Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on October 20, 2004
Human Molecular Genetics 2004 13(24):3079-3088; doi:10.1093/hmg/ddh327

This Article Abstract FREE Full Text (PDF) Supplementary Data All Versions of this Article: 13/24/3079    most recent ddh327v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by Jiang, H. Articles by Thornton, C. A. Search for Related Content PubMed PubMed Citation Articles by Jiang, H. Articles by Thornton, C. A. Social Bookmarking          What's this?

Human Molecular Genetics, Vol. 13, No. 24 © Oxford University Press 2004; all rights reserved

Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons

Hong Jiang¹, Ami Mankodi¹, Maurice S. Swanson², Richard T. Moxley¹ and Charles A. Thornton^1,*

¹Department of Neurology, University of Rochester School of Medicine and Dentistry, PO Box 673, 601 Elmwood Avenue, Rochester, NY 14642, USA and ²Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL 32610, USA

Received July 16, 2004; Accepted October 11, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene. In skeletal muscles, DM1 may involve a novel, RNA-dominant disease mechanism in which transcripts from the mutant DMPK allele accumulate in the nucleus and compromise the regulation of alternative splicing. Here we show evidence for a similar disease mechanism in brain. Examination of post-mortem DM1 tissue by fluorescence in situ hybridization indicates that the mutant DMPK mRNA, with its expanded CUG repeat in the 3'-untranslated region, is widely expressed in cortical and subcortical neurons. The mutant transcripts accumulate in discrete foci within neuronal nuclei. Proteins in the muscleblind family are recruited into the RNA foci and depleted elsewhere in the nucleoplasm. In parallel, a subset of neuronal pre-mRNAs show abnormal regulation of alternative splicing. These observations suggest that CNS impairment in DM1 may result from a deleterious gain-of-function by mutant DMPK mRNA.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Myotonic dystrophy type 1 (dystrophia myotonica, DM1) is the most common inherited disease of skeletal muscle in adults. Muscle wasting and delayed relaxation after muscle contraction (myotonia) are the prominent features of this illness. However, the effects of DM1 on the nervous system, heart and ocular lens, while less obvious, also pose a major threat to function and survival. The CNS symptoms of DM1 may include cognitive impairment, hypersomnolence, heightened sensitivity to anesthetic agents, central hypoventilation, neuroendocrine dysfunction and effects on personality and behavior (reviewed in 1,2). Some of these effects, such as mental retardation in individuals with congenital DM1, occur during development (3). Other symptoms, such as hypersomnolence, appear during adult life. The mechanism and neuropathologic correlates for CNS involvement in DM1 are unknown.

The genetic basis for DM1 is a dominantly inherited unstable expansion of CTG triplet repeats in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene (4). The severity of DM1 roughly correlates with the length of the CTG repeat tract in peripheral blood (5). For example, individuals with minimal expansions of 50–100 repeats generally have mild, late-onset symptoms, whereas those with 1000 or more repeats usually have severe disease in infancy. In many tissues, including skeletal muscle and brain, somatic instability leads to expansions that typically are 1000–4000 repeats in length (6,7).

Recent evidence suggests that the pathogenesis of skeletal myopathy in DM1 involves a toxic gain-of-function by mutant RNA. The CUG repeat in the mutant transcript is highly expanded but the portion of the mRNA encoding DMPK remains intact. CUG-expanded transcripts are retained in muscle nuclei in focal inclusions (8,9). Accumulation of mutant RNA in the nucleus compromises the regulation of alternative splicing for a subset of muscle transcripts (10–13), and may also disrupt regulation of transcription (14). Several models for this RNA-mediated disease mechanism have been proposed. First, the CUG expansion RNA may alter the activity or cellular distribution of CUG binding protein 1 (CUGBP1), a regulator of RNA splicing and translation (10,15). Second, the CUG expansion RNA may form hairpin loop structures that interact with double-stranded RNA binding proteins (dsRBPs) (16). Third, the CUG expansion RNA may interact with transcription factors, such as, retinoic acid receptor- and Sp1, depleting these factors from active chromatin (14).

