI et al. (1996) also reported that defective expression of the slow/cardiac isoform of the Caz+-binding protein calsequestrin in DM skeletal muscle and DM slow-twitch muscle fibers produced a maturationalrelated abnormality with or without an altered modulatory mechanism of SR Ca2+transport. DMPK localizes in the terminal cisternae of both normal and DM muscles, and DMPK-positive cells are susceptible to degeneration. It has also been shown that a decreased level of DMPK leads to apoptosis in muscle cells in vitro (BHAGWATI et al. 1997). These findings indicate that the decreased DMPK level may be associated with the progression of muscle degeneration in adult-type DM. Concerning the pathological changes of DM, abnormalities in the neuromuscular junction were previously reported in biopsied materials of extrafusal DM muscle fibers (Mc DERMOT 1961; ENGEL and BROOKE 1966; COERS et al. 1973) and intrafusal fibers (DANIEL and STRICH 1964). Previously we reported that the nerve-muscle contacts on innervated DM muscle cells seemed less mature than equally long-term innervated cultured control muscle cells, as evidenced by impairment of the normal progression from multifocal to unifocal innervation and less well organized acetylcholinesterase patches (KOBAYASHI et al. 1987). Because DMPK is localized in both normal and DM neuromuscular junctions, DMPK may play an important role in synaptic transmission and signal transduction. To date, no ultrastructural studies of DMPK localization in neuromuscular junctions have been performed. Fig. 19. Immunohistochemical photomicrographs of degenerated DM muscle fibers. (a)-(f) confocal views of immunofluorescent staining of DMPK; (g) Immunoelectron micrograph of a DM skeletal muscle fiber immunostained with anti-DMPK antibody and visualized by HRPDAB technique. During the degenerative process of DM muscle fibers, DMPK staining in a muscle fiber becomes heterogeneous (a). Some areas have densely DMPK-positive staining with various DMPKpositive granules. In longitudinal sections (b), granular DMPK-positive materials appear in the region still containing cross-striated patterns, and in cross-section (c), they appear also as many granules. In more degenerated DM muscle fibers, DMPK appears in or beneath the plasmalemma (d), DMPK-positive granular materials become more irregular and various in size (d-f), and no DMPK-positive areas are present in the sarcoplasm (d, e). In a more advanced stage, various DMPK-positive granules become fewer and sparser (f). DMPK immunoreactivity in the SR (arrowheads in g) is recognized in the site facing the T-tubule (T) in very slight changes of DM skeletal muscle. TC, terminal cisternae; T, transverse tubule; Z, Z line. Bar, SO ftm in (a-f) and 200 nm in (g). (From VEDA et al. 1999)

Myotonic dystrophy/Myotonic dystroph y protein kinase' 137

':\·:/l ::~.:;":1,~

,,:).

'~Jo':.,

(~fj\, ...

.-." ~.

,, ,

f'

'.

238 . H. Ueda et al.

6.2 Pathological aspects of congenital DM muscle Usually, normal individuals have between 5-35 CTG repeats, mildly affected patients have 50-150 repeats, adult onset (classical) patients have 100-1000 repeats and congenital DM (cDM) patients have 2,000 repeats. cDM has been considered to be exclusively maternally inherited (HOFFMANN-RADVANYI et al. 1993a), but a case of paternally inherited cDM has been recently reported (NAKAGAWA et al. 1994). Therefore, transmission of a large allele from a male can occur, although it is a very rare event. This case eliminates the possibility that cDM is caused by maternal imprinting, mitochondrial inheritance or transplacental factors. cDM patients have quite different clinical features from adult DM. They have severe hypotonia with decreased electrophysiological activity, facial dysplegia, respiratory distress and severe mental retardation. MARTORELL et al. (1997) investigated the timing of instability of (CTG)n repeats in a series of cDM fetuses and neonates. They found that the repeat is apparently stable during the first trimester and that instability only becomes detectable during the second and third trimesters. According to their study, repeat instability is apparent from 13- to 16-week gestational age. The CTG-repeat heterogeneity appeared in a time specific pattern, and the heart showed the largest expansion. The degree of heterogeneity is not correlated with initial expansion size as gauged by chorionic villus and blood (CTG)n repeat sizes. Recently, STEINBACH et al. (1998) reported that the DMPK gene of cDM patients was hypermethylated proximal to the largely expanded CTG repeat, whereas that of normal individuals and adult type of DM1 was unmethylated. This difference may explain the unique features of cDM such as mental retardation, hypotonia and respiratory distress. As shown in Fig. 20, DMPK is developmentally expressed in only large muscle cells before birth, and the expression of an almost adult pattern, which shows a mosaic pattern with three different intensities, is observed in 2-year-old skeletal muscle, although the diameter of each muscle fiber is quite a bit smaller than that of an adult one. The muscle of one of the most severe cDM floppy infants, who died at birth with respiratory distress and no muscle movement, looks quite immature and DMPK is mainly expressed in large round muscle fibers that look like the control muscle obtained from a 28-week fetus. However, in another case of severe cDM, who had muscle movement but died at the age of 11 months, a mosaic pattern of DMPK expression with three different intensities similar to two-year-old skeletal muscle is observed. The diameter of each muscle fiber is much smaller than that of normal two-year-old and adult fibers. Interestingly, two groups of severe cDM patients, who died at birth (two patients) and 11 months (two patients) had almost the same number of expanded (CTG)n repeats in their muscles (15-18 kb and 15-17 kb, respectively), but the expression of DMPK was quite different. Our studies indicate that DMPK is expressed even in most severely affected cDM skeletal muscle, and DMPK expression is developmentally regulated even after birth.

Myotonic dystrophy/Myotonic dystrophy protein kinase' 239

Fig. 20. Immunohistochemical photomicrographs of cDM and control skeletal muscles immunostained with anti-DMPK antibody and visualized by HRP-DAB technique. a: control adult skeletal muscle; b: skeletal muscle with William's syndrome at the age of 3 years; c: skeletal muscle with suffocation at 12 weeks of gestation; d: skeletal muscle of a cDM patient who died at the age of 0 months, e: skeletal muscle of a cDM patient who died at the age of 11 months. Adult skeletal muscle and 3-year-old skeletal muscle with William's syndrome have a mosaic pattern of DMPK expression, although the diameter of 3-year-old skeletal muscle is smaller than that of adult one, wherears DMPK-positive large round muscle fibers are observed in 12week-gestation muscle. In cDM, DMPK-positive large round fibers are detected in O-month-old muscle, but DMPK-positive fibers become mosaicized in l1-month-old muscle. Bars: 10 f!m.

Conflicting results in cDM studies have been reported. Two studies have reported a decrease or absence of expanded alleles (Fu et aI, 1993; HOFFMANN-RADVANYI et al. 1993), whereas SABOURIN et al. (1993) reported an increased expression of this mutant allele in brain tissue. Significant differences in methods employed for RNA quantitation are obvious in these studies (HOFFMANN-RADVANYI and JUNIEN, 1993a). The published results were determined with DM and control 20-week-old foetuses (HOFFMANNRADVANYI et aL 1993a) or cDM infants compared with an adult control (Fu et al. 1993). SABOURIN et al. (1993) reported that DMPK mRNA steady-state levels were markedly increased in cDM tissues and that they might be due to elevated levels of transcripts derived from the expanded DMPK allele. However, mRNA from healthy adults and foetal tissues were used in their study, so the different DMPK gene expression during development may cause these discrepancies. To evaluate the expression of DMPK transcript and protein in newborn and infant controls of the exact same age will be needed to reach a final conclusion. cDM is well known to be associated with an arrest of muscle development and mental retardation, suggesting that other genes might play more crucial roles during the differentiation and maturation of muscle and the central nervous system.