A particularly attractive model for the disease mechanism in DM1 emerges from recent studies of proteins in the muscleblind (MBNL) family (17). These proteins bind to expanded CUG repeats in vitro and they are strongly recruited into RNA foci in muscle nuclei, raising the possibility that sequestration of MBNL proteins leads to muscle disease in DM1 (17–20). In support of this model, disruption of murine Mbnl1 produces a phenotype of myopathy (myotonia, defective regulation of alternative splicing) and lens cataracts that closely resembles human DM1 (21).

However, it is unclear whether RNA gain-of-function can explain all manifestations of DM1 in skeletal muscle or whether this mechanism can account for the non-muscle symptoms. Other effects of the DM1 mutation, such as decreased levels of DMPK protein (due to nuclear retention of RNA from the mutant allele) (8,9) or silencing of the neighboring gene SIX5 (due to effects of the repeat expansion on chromatin structure) (22–24), may also contribute to pathogenesis. Indeed, studies of knockout mice suggest that reduced expression of genes at the DM1 locus contributes to the cardiac (in the Dmpk knockouts) (25) or ocular (in the Six5 knockouts) (26,27) signs of DM1.

Presently it is unclear whether any steps in the pathogenic sequence of poly(CUG) expression, formation of RNA inclusions, sequestration of RNA binding proteins and disruption of alternative splicing can take place in the CNS. There is controversy about which cells in the mature brain, if any, express DMPK (28). To address these questions we have examined the expression and distribution of expanded poly(CUG) RNA in relation to putative binding proteins in post-mortem brain tissue, and screened for abnormalities of alternative splicing.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Fluorescence in situ hybridization (FISH) of brain sections with CAG repeat probes revealed nuclear RNA foci in every individual with DM1 (n=10, Fig. 1A) but not in controls with (n=7) or without (n=6) neurologic disease. RNA foci were not observed with CUG (sense) or GUC repeat probes. The hybridization of CAG probes to nuclear foci in DM1 did not require denaturation of genomic DNA. These results indicate that CAG repeat probes recognize CUG expansion RNA rather than a cross-reactive RNA or DNA.

View larger version (58K): [in this window] [in a new window]   Figure 1. FISH (left panels) and immunofluorescence (IF, middle panels) on frozen sections of DM1 brain shows nuclear foci of mutant DMPK mRNA. FISH, IF and nuclear stain (DAPI, blue) images are merged in panels on the right. (A). FISH (without IF) using Texas Red-labeled CAG repeat probe shows an RNA inclusion in a frontal cortical neuron. Autofluorescence from lipofuscin occurs at broad spectrum of wavelengths. It appears in every color channel and as yellow–brown perinuclear material in the merged image. RNA inclusions in cerebral cortex are confined to neurons identified by IF for NeuN (B) or MAP2 (C). Small foci are present in cerebellar Purkinje cells (D) or oligodendrocytes of the centrum semiovale (E) identified by IF for calbindin or CNPase, respectively. (F) RNA foci do not colocalize with PML bodies in cortical neurons. Bar, 5 µm, applies to all panels.

 
Nuclear RNA foci ranged in diameter from 0.2 to 2 µm. Resolution of these small structures required direct fluorescence detection methods. However, the autofluorescent material in brain (lipofuscin) was a complicating factor. We found that RNA foci were clearly distinguished from lipofuscin when the epifluorescence from three color channels was merged in a single image. As shown in Figure 1A, the nuclear foci appeared in a single channel determined by the probe label (Texas Red). Lipofuscin, which excites and emits at a broad spectrum of wavelengths, generated signal in all channels and appeared as a different color (typically yellow–brown) in the merged image. These observations formed the basis for distinguishing RNA foci in subsequent experiments. RNA foci were red, sharply demarcated structures in the nucleus. Lipofuscin was yellow–brown perinuclear material with indistinct margins.

To determine which cells express mutant DMPK and form RNA inclusions, we surveyed different brain regions using FISH in combination with antibodies that mark specific cell types. In cerebral cortex, the nuclear foci were distributed throughout all cortical layers and were confined to neurons, as determined by immunofluorescence (IF) of neuronal markers NeuN (Fig. 1B) or MAP2 (Fig. 1C). Counts of 100 NeuN-positive cells from temporal and frontal cortex of four patients with classical DM1 (selected for best relative preservation of cortical architecture) showed RNA foci in >85% of cortical neurons in each case. More than one focus was visible in 30% of cortical neurons, and occasional neurons had up to 15 small foci. In contrast, the individual having a small CTG repeat expansion (77 repeats) and mild phenotype (cataracts, mild weakness and cognitive impairment after age 60 years) had foci in only 39% of NeuN-positive neurons in temporal cortex.

RNA foci were widely distributed in other neuronal populations, including the hippocampus (all sectors), dentate gyrus, thalamus and also the substantia nigra and brain stem tegmentum (in each of four patients examined) (Supplementary Material, Fig. S1). The main exception was in cerebellar cortex, where small foci were detected in some Purkinje cells but not in neurons of the molecular or granular cell layers (n=6 patients examined) (Fig. 1D). RNA foci were also present in the subcortical white matter and corpus callosum in occasional cells expressing 2'3'-cyclic nucleotide-3'-phosphodiesterase (CNPase), a marker for oligodendrocytes (Fig. 1E). However, these foci were smaller and less intense than those in cortical neurons. In sections processed on the same slide and imaged under the same exposure settings, quantitation of FISH signals indicated that the amount of CUG expansion RNA in frontal cortical neurons was 2.9-fold greater (areaxintensity) than in Purkinje cells (P<0.001) and 18-fold greater than in oligodendrocytes (P<0.001) within the same individual (n=3 patients, 60 nuclei per patient).

Paired samples of frontal cortex and biceps muscle were available for three patients. When sections of skeletal muscle and cerebral cortex from the same patient were processed on the same slide and imaged under the same exposure settings, the RNA inclusions were larger and more intense (3.1-fold greater, areaxintensity) in frontal cortical neurons than in skeletal muscle from the same individual (P<10^–10, Supplementary Material, Fig. S2).

To determine whether mutant RNA resides in a previously identified nuclear domain, we tested for colocalization of mutant RNA with proteins that mark different nuclear compartments. These and subsequent experiments localizing protein relative to expanded poly(CUG) RNA were performed on a subset of four DM1 and three non-disease control samples showing the best preservation of cortical architecture. In contrast to nuclear inclusions of polyglutamine proteins (29), RNA foci did not colocalize with PML bodies (Fig. 1F). We also did not find colocalization of mutant RNA with the nucleolus (visualized by DNA staining or antibodies to C23 nucleolin), perinucleolar compartment (antibodies to polypyrimidine tract binding protein), or ‘speckles’ (antibodies to hnRNP C) (data not shown). We cannot eliminate the possibility of colocalization with Cajal bodies because p80 coilin antibodies did not consistently identify Cajal bodies in cortical neurons stained by our methods.

The proteasome and exosome are multisubunit complexes responsible for protein and RNA degradation, respectively. To determine whether these complexes are recruited to nuclear RNA foci, we combined FISH with immunofluorescence using antibodies to components of the proteasome or exosome. Three components of the proteasome (20S, 11S and 11S subunits) were recruited to RNA foci in cortical neurons (Fig. 2A and Supplementary Material, Fig. S3A). We did not, however, find evidence for ubiquitination or sumoylation of the foci (data not shown). In contrast, antibodies to the PM/Scl75 or PM/Scl100 components of the exosome did not colocalize with RNA foci (Fig. 2B)

View larger version (89K): [in this window] [in a new window]   Figure 2. FISH and IF on sections of temporal or frontal cortical neurons show colocalization of mutant DMPK mRNA [(CUG)n] with 20S -subunit of proteasome (A), MBNL2 (E) and hnRNP F (F). There is a marked redistribution of MBNL1 into RNA foci in DM1 cortical neurons (G), compared with the distribution in the nucleus (excluding nucleolus) and cytoplasm of normal neurons (H). Mutant DMPK mRNA does not colocalize with the PM/Scl100 (nuclear) component of the exosome (B), CUGBP1 (C) or NF90 (D). RAR does not colocalize with RNA foci in DM1 cortical neurons (I). The distribution of RAR in the DM1 (I) and non-neurologic-disease (J) neuronal nucleus is similar. Bar, 5 µm, applies to all panels.