240 . H. Veda et al.

7 Conclusions DM is the most common disease with multisystemic disorders among the muscular dystrophies. DMPK was designated the gene responsible for DM on chromosome 19, which is now designated as DM1, because the gene product has extensive homology to protein kinase catalytic domains. The genetic basis of DM1 is identified as a meiotically and mitotically unstable mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'UTR of DMPK. Full-length DMPK and various isoforms have been reported in skeletal and cardiac muscles and central nervous tissues. Regarding the molecular pathology of DM1, DMPK gene is located in a gene-rich region of the genome and probably simultaneously renders several genes dysfunctional. The somatic heterogeneity of the triplet repeats and the involvement of several genes accounts for the pleiotropy observed in the phenotype. DMPK is expressed predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in skeletal muscle. In cardiac muscle it is localized in intercalated discs and Purkije fibers. Electron microscopically it is detected in the the terminal cisternae of SR in skeletal muscle and the junctional and corbular SRs in cardiac muscle. In the central nervous system, it is expressed in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rER. The functional role of DMPK is not fully understood, but it may play an important role in Ca2+ homeostasis and the signal transduction system. Several molecular pathogenetical mechanisms of DM1 have been hypothesized, such as loss of function of DMPK, dysfunction of surrounding genes by structural change of the chromosome due to long trinucleotide repeats or the trans-gain of function of CUG-binding proteins. Decreased amounts of DMPK may be associated with the degeneration of skeletal muscle in adult type DM1, but other genes may play more important roles in the muscle developmental arrest, respiratory distress and mental retardation in cDM. Further studies of the exact function of DMPK and the molecular pathological roles of DMPK and surrounding genes will lead to new fundamental sights into the molecular relationships of transcription and translation of DMPK and surrounding genes and treatment for these complicated multisystem dysorders.

Myotonic dystrophy/Myotonic dystrophy protein kinase' 241

References ABE, T., SATO, M., KUBOKI, J., KANO, T., TAMAI, M.: Lens epithelial changes and mutated gene expression in patients with myotonic dystrophy. - Brit. J. Ophthalmol. 83, 452-457 (1999). ANDERSON, J. T., WILSON, S. M., DATAR, K. SWANSON, M. S.: NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability. - Mol. Cell BioI. 13, 2730-2741 (1993). ANVRET, M., AHLBERG, G., GRANDELL, U., HEDEBERG, B., JOHNSON, K., EDSTROM, L.: Larger expansions of the CTG repeat in muscle compared to lymphocytes from patients with myotonic dystrophy. - Hum. Mol. Genet. 2, 1397-1400 (1993). ASHIZAWA, T.: New nomenclature for myotonic dystrophies and DNA testing guidelines for myotonic dystrophy type 1. The International Myotonic Dystrophy Consortium (IDMC). Neurology 54,1218-1221 (2000). ASHIZAWA, T., DUBEL, J. R., DUNNE, P. W., DUNNC.]., Fu, Y-H., PIZZUTI, A., CASKEY, C. T., BOERWINKLE, E., PERRYMAN, M. B., EpSTEIN, H. E, HEJTMANCIK, J. E: Anticipation in myotonic dystrophy. II. Complex relationship between clinical findings and structure of the GCT repeat. Neurology 42,1877-1883 (1992b). ASHIZAWA, T., DUNNE, C.J., DUBEL, J.R., PERRYMAN, M.B., EpSTEIN, H.E, BOERWINKLE, E., HE]TMANCIK, J. E: Anticipation in myotonic dystrophy. I. Statistical verification based on clinical and haplotype findings. Neurol. 42,1871-1877 (1992a). ASHIZAWA, T., ZOGHBI, H.: Diseases inherited with trinucleotide repeat expansion. - Current Neurol.17, 79-135 (1997). ASKANAS, K WAN, H., ALVAREZ, R. B., ENGEL, W. K., KOBAYASHI, T., MARTINUZZI, A., HAWKINS, E. E: De novo neuromuscular junction formation on human muscle fibers cultured in monolayer and innervated by feotal rat spinal cord: Ultrastructural and ultrastructural-cytochemical studies. - J. Neurocytol.16, 423-537 (1987). ASLANDIS, C., JANSEN, G., AMEMIYA, C., SHUTLER, G., MAHADEVAN, M., TSILFIDIS, C., CHEN, C., ALLEMAN,]., WORMSKAMP, N.G.M., VOOIJS, M., BUXTON,]., JOHNSON, K., SMEETS, H.J.M., LENNON, G., CARRANA, A. KORNELUK, R. G., WIERINGA, A. B., DE JONG, P.J.: Cloning of the essential myotonic dystrophy region and mapping of the putative defect. - Nature 355, 548-551 (1992). BABITZKE, P.: Regulation of tryptophan biosynthesis: Trp-ing the TRAP or how Bacillus subtilis reinvented the wheel. - Mol. Microbiol. 26, 1-9 (1997). BALASUBRAMANYAM, A., hER, D., STRINGER, J. L., BEAULIEU, C., POTVIN, A., NEUMEYER, A. M., AVRUCH, H.]., EpSTEIN, H. E: Developmental changes in expression of myotonic dystrophy protein kinase in the rat central nervous system. - J. compo Neurol. 394, 309-325 (1998). BENDERS, A. A., GROENEN, P.J., OERLEMANS, E T., VEERKAMP, J. H., WIERINGA, B.: Myotonic dystrophy protein kinase is involved in the modulation in the Ca2 + homeostasis in skeletal muscle cells. - J. clin. Invest. 100, 1440-1447 (1997). BENDERS, A. A., TIMMERMANS, J. A., OOSTERHOF, A., TER LAAK, H.]., VAN KUPPERVELT, T. H., WEYERS, R.A., VEERKAMP, J.H.: Deficiency of Na+/K+-ATPase and sarcoplasmic reticulum Ca2 +-ATPase in skeletal muscle and cultured muscle cells of myotonic dystrophy patients.Biochem. J. 293, 269-274 (1993). BHAGWATI, S., GHATPANDE, A., LEUNG, B.: Normal levels of DM RNA and myotonin protein kinase in skeletal muscle from adult myotonic dystrophy (DM) patients. - Biochem. biophy. Acta. 131l, 155-157 (1996). BHAGWATI, S., LEUNG, B., SHAFIO, S. A., GHATPANDE, A.: Myotonic dystrophy levels of myotonic protein kinase (Mt-PK) leads to apostosis in muscle cells. - Exp. Neurol.146, 277-281 (1997). BOUCHER, C. A., KING, S. K., CAREY, N., KRAHE, R., WINCHESTERM, C. L., RAHMAN, S., CREAVIN, T., MEGHJI, P., BAILEY, M. E. S., CHARTER, E L., BROWN, S. D., SICILIANO, M.l, JOHNSON, K.J.:

v.,

v.,

v.,

242 . H. Veda et al.

A novel homeodomain-encoding gene is associated with a large CpG island interrupted by the myotonic dystrophy unstable (CTG)n repeat. - Hum. Mol. Genet. 4, 1919-1825 (1995). BREWSTER, B. S., JEAL, S., STRONG, P. N.: Identification of a protein product of the myotonic dystrophy gene using peptide specific antibodies. - Biochem. Biophys. Res. Commun. 194, 12561260 (1993). BROOK,]. D., MCCURRACH, M. E., HARLEY, H. G., BUCKLER, A.J., CURCH, D., ABURATANI, H., HUNTER, K., SANTON, v.P., THIRION, ].-P., HUDSON, T., SOHN, R., ZEMELMANN, B., SNELL, R. G., RUNDLE, S. A., CROW, S., DAVIES, J., SHELBOURNE, P., BUXTON,]., JONES, C., JUVONEN, v., JOHNSON, K., HARPER, P. S., SHAW, D.J., HOUSEMANN, D. E.: Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3' end of a transcript encoding a protein kinase family member. - Cell 68, 799-808 (1992) [erratum appears in Cell 69, 385 (1992)]. BRUNNER, H.G., SMEETS, R.P., LAMBERMON, N.H.M., COEWINKEL-DRIESSEN, M., VAN OOST, B. A., WIERINGA, B., ROPERS, H. H.: A mutation point linkage map around the locus for myotonic dystrophy on chromosome 19. - Genomics 5, 589-95 (1989). BUNGENER, R. C.,JOUVENT, R., DELAPORTE, c.: Psychological and emotional deficits in myotonic dystrophy. - ]. Neurol. Neurosurg. Psychiat. 65, 353-356 (1998). BUSH, E. W., TAFT, C. S., MEIXELL, G. E., PERRYMAN, M. B.: Overexpression of myotonic dystrophy kinase in BC3H1 cells induces the skeletal muscle phenotype. - ]. bioI. Chern. 271,548-552 (1996 ). BUXON,]., SHELBOURNE, P., DAVIES,J.,JONES, C., VAN TONGEREN, T., ASLANIDIS, C., DEJONG, P., JANSEN, G., ANVRET, M., RILEY, B., WILLIAMSON, R.,J OHNSON, K.: Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. - Nature 355, 547-548 (1992). CARANGO, P., NOBLE,]. E, MARKS, H. G., FUNANGE, V. L.: Absence of myotonic dystrophy protein kinase (DMPK) mRNA as a result of a triplet expansion in myotonic dystrophy. - Genomics 18, 340-348 (1993). CASKEY, C. T., SWANSON, M. S., TIMCHENKO, L. T.: Myotonic dystrophy: Discussion of molecular mechanism. - Cold Spring Harbor Symposia on Quantitative Biology LXI, 607-614 (1996). CHAHNINE, M., GEORGE, A. L. JR.: Myotonic dystrophy kinase modulates skeletal muscle but not cardiac voltage-gated sodium channels. - FEBS Lett. 412,621-624 (1998). CHANGEUX,J. P., COURREGE, P., DAN CHIN, A., LASRY,]. M.: A biochemical mechanism for the epigenesis of the neuromuscular junction. - C. R. des Seances de lere Academie des Sciences-Serie Iii (Sciences de la Vie) 292,449-453 (1981). COERS, c., TELERMAN-TOPPET, N., GERARD, J. M.: Terminal innervation ratio in neuromuscular disease. II. Disorders of lower motor neuron, peripheral nerve, and muscle. - Arch. N eurol. 29, 215-222 (1973). CONTI-TRONCOPNI, B. M., RAFTERY, M. A.: The nicotinic cholinergic receptor: correlation of molecular structure with functional properties. Ann. Rev. Biochem. 51, 491-530 (1982). DAMIANI, E., ANGELINI, c., PELOSI, M., SACCHETTO, R., BORTOLOSO, E., MARGRETH, A.: Skeletal muscle sarcoplasmic reticulum phenotype in myotonic dystrophy. - Neuromuscul. Disord. 6, 33-47 (1996). DANIEL, P. M., STRICH, S.].: Abnormalities in the the muscle spindle in dystrophia myotonica. Neurology 14,310-316 (1964). DAVIS, B.M., Mc CURRACH, M.E., TANEJA, K. L., SINGER, R.H., HOUSMAN, D.E.: Expansion of a CUG trinucleotide repeat in the 3'untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. - Proc. Natl. Acad. Sci. USA 94, 73887393 (1997). DAY,]. W., ROELOFS, R., LEROY, B., PECH, I., BENZOW, K., RANUM, L. P.: Clinical and genetic characteristics of a five-generation family with a novel form of myotonic dystrophy (DM2). - Neuromuscul. Disord. 9, 19-27 (1999).

Myotonic dystrophy/Myotonic dystrophy protein kinase' 243

DELAPORTE, c.: Personality patterns in patients with myotonic dystrophy. - Arch. Neurol. 55, 635-640 (1998). DUBNAU,]., STRUHL, G.: RNA recoginition and translational regulation by a homeodomain protein. - Nature 379,694-699 (1996) [erratum appears in Nature 388:697 (1997)]. DUNNE, P. W., WALCH, E. T., EpSTEIN, H. F.: Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition. - Biochemistry 33, 10809-10814 (1994). DUNNE, P. W., MA, L., CASEY, D., HARATI, Y., EpSTEIN, H. F.: Localization of myotonic dystrophy protein kinase in skeletal muscle and its alteration with disease. - Cell Motil. Cytoskel. 33, 52-63 (1996). ENGEL, W. K., BROOKE, M. H.: Histochemistry of the myotonic disorders. Progressive Muskeldystrophie, M yotonie, M yathenie, - Springer-Verlag NY Inc. New York 1966. EtoNGUE-MAYER, P., FAURE, R., BOUCHARD,]. P., THIBAULT, M. c., PUYMURAT,].: The myotoninprotein kinase phosphorylates tyrosine residues in normal human skeletal muscle. - Biochem. Biophys. Res. Commun.199, 89-92 (1994). ETONGUE-MAYER, P., FAURE, R., BOUCHARD, ].-P., PUYMURAT,].: Characterization of a 54-kilodalton human protein kinase recognized by an antiserum raised against the myotonin kinase. Muscle Nerve 21,8-17 (1998). FLUCHER, B. E., TAKAKURA, H., FRANZINI-ARMSTRONG, c.: Development of the excitationcontraction coupling apparatus in skeletal muscle: association of sarcoplasmic reticulum and transverse tubules with myofibrils. - Develop. BioI. 60, 135-147 (1993). FORBES, M. S., VAN NIEL, E. E.: Membrane systems of guinea pig myocardium: Ultrastructure and morphometric studies. - Anat. Rec. 222, 362-379 (1988). FRANZINI-ARMSTRONG, c., JORGENSEN, A. 0.: Structure and development of E-C coupling units in skeletal muscle. - Ann. Rev. Physiol. 56, 509-34 (1994). Fu, Y.-H.: Identification of a novel protein, DMAP, which interacts with the myotonic dystrophy protein kinase and shows strong homology to D1 snRNP. - Genetica 97, 117-125 (1996). Fu, Y.-H., FRIEDMAN, D. L., RICHARDS, S., PEARLMAN,]. A., GIBBS, R. A., PIZZUTI, A., ASHIZAWA, T., PERRYMAN, M. B., SCARLATO, G., FENWICK, R. G. jR, CASKEY, C. T.: Decreased expression of myotonin-protein kinase messenger RNA and protein in adult form of myotonic dystrophy.Science 260, 235-238 (1993). Fu, Y.-H., PIZZUTI, A., FENWICK, R. G. F. jR, KING,]., RAJNARANAYAN, S., DUNNE, P. W., DUBEL, ]., NASSER, G.A., ASHIZAWA, T., DEJONG, P., WIERINGA, B., KORNELUK, R., PERRYMAN, M.B., EpSTEIN, H. F., CASKEY, C. T.: An unstable triplet repeat in a gene related to myotonic muscular dystrophy. - Science 255, 1256-1258 (1992). FURUYA, A., KOBAYASHI, T., KAMEDA, N., TSUKAGOSHI, H.: Human myasthenia gravis thymic myoid cells: De novo immunohistochemical and intracellular electrophysiological studies. ]. Neurol. Sci.10l, 208-220 (1991). GORDENIN, D. A., KUNKEL, T. A., RESNICK, M. A: Repeat expansion-all in a flap? - Nature Genet. 16,116-118 (1997). GOURDON, G., RADVANYI, F., LIA, A.-S., DUROS, C., BLANCHE, M., ABIBOL, M., ]UNIEN, C., HOFMANN-RADVANYI, H.: Moderate intergenerational and somatic instability of a SS-CTG repeat in transgenic mice. - Nature Genet. 15, 190-192 (1997). HAMSHERE, M. G., NEWMAN, E. E., ALWAZZAN, M., ATHWAL, B. S., BROOK,]. D.: Transcriptional anbormatily in myotonic dystrophy affects DMPK but not neighboring genes. - Proc. natl. Acad. Sci. USA. 94, 7394-7399 (1997). HARLEY, H. G., BROOK,]. D., FLOYD,]., RUNDLE, S.A., CROW, S., WALSH, K. v., THIBAULT, M. c., HARPER, P. S., SHAW, D.].: Detection of linkage disequilibrium between the myotonic dystrophy locus and a new polymorphic DNA marker. - Amer. J. Hum. Genet. 49, 68-75 (1991).