 
Monoclonal antibody 3B1 shows strong expression of CUGBP1 in cortical neurons (Fig. 2C). The distribution of this protein in neuronal nucleus and cytoplasm appears similar in DM1 patients and controls, and FISH/IF analysis shows that CUGBP1 is not recruited into RNA foci. Polyclonal antibodies to other members of the CUGBP1 family, ETR3 and CELF4, also fail to colocalize with foci (data not shown). None of six different dsRNA binding proteins in neuronal nuclei (staufen, NF90, ADAR1, PACT, PKR, RNA helicase A) colocalize with RNA foci (representative images for NF90 are shown in Fig. 2D and ADAR1 in Supplementary Material, Fig. S3B). The RNA binding proteins hnRNP A1, hnRNP I, hnRNP M, KSRP and HuR did not colocalize with RNA foci (representative image for hnRNP M is shown in Supplementary Material, Fig. S3D). In contrast, hnRNPs H and F colocalized with foci in cortical neurons to a limited extent (Fig. 2F and Supplementary Material, Fig. S3C), and these results were verified using two different polyclonal antibodies for each protein. The intensity of immunofluorescence for these proteins was greatest at the site of RNA foci; however, there did not appear to be significant depletion of hnRNP H or hnRNP F elsewhere in the neuronal nucleoplasm.

Mutant DMPK mRNA is reported to interact with transcription factors retinoic acid receptor gamma (RAR) and Sp1 (14). In cortical neurons, these transcription factors were readily detected by immunofluorescence but they did not colocalize with RNA foci (Fig. 2I and Supplementary Material, Fig. S3E) and their distribution was similar in DM1 patients and controls (Fig. 2I and J).

Polyclonal antisera recognizing all members of the muscleblind family (MBNL1, MBNL2 and MBNL3) showed strong colocalization with RNA foci (data not shown). We used monoclonal antibodies raised against epitopes specific for MBNL1 or MBNL2 to determine which muscleblind proteins interact with CUG expansion RNA in neurons. MBNL3 was not examined because its expression in adults is mainly restricted to placenta (20,30). In normal controls, monoclonal antibody 3A4 showed expression of MBNL1 in nuclei and cytoplasm of cortical neurons (Fig. 2H). In DM1, MBNL1 was strongly recruited into RNA foci whereas staining elsewhere in the nucleus was markedly reduced (Fig. 2G). Quantitative analysis was performed on three DM1 patients and non-neurologic disease controls having the shortest post-mortem intervals and best preservation of cortical architecture (Fig. 3). The mean immunofluorescence intensity for MBNL1 in the nucleoplasm (excluding RNA foci and nucleoli) was 2.3-fold lower in DM1 neurons than in non-disease controls (26±9 areaxintensity units in DM1 patients versus 61±17 in controls, 20 neuronal nuclei per subject, P<0.00001). Monoclonal antibody 2D9 showed that MBNL2 was also recruited into RNA foci (Fig. 2E). However, immunofluorescence signals in neurons with MoAb 2D9 were lower in relation to background staining in the neuropil, precluding a reliable quantification of its distribution.

View larger version (15K): [in this window] [in a new window]   Figure 3. MBNL1 is decreased in nucleoplasm of DM1 cortical neurons. Immunofluorescence (area x intensity) for MBNL1 in the nucleus, excluding nucleolus and RNA foci, was determined for 20 neurons in sections of temporal cortex from three individuals with DM1 and three controls without neurologic disease (C).

 
To determine whether DM1 is associated with altered regulation of alternative splicing in brain, we examined 45 exons (in 31 genes) known to undergo alternative splicing in brain (Supplementary Material, Table S2). For each exon, the ratio of inclusion versus exclusion isoforms was determined by reverse transcriptase–polymerase chain reaction (RT–PCR) using primers flanking the regulated exon. An initial screen was performed using total RNA extracted from superior temporal cortex from two controls without neurological disease and four DM1 patients. Among 45 exons screened, four appeared to show a change in the ratio of exon inclusion/exclusion splice products in DM1. These differences were then confirmed and quantified in triplicate assays using temporal cortex RNA from seven patients with DM1 and five controls (Fig. 4). DM1 was associated with decreased inclusion of amyloid precursor protein exon 7 (10±1% in DM1, 30 ±11% in controls, P<0.001), increased inclusion of NMDA NR1 receptor exon 5 (33±11% in DM1, 11±5% in controls, P<0.01), decreased inclusion of tau exon 2 (5±1% in DM1, 36±10% in controls, P<0.00001) and decreased inclusion of tau exon 10 (21±1% in DM1, 41±5% in controls, P<0.000001).