244 . H. Veda et al.

HARLEY, H. G., BROOK, j. D., RUNDLE, S. A., CROW, S., READON, W., BUCKLER, A.J., HARPER, P. S., HOUSMAN, D. E., SHAW, D.J.: Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy. - Nature 355,545-546 (1992). HARPER, P.: Myotonic dystrophy. 2nd ed. W. B. Saunders Co., Philadelphia 1989. HARPER, P., RUDEL, R.: Myology. 2 nd ed. - McGraw-Hill Co., New York 1994. HARRIS, S., MONCRIEFF, C.,jOHNSON, K.: Myotonic dystrophy: will the real gene please step forward! - Hum. Mol. Genet. 5,1417-1423 (1996). HIROMASA, S., IKEDA, T., KUBOTA, K., HATTORI, N., COTO, H., MALDONADO, C., KUPERSMITH, J.: Ventricular tachycardia and sudden death in myotonic dystrophy. - Amer. Heart J. 115, 914-915 (1988). HOFMANN-RADVANYI, H., LAVEDAN, C., RABES,]., SAVOY, D., DUROS, C., JOHNSON, K., jUNIEN, Myotonic dystrophy: Absence of CTG enlarge transcript in congenital forms and low expression of the normal allele. - Hum. Mol. Genet. 2, 1263-1266 (1993a). HOFMANN-RAvANYI, H., jUNIEN, Myotonic dystrophy: Over-expression and/or under expression? A critical review on a controversial point. - Neuromuscul. Disord. 3,497-501 (1993b). HUND, E., jANsENO, 0., KOCH, M. C., RICKER, K., FOGEL, W., NIEDER-MAIER, N., OTTO, M., KUHN, E., MEINOK, H. M.: Proximal myotonic myopathy with MRI white matter abnormalities of the brain. - Neurology 48, 33-37 (1997). HUNTER, A. G. W., TSILFIDIS, C., METTLER, G., JACOB, P., MAHADEVAN, M., SURH, L. C., KORNELUK, R. G.: The correlation of age at onset with CTG trinucleotide repeat amplification in myotonic dystrophy. -J. Med. Genet. 29,774-779 (1992). ISHIZAKI, Y. M., MAEKAWA, K., FUJISAWA, K., OAKAWA, A., IWAMATSU, A., FUJITA, M., WATANABE, Y., SAITO, A., KAKIZUKA, M., MORII, M., NARUMYIA, S.: The small CTG-binding protein Rho binds to and activates a 160 kDa SeriThr protein kinase homologous to myotonic dystrophy kinase. - EMBO J. 15, 1885-1893 (1996). JACOBS, A. E. M., BENDERS, R. A. G. M., OOSTERHOF, A., VEERKAMP, J. H., MIER, P. v., WEVERS, R. A.,joosTEN, E. M. G.: The calcium homeostasis and the membrane potential of cultured muscle cells from patients with myotonic dystrophy. - Biochem. biophys. Acta 1096, 14-19 (1991). JANSEN, G., BACHNER, D., COERWINKEL, M., WORMSKAMP, N., HAMEUSTER, H., WIERINGA, B.: Structural organization and developmental expression pattern of the mouse Wd-repeat gene Dmr-N9 immediately upstream of the myotonic dystrophy locus. - Human Mol. Genet. 4, 843-852 (1995). JANSEN, G., GROENEN, P.j. T. A., BACHNER, D., JAP, P. H. K., COERWINKEL, M., OERLEMANS, E, VAN DEN BROEK, W., GOHLSCH, B., PETTE, D., PLOMP, J.J., MOLENAAR, P. C., NEDERHOFF, M.G.]., VAN ECHTELD, C.J.A., DEKKER, M., BERNS, A., HAMEISTER, H., WIERINGA, B.: Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nature Genet. 13, 316-324 (1996). JANSEN, G., MAHADEVAN, M., AMEMYIA, C., WORMSKAMP, N., SEGERS, B., HENDRIKS, W., O'HoY, K., BAIRD, S., SABOURIN, L., LENNON, G., JAP, P. L., ILEs, D., COERWINKEL, M., HOFKER, M., CARRANO, A. V., DEJONG, P.]., KORNELUK, R. G., WIERINGA, B.: Characterization of the myotonic dystrophy region predicts multiple protein isoform-encoding mRNAs. - Nature Genet. 1,261-266 (1992). JANSEN, G., WILLEMS, P., COERWINKEL, M., NILLESEN, W., SMEETS, H., VITS, L., HOWELER, C., BRUNNER, H., WIERINGA, B.: Gonosomal mosaicism in myotonic dystrophy patients: involvement of mitotic events in (CTG)n repeat variation and selection against extreme expansion in sperm. Am.J. Genet. 54, 575-585 (1994). jINNAI, K. SUGIO, T., MITANI, M., HASHIMOTO, K., TAKAHASHI, K.: Elongation of (CTG)n repeats in myotonic dystrophy protein kinase gene in tumors associated with myotonic dystrophy patients. - Muscle Nerve 22, 1271-1274 (1999).

c.:

c.:

Myotonic dystrophy/Myotonic dystrophy protein kinase' 245

JOHNSON, K., SHELBOURNE, P., DAVIES,J., BUXTON,J., NIMMO, E., SICILIANO, M.J., BACHINSKI, L. L., ANVRET, M., HARLEY, H., RUNDLE, S.: A new polymorphic probe which defines the region of chromosome 19 containing the myotonic dystrophy locus. - Amer.J. Hum. Genet. 46, 1073-1081 (1990). JOHNSTON, L.H., EBERLY, S.L., CHAPMAN, H., ARAKI, H., SUGINO, A.: The product of the Saccharomyces cervisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle. - Mol. Cell BioI. 10, 1358-1366 (1990). JORGENSEN, A. 0., JONES, L. R.: Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle. J. Cell BioI. 104, 1343-1352 (1987). JORGENSEN, A. 0., SHEN, A. c.- Y, ARNOLD, W., MCPHERSON, P. S., CAMPBELLY, K. P.: The Ca2+release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle. - J. Cell BioI. 120, 969-980 (1993). JORGENSEN, A. 0., SHEN, A. c.-y, CAMPBELLY, K. P.: Ultrastructural localization of calsequestrin in adult rat atrial and ventricular muscle cells. - J. Cell BioI. 101, 257-268 (1985). JORGENSEN, A. 0., SHEN, A. c.-y, DALY, P., MACLENNAN, D. H.: Localization of Ca2 + + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle. - J. Cell BioI. 93, 883-892 (1982). JUSTICE, R. W., ZILIAN, 0., WOODS, D. E, NOLL, M., BRYANT, P. J.: The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation. - Genes Dev. 9, 534-546 (1995). KAMEDA, N., KOBAYASHI, T., PARK-MATSUMOTO, Y., TSUKAGOSHI, H., SHIMIZU, T.: Developmental studies of the expression of myosin heavy chain isoforms in cultured human muscle aneurally and innervated with fetal rat spinal cord. - J. Neurol. Sci. 11, 85-98 (1993). KAMEDA, N., UEDA, H., OHNO, S., SHIMOKAWA, M., USUKI, E, ISHIURA, S., KOBAYASHI, T.: Developmental regulation of myotonic dystrophy protein kinase in human muscle cells in vitro. - Neuroscience 85, 311-322 (1998). KANG, S.,JAWORSKI, A., OHSHIMA, K., WELLS, R. D.: Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coli. - Nature Genet. 10,213-218 (1995a). KANG, S., OHSHIMA, K., SHIMIZU, M., AMIRHAERI, S., WELLS, R. D.: Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes. J. bioI. Chern. 270, 27014-27021 (1995b). KAWAKAMI, K., OHTO, H., IKEDA, K., ROEDER, R. G.: Structure, function and expression of a murine homeobox protein AREC3, a homologue of Drosophila sine oculis gene product, and implication in development. - Nucleic Acids Res. 24,303-310 (1996). KLESERT, T. R., OTTEN, A. D., BIRD, T. D., TAPSCOTT, S. J.: Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. - Nature Genet. 16, 402-406 (1997). KNUDSON, A. G.: Anti-oncogenes and human cancer. - Proc. Nat! Acad. Sci. USA 90, 1091410921 (1993). KOBAYASHI, T., ASKANAS, V., ENGEL, W. K.: Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation. - J. Neurosci. 7,3131-3141 (1987). KOBAYASHI, T., ASKANAS, SAITO, K., ENGEL, W. K., ISHIKAWA, K.: Abnormalities of aneural and innervated cultured muscle fibers from patients with myotonic atrophy (dystrophy). - Arch. Neurol. 47, 893-896 (1990a). KOBAYASHI, T., MICHIKAWA, M., MIYAZAKI, H., TSUKAGOSHIK, H.: The effect of Ba ion on human muscle cultured in monolayer and innervated with fetal rat spinal cord. - N eurosci. Lett. 111, 157-163 (1990b). KOBAYASHI, T., OHNO, S., PARK-MATSUMOTO, Y c., KAMEDA, N., BABA, T.: Developmental studies of dystrophin and other cytoskeletal proteins in cultured muscle cells. - Microsc. Res. Tech. 30,437-457 (1995).

v.,

246 . H. Ueda et al.

KOBAYASHI, T., SHIMOKAWA, M., ISHIURA, S., YAMAMOTO, M., KAMEDA, N., TANAKA, H., MIzuSAWA, H., VEDA, H., OHNO 5.: Myotonic dystrophy protein kinase is a sarcoplasmic reticulum protein specifically localized in type I muscle fibers without colocalization of SERCA II ATPase. Basic appl. Myol. 7, 311-316 (1997). KOGA, R., NAKAo, Y., KURANO, Y., TSUKAHARA, T., NAKAMURA, A., ISHIURA, 5., NONAKA, I., ARAHATA, K.: Decreased myotonin-protein kinase in the skeletal and cardiac muscles in myotonic dystrophy. - Biochem. Biophys. Res. Commun. 202, 577-585 (1994). KOIKE, H., SAITOH, N., SASAGAWA, N., WATANABE, T., SHIMOKAWA, M., SORIMACHI, H., ARAHATA, K., ISHIURA, 5., SUZUKI, K.: Identification and purification of myotonin protein kinase (MtPK) from rat skeletal muscle sarcoplasmic reticulum. - Biomed. Res. 19, 93-99 (1998). KORADE-MIRNIcs, Z., BABITZKE, P., HOFFMAN, E.: Myotonic dystrophy:molecular windows on a complex etiology. - Nucleic Acids Res. 26, 1363-1368 (1998). KRAHE, R., ASHIZAWA, T., ABBRUZESE, C., ROEDER, E., CARRANGO, P., GIACANELLI, M., FUNANAGE, V. L., SICILIANO, M.].: Effect of myotonic dystrophy trinucleotide repeat expansion on DMPK transcription and processing. - Genomics 28, 1-14 (1995). KUNKEL, T. A., RESNICK, M. A., GORDENIN, D. A.: Mutator specificity and disease: looking over the FENce. - Cell 88, 155-158 (1997). LAURENT, A., PINSET, c., AURADE, F., VIDAUD, M., MONTARRAS, D.: Expression of myotonic dystrophy protein kinase gene during in vivo and in vitro mouse myogenesis. - Cell. Mol. BioI. 43, 881-888 (1997). LAVEDAN, C., HOFMANN-RADVANYI, H., SHELBOURNE, P., RABES,]. P., DUROS, C., SAVOY, D., DEHAUPAS, I., LUCKE, S., JOHNSON, K., JUNIEN, c.: Myotonic dystrophy size- and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism. - Amer. ]. Hum. Genet. 52, 875-883 (1993). LEUNG, T. E., MANSER, L., LIM, L.: A novel serine/threonin kinase binding the Ras-related PhoA GTPase which trans locates the kinase to peripheral membranes. -]. bioI. Chern. 270, 2905129054 (1995). MAEDA, M., TAFT, C. 5., BUSSH, E. W., HOLDER, E., BAILEY, W. M., NEVILLE, H., PERRYMAN, M. B., BIEs, R. D.: Identification, tissue-specific expression, and subcellular localization of the 80- and 71-kDa forms of myotonic dystrophy kinase protein. - ]. bioI. Chern. 270, 20246-20249 (1995). MAHADEVAN, M., TSILFIDIS, C., SABOURIN, L., SHUTLER, G., AMEMYIA, C., JANSEN, G., NEVILLE, C., NARANG, M., BARCELO, l, O'Hoy, K., LEBLOND,S., EARLE-MACDONALD, l, DE JONG, P.l, WIERINGA, B., KORNELUK, R. G.: Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene. - Science 255, 1253-1255 (1992). MAHADEVAN, M.S., AMEMYlA, c., JANSEN, G., SABOURIN, L., BAIRD, S., NEVILLE, C. E., WORMSKAMP, N., SEGERS, B., BATZER, M., LAMERDIN, l, DE JONG, P., WIERINGA, B., KORNELUK, R. G.: Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene. - Hum. Mol. Genet. 2, 299-304 (1993). MARTINUZZI, A., ASKANAS, V., KOBAYASHI, T., ENGEL, W. K.: Asynchronous regulation of muscle specific isoenzyme of creatine kinase, glycogen phosphorylase, lactic dehydrogenase, and phosphoglycerate mutase in innervated and noninnervated cultured human muscle. - Neurosci. Lett. 89, 190-205 (1988). MARTINUZZI, A., ASKANAS, v., KOBAYASHI, T., ENGEL, W. K., DIMAURO, S.: Expression of muscle gene specific isoenzyme of phosphorylase and creatine kinase in innervated cultured human muscle. -]. Cell BioI. 103, 1423-1429 (1986). MARTINUZZI, A., ASKANAS, V., KOBAYASHI, T., ENGEL, W. K., GORSKI,]. E.: Developmental expression of the muscle specific isoenzyme of phophoglycerate mutase in human cultured monolayer and innervated by fetal rat spinal cord. - Exp. Neurol. 96, 365-375 (1987).