View larger version (26K): [in this window] [in a new window]   Figure 4. Alternative splicing of NMDA NR1 receptor (NMDAR1), amyloid beta precursor protein (APP) and microtubule-associated protein tau (MAPT) is abnormally regulated in DM1. (A) Splice products obtained by RT–PCR amplification of RNA isolated from non-disease control (n=5) or DM1 (n=7) temporal cortex. Exon utilization for each splice product is shown in diagram. (B) Quantification of RT–PCR splicing assay (triplicates); ex, exon.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
The DM1 mutation has complex effects on genome function. Expansion of the CTG repeat in DMPK leads to reduced expression of DMPK protein (due to nuclear retention of the mutant mRNA), trans-interference with alternative splicing (10) and partial silencing of genes at the DM1 locus, such as the transcription factor SIX5 (due to effects on chromatin structure) (22–24). Following a reductionist approach, each of these effects has been separately modeled in mice to assess their respective roles in pathogenesis (26,27,31–34).It is unclear, however, whether any models have reproduced the CNS symptoms of DM1. Neurobehavioral studies have not been reported for Dmpk, Six5 or Mbnl1 knockout mice. Dmpk knockout mice show abnormalities of hippocampal physiology (35). However, this finding has no known correlate in DM1, in which there is only partial DMPK deficiency. Transgenic mice expressing CUG expansion RNA in the brain show an abnormal distribution of tau protein isoforms (34). However, the pattern of transgene expression in the CNS has not been determined, and it is unknown whether these mice develop RNA foci in neurons, abnormal splicing of tau pre-mRNA or CNS impairment.

Neuronal intranuclear inclusions are characteristic of several neurological disorders. In the polyglutamine disorders, the core component of the inclusion is mutant protein or a cleavage product containing the polyglutamine tract (36,37). In neuronal intranuclear inclusion disease, the main component of the inclusion has not been identified (38). In Fragile X tremor ataxia syndrome (FXTAS), FMR1 mRNA having an expanded CGG repeat leads to formation of nuclear inclusions (39). Our results indicate that DM1 should be added to the list of disorders characterized by neuronal intranuclear inclusions. In general terms, it is uncertain whether nuclear inclusions directly contribute to neuronal dysfunction in any of these disorders (40,41). In DM1 muscle tissue, however, evidence suggests that RNA inclusions are directly involved in disease pathogenesis, through a mechanism that involves sequestration of muscleblind proteins and misregulation of alternative splicing (21,33). Our observations showing strong expression of expanded poly(CUG) RNA in DM1 neurons, formation of RNA inclusions, redistribution of muscleblind proteins and altered regulation of alternative splicing raise the possibility that CNS symptoms of DM1 also are triggered by RNA inclusions.

The phenotypes that distinguish different CAG·CTG expansion disorders reflect distinct patterns of cell vulnerability. For example, the polyglutamine proteins in Huntington's disease or the spinocerebellar ataxias are widely expressed, yet the phenotypes are almost exclusively due to neuronal dysfunction. DM1 presents a different pattern. Despite evidence that mutant DMPK RNA accumulates to higher levels in cortical neurons (Supplementary Material, Fig. S2), the cell degeneration is more severe in muscle. Our initial studies also suggest that splicing abnormalities are less frequent and less severe in cerebral cortex than in skeletal muscle (Fig. 4 and C. Thornton, A. Mankodi and X. Lin, unpublished data), raising the possibility that muscleblind proteins are more effectively sequestered in muscle nuclei, or that compensation for muscleblind deficiency is more effective in neurons, perhaps due to expression of additional RNA binding proteins. The exact determinants of cell vulnerability in DM1 are unknown but the stoichiometry of CUG expansion RNA in relation to muscleblind proteins is likely to play an important role. For this reason, differences in the size of foci in different cells do not necessarily predict severity of an RNA-mediated disease process.

In spinocerebellar ataxia type 8 