Myotonic dystrophy/Myotonic dystrophy protein kinase· 247

MARTORELL, L., ILLA, 1., ROSELL, J., BENITEZ, J., SEDANO, M.]., BAIGET, M.: Homozygous myotonic dystrophy: clinical and molecular studies of three unrelated cases. - J. Med. Genet. 33, 783-785 (1996). MARTORELL, L., JOHNSON, K., BOUCHER, C. A., BAIGET, M.: Somatic instability of the myotonic dystrophy (CTG)n repeat during human fetal development. - Hum. Mol. Genet. 6, 877-880 (1997). MARTORELL, L., MARTINEZ,]. M., CAREY, N., JOHNSON, K., BAIGET, M.: Comparison of CTG repeat length expansion and clinical progression of myotonic dsytrophy over a five year period. - ]. Med. Genet. 32, 593-596 (1995). McDERMOT, Y.: The histology of the neuromuscular junction in dystrophia myotonica. - Brain 84,75-84 (1961). MELACINI, P., VILLANOVA, C., MENEGAZZO, E., NOVELLI, G., DANIELI, G., RIZZOLI, G., FASOLI, G., ANGELINI, C., BUJA, G., MIORELLI, M., DALLAPICOLA, B., VOLTA, S. D.: Correlation between cardiac involvement and CTG trinucleotide repeat length in myotonic dystrophy. ]. Amer. Coll. Cardiol. 25, 239-245 (1995). MEOLA, G., SANSON, Y., RAmcE, S., SKRADSKI, S., PTACEK, L.: A family with an unusal myotonic and myopathic phenotype and no CTG expansion (proximal myotonic myopathy syndrome): a challenge for future molecular studies. - Neuromusci. Disord. 6, 143-150 (1996). MICHIKAWA, M., KOBAYASHI, T., TSUKAGOSHI, H.: Early events of chemical transmissions of newly formed neuromuscular junctions in monolayers of human muscle cells co-cultured with fetal rat spinal cord explants. - Brain Res. 538,79-85 (1991). MISHINA, M., TAKAI, T., IMOTo, K., NODA, M., TAKAHASHI, T., NUMA, S.: Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. - Nature 321, 1-6 (1986). MIYAKAZI, H., KOBAYASHI, T., TSUKAGOSHI, H.: Influence of innervation on the development of tetrodotoxin-sensitive sodium channels in cultured human muscle cells. - ]. Neurol. Sci. 98, 223 (1990). MONCKTON, D. G., COOLBAUGH, M. 1., ASHIZAWA, K. T., SICILIANO, M.J., CASKEY, C. T.: Hypermutable myotonic dystrophy CTG repeats in transgenic mice. - Nature Genet. 15, 193-196 (1997). MONCKTON, D. G., WONG, L.J. c., ASHIZAWA, T., CASKEY, C. T.: Somatic mosaicism, germline expansions, germline reservations and intergenerational reductions in myotonic dystrophy males - small pool PCR analyses. - Hum. Mol. Genet. 4, 1-8 (1995). MORRONE, A., PEGORARO, E., AGNELINI, C., ZAMMARCHI, E., MARCONI, G., HOFFMAN, E. P.: RNA metabolism in myotonic dystrophy: patient muscle shows decreased insulin receptor RNA and protein consistent with abnormal insulin resistance. - J. clin. Invest. 99, 1691-1698 (1997). MOTTA, J., GUILLEMINAULT, c., BILLINGHAM, M., BARRY, W., MASON, ].: Cardiac abnormalities in myotonic dystrophy: electrophysiologic and histopathologic studies. - Amer.]. Med. 67, 467-473 (1979). MOUNSEY,]. P., Xu, P., JOHN,J. E. 3RD, HORNE, L. T., GILBERT,]., ROSES, A. D., MOORMAN,J. R.: Modulation of skeletal muscle sodium channnels by human myotonin protein kinase. - ]. clin. Invest. 95, 2379-2384 (1995). MUSSINI, 1., DIMAURO, S., ANGELINI, c.: Early ultrastructural and biochemical changes in muscle in dystrophia myotonica. - ]. Neurol Sci. 10, 585-604 (1970). NAKAGAWA, M., YAMADA, H., HIGUCHI, 1., KAMINISHI, Y., MIKI, T., JOHNSON, K.J., OSAME, M.: A case of paternally inherited congenital myotonic dystrophy. - ]. Med. Genet. 31, 397-400 (1994). NOVELLI, G., GENNARELLI, M., ZELANO, G., PIZZUTI, A., FATTORINI, C., CASKEY, C. T., DALLAPICCOLA, B.: Failure in detecting mRNA transcripts from the mutated allele in myotonic dystrophy muscle. - Biochem. Mol. BioI. Int. 29, 291-297 (1993).

248 . H .Veda er al.

OGATA, T, YAMASAKI, Y.: High-resolution scanning electron microscopic studies on the threedimensional structure of the transverse-axial tubular system, sarcoplasmic resiculum and intercalated disc of the rat myocardium. - Anat. Rec. 228, 277-287 (1990). OTTEN, A. D., TAPSCOTT, S.J.: Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure. - Proc. natl. Acad. Sci. USA 92, 5465-5469 (1995). PARK-MATSUMOTOI, Y. c., KAMEDA, N., KOBAYASHI, T, TSUKAGOSHI, H.: Developmental study of the expression of dystrophin in cultured human muscle aneurally and innervated with fetal rat spinal cord. - Brain Res. 565, 280-289 (1991). PARSONS, M. A., SINDEN, R. R., IZBAN, M. G.: Transcriptional properties of RNA polymerase II within triplet repeat-containing DNA from the human myotonic dystrophy and fragile loci. J. bioI. Chem. 273, 26998-26700 (1998). PEARSON, C. E., SINDEN, R. R .: Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci. - Biochemistry 35, 5041-5053 (1996). PEARSON, C. E., WANG, Y. H ., GRIFFITH, J. D., SINDEN, R. R.: Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus. Nucleic Acids Res. 26, 816-823 (1998). PHAM, Y. c., MAN, N., LAM, L. T, MORRIS, G. E.: Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies. - Human Mol. Genet. 7, 1957-1965 (1998). PHILIPS, A. v., TIMCHENKO, L. T., COOPER, T A.: Disruption of splicing regulated bya CUGbinding protein in myotonic dystrophy - Science 280,737-741 (1998). PHILLIPS, M . E, HARPER, P. S.: Cardiac disease in my otonic dystrophy. - Cardiovasc. Res. 33, 13-22 (1997). PRYSTOWSKI, E. N., PRITCHETT, E . L. c., ROSES, A. D., GALLAGHER, J.: The natural history of conduction system disease in myotonic dystrophy as determined by serial electrophysiologic studies. - Circulation 60, 1360-1364 (1979). RAN UM, L. P., RASMUSSEN, P. E, BENZOW, K. A., KOOB, M. D., DAY, J. W.: Genetic mapping of a second myotonic dystrophy locus. - Nature Genet. 19, 196-198 (1998). REDDY, S., SMITH, D. B.J., RICH, M . M., LEFEROVICH, J. M., REILLY, P., DAVIS, B. M., TRAN, K., RAYBURN, H., BRONSON, R., CROS, D., BALICE-GORDON, R., HOUSMAN, D.: Mice lacking the my otonic dystrophy protein kinase develop a late onset progressive myopathy. - Nature G enet. 13, 325-335 (1996). RENAUD, J. E, DESNUELLE, C., ANTONARCHI, H. S., HUGUSS, M., SERRATRICE, G., LAZDUNSKI, M .: Expression of apamin receptor in muscles of patients with myotonic muscular dystrophy. Nature 319,281-287 (1986). RICKER, K.: Myotonic dystrophy and proximal myotonic myopathy. - J. Neurol. 246, 334-338 (1999). RICKER, K., KOCH, M. C., LEHMANN-HoRN, E, PONGRATZ, D., OTTO, M., HEINE, R., MOXLEY, R. T. 3RD: Proximal myotonic myopathy: A new dominant disorder with myotonia, muscle weakness, and cataracts. - Neurology 44,1448-1452 (1994). RI CKER, K., KOCH, M. c., LEHMANN-HoRN, E, PONGRATZ, D., OTTO, M., HEINE, R., MOXLEY, R. T. 3RD: Proximal myotonic myopathy. Clinical features of a multisystem disorder similar to myotonic dystrophy. - Arch. Neurol. 52, 25-31 (1995). RICKER, K., GRIMM, T., KOCH, M . C., SCHNEIDER, C., KRESS, W., REIMERS, C. D., SCHULTEMATTLER, W., MUELLER-MYHSOK, B., TOYKA, K. V., MU ELLER, C. R .: Linkage of proximal myotonic myopathy to chromosome 3q. - Neurology 52, 170-171 (1999). ROBERTS, R., TIMCHENKO, N . A., MILLER, J. W., REDDY, S., CASKEY, C. T, SWANSON, M., TIMCHENKO, L. T.: Altered phosphorylation and intracellular distribution of a (CUG)n triplet

Myotonic dystrophy/ Myotonic dystrophy protein kinase' 249

RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase.Proc. Natl. Acad. Sci. USA 94, 13221-13226 (1997). SABOURIN, L. A., MAHADEVAN, M . S., NARANGE, M., LEE, D. S. C., SURTH, L. C., KORNELUK, R. G.: Effect of myotonic dystrophy (DM) mutation on mRNA levels of the DM gene. - Nature Genet 4, 233-238 (1993). SAITO, K., KOBAYASHI, T., ASKANAS, v., ENGEL, W. K., ISHIKAWA, K.: Electrical properties of human muscle cultured in monolayer aneurally and co-cultured with fetal rat spinal cord. Biomed. Res. 11, 19-28 (1990). SAITOH, N., SASAGAWA, N., KOIKE, H ., SHIMOKAWA, M., SORIMACHI, H ., ISHIURA, S., SUZUKI, K.: Immunocytochemical localization of a full-length myotonin protein kinase in rat L6 myoblasts. - Neurosci. Lett. 218, 214-216 (1996). ' : SALVATORI, S., BIRAL, D., FURLAN, S., MARIN, 0.: Identification and localization of the myotonic dystrophy gene product in skeletal and cardiac muscles. - Biochem. Biophys. Res. Commun. 203,1365-1370 (1994). SALVATORI, S., BlRAL, D., FURLAN, S., MARIN, 0.: Evidence for localization of the myotonic dystrophy protein kinase to the terminal cisternae of the sarcoplasmic reticulum. - J. Muscle Res. Cell Mot. 18, 429-440 (1997). SASAGAWA, N., SAITOH, N., SHIMOKAWA, M., SORIMACHI, H., MARUYAMA, K., ARAHATA, K., ISHIURA, S., SUZUKi, K.: Effect of artificial (CTG) repeat expansion on the expression of myotonin protein kinase (MtPK) in COS-l cells. - Biochem. biophys. Acta. 203,1365-1370 (1996). SASAGAWA, N., SORIMACHI, H., MARUYAMA, K., ARAHATA, K., ISHIURA, S., SUZUKI, K.: Expression of a novel human myotonin protein kinase (MtPK) eDNA clone which encodes a protein with a thymopoietin-like domain in COS cells. - FEBS Lett 351, 22-26 (1994). SHAW, D.J., MCCURRACH, M., RUNDLE, S. A ., HARLEY, H. G ., CROW, S. R., SOHN, R., THIRRIO N, J.-P., HAMSHERE, M. G., BUCKLER, A.J., HARPER, P. S., HOUSMAN, D. E., BROOK, J. D.: Genomic organization and transcriptional units at the myotonic dystrophy locus. - Genomics 18, 673-679 (1993). SHELBOURNE, P., WINQVIST, R., KUNERT, E., DAVIES, J., LEISTI, J., THIELE, H., BACHMANN, H., BUXTON, J., WILLIAMSON, B., JOHNSON, K.: Unstable DNA may be responsible for the incomplete penetrance of the myotonic dystrophy phenotype. - Hum. Mol. Genet. 1, 467-473 (1992). SHIMOKAWA, M., ISHIURA, S., KAM EDA, N., YAMAMOTO, M., SASAGAWA, N., SAITOH, N., SORIMACHI, H ., UEDA, H., OHNO, S., SUZUKI, K., KOBAYASHI, T.: Novel isoform of myotonin protein kinase gene product of myotonic dystrophy is localized in the sarcoplasmic reticulum of skeletal muscle. - Amer. J. Pathol. 150, 1285-1295 (1997). SINGER, R.: Triplet-repeat transcript: a role for RNA in disease. - Science 280, 696-697 (1998). SOMMER, J. R., JOHNSON, E. A.: Cardiac muscle. A comparative study of Purkinje fibers and ventricular fibers. - J. Cell BioI. 36,497-526 (1968). STEINBACH, P., GLASER, D., VOGEL, W., WOLF, M., SCHWEMMLE, S.: The DMPK gene of severely affected myotonic dystrophy patients is hypermethylated proximal to the largely expanded CTG repeat. - Amer. J. Hum. Genet. 62, 278-285 (1998). ST. JOHNSTON, D.: The intracellular localization of messenger RNAs. - Cell 81 , 161-170 (1995). SUZUKI, A., SUGIYAMA, Y., HAYASHI, Y., Nyu-I., N., YOSHIDA, M., NONAKA, I., ISHIURA, S., ARAHATA, K., OHNO, S.: MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase. - J. Cell BioI. 140, 1113-1124 (1998). SWASH, M .: The morphology and innervation of the muscle spindle in dystrophia myotonica.Brain 95, 357-368 (1972). T ACHI, N., KOZUKA, N., OHYA, K., CHIBA, S., KIKUCHI, K.: Expression of myotonic dystrophy protein kinase in biopsied muscles. - J. Neurol. Sci 132,61-64 (1995).

250 . H. Veda et al.

TAKAI, T., NODA, M., MISHINA, M., SHIMIZU, S., FURUTANI, Y., KAYANO, T., IKEDA, T., KUBO, T., TAKAHASHI, T., KUNO, M., NUMA, S.: Cloning, sequencing and expression of eDNA for a novel subunit of acetylcholine receptor from calf muscle. - Nature 315, 761-764 (1985). TANEJA, K. L., MCCURRACH, M., SCHALLING, M., HOUSMAN, D., SINGER, R. H.: Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. - J. Cell BioI. 128, 995-1002 (1995) THORNTON, C A., GRIGGS, R. C, MOXLEY, R. T. 3RD: Myotonic dystrophy with no trinucleotide repeat expansion. - Ann. Neurol. 35, 269-272 (1994). THORNTON, C, WYMER, J., SIMMONS, Z., Mc CLAIN, C, MOXLEY, R. T. 3RD: Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. - Nature Genet. 16, 407-409 (1997). THORNTON, CA., ASHIZAWA, T.: Getting a grip on the myotonic dystrophies. - Neurology 52, 12-13 (1999). TIMCHENKO, L. T., MILLER, J. W., TIMCHENKO, N. A., DEVORE, D. R., DATAR, K. v., LIN, L., ROBERTS, R., CASKEY, C T., SWANSON, M. S.: Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. - Nucleic Acids Res. 24, 4407-4414 (1996b). TIMCHENKO, L., NASTAINCZYK, W., SCHENEIDER, T., PATEL, B., HOFMANN, E, CASKEY, C. T.: Fulllength myotonin protein kinase (72kDa) dysplays serine kinase activity. - Proc. Natl. Acad. Sci. USA 92,5366-5370 (1995). TIMCHENKO, L. T., TIMCHENKO, N. A., CASKEY, C T., ROBERTS, R.: Novel proteins with binding specificity for DNA CTG repeats and RNA CUG repeats: implications for myotonic dystrophy. - Hum. Mol. Genet. 5, 115-121 (1996a). TISHKOFF, D. X., FILOSI, N., GAIDA, G. M., KOLODNER, R. D.: A novel mutation avoideance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair. - Cell 88, 253-263 (1997). TOYN,J. H., ARAKI, H., SUGINO, A., JOHNSTON, L. H.: The cell-cycle regulated budding yeast gene DBF2, encoding a putative protein kinase, has a homologue that is not under cell-cycle control. - Gene 104, 63-70 (1991). TOYN, H., JOHNSTON, L. H.: The Dbf2 and Dbf20 protein kinases of budding yeast are activated after the metaphase to anaohase cell cycle transition. - EMBO J. 13, 1103-1113 (1994). TSIFIDIS, C., MAC KENZIE, A. E., METTLER, G., BARCELO, J., KORNELIK, R. G.: Correlation between CTG trinucleotide repeat length and frequency of severe congenital myotonic dystrophy. - Nature Genet. 1, 192-195 (1992). UDD, B., KRAHE, R., WALLGREN-PETTERSSON, C., FALCK, B., KALIMO, H.: Proximal myotonic dystrophy - a family with autosomal dominant muscular dystrophy, cataracts, hearing loss and hypogonadism: heterogeneity of proximal myotonic syndromes? - N euromuscul. Disord. 7, 217-228 (1997). UEDA, H., KAMEDA, N., BABA, T., TERADA, N., SHIMOKAWA, M., YAMAMOTO, M., ISH IURA, S., KoBAYASHI, T., OHNO, S.: Immunolocalization of myotonic dystrophy protein kinase in corbular and junctional sarcoplasmic reticulum of human cardiac muscle. - Histochem. J. 30, 245-251 (1998). UEDA, H., SHIMOKAWA, M., YAMAMOTO, M., KAMEDA, N., MIZUSAWA, H., BABA, T., TREADA, N., FUJII, Y., OHNO, ISHIURA, S., KOBAYASHI, T.: Decreased expression of myotonic dystrophy protein kinase and disorganization of sarcoplasmic reticulum in skeletal muscle of myotonic dystrophy. - J. Neurol. Sci., 162, 38-50 (1999). VAN DER VEN, P.EM., JANSEN, G., VAN KUPPEVELT, T.H.S.M., PERRYMAN, M.B., LUPA, M., DUNNE, P. W., TER LAAK, H.J., JAP, P. H. K., VEERKAMP, J. H., EpSTEIN, H. E, WIERUNGA, B.: Myotonic dystrophy kinase is a component of neuromuscular junctions. - Human Mol. Genet. 2,1889-1894 (1993).

Myotonic dystrophy/Myotonic dystrophy protein kinase· 251

WANG,]., PEGORARO, E., MENEGARRO, E., GENNARELLI, M., Hoop, R., ANGELINI, C., HOFFMAN, E. P.: Myotonic dystrophy:evidence for a possible dominant-negative RNA mutation. - Hum. Mol. Genet. 4, 599-606 (1995). WANG, Y. H., AMIRHAERI, S., KANG, S. WELLS, R., GRIFFITH, J. D: Preferential nucleosome assembly at DNA triplet repeats from the myotonic dystrophy gene. - Science 265, 669-671 (1994). WANG, Y. H., GRIFFITH, J.: Expanded CTG triplet blocks from the myotyonic dystrophy gene create the strongest known natural nucleosome positioning elements. - Genomics 25, 570-573 (1995). WARING,J.D., HAQ, R., TAMAI, K., SABOURIN, L.A., IKEDA,J.E., KORNELUK, R. G.: Investigation of myotonic dystrophy kinase isoform translocation and membrane association. - J. bioI. Chern. 271,15187-15193 (1996). WATANABE, G., SAITO, Y., MADAULE, P., ISHIZAKI, T., FUJISAWA, N., MORII, N., MUKAI, H., ONO, Y., KAKIZUKA, A., NARIMYIA, A. S.: Protein kinase N (PKN) and PKN -related protein rhophilin as targets of small GTPase Rho. - Science 271,645-648 (1996). WATSON, K. L.: Drosophilia WARTS-tumor suppressor and member of the myotonic dystrophy protein kinase family. - Bioessays 17, 673-676 (1995). WATSON, K. L., JUSTICE, R. W., BRYANT, P.J.: The fisrt fifty tumour suppressor genes. - J. Cell Sci. (Suppl.) 18,11302-11306 (1994). WELLS, R.: Molecular basis of genetic instability of triplet repeats. - J. bioI. Chern. 271, 2875-2878 (1996). WELLS, R., PARNIEWSK, P., PLUCIENNIK, A., BACOLLA, A., GELLIBOLIANT, R.,JAWORSKI, A.: Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases. - J. bioI. Chern. 273, 19532-19541 (1998). WHITING, E.J., WARING,J.D., TAMAI, K., SOMERVILLE, M.J., HINCKE, M., STAINES, W.A., IKEDA, J.-E., KORNELUK, R. G.: Characterization of myotonic dystrophy kinase (DMK) protein in human and rodent muscle and central nervous tissue. - Human Mol. Genet. 4, 1063-1072 (1995). WIERINGA, B.: Commentary: Myotonic dystrophy reviewed: back to the future? - Hum. Mol. Genet. 3, 1-7 (1994). WILSON, S. M., DATAR, K. v., PADDY, M. R., SWEDLOW, J. R., SWANSON, M. S.: Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces serevisiae. - J. Cell BioI. 127, 1173-1184 (1994). WITZEMANN, v., BARG, B., NISHIKAWA, Y., SAKMANN, B., NUMA, S.: Differential regulation of muscle acetylcholine receptor y- and E-subunit mRNAs. - FEBS Lett. 223, 104-112 (1987). WITZEMANN, V., BRENNER, H. R., SAKMANN, B.: Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses.- J. Cell BioI. 114, 125-141 (1991). WOHRLE, D., KENNERKNECHT, I., WOLF, M., ENDERS, H., SCHWEMMLE, S., STEINBACH, P.: Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a causal involvement of methyl-directed DNA mismatch repair in triplet repeat stability. - Hum. Mol. Genet. 7, 1147-1153 (1995). YARDEN, 0., PLAMANN, M., EBB OLE, D.J., YANOFSKY, cot-l, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. - EMBO J. 11, 2159-2166 (1992). ZHAO, Y., LOYER, P., LI, H., VALENTINE, v., KIDD, V., KRAFT, A. S.: Cloning and chromosomal location of a novel member of the myotonic dystrophy family of protein kinases. - J. BioI. Chern. 272,10013-10020 (1997).

c.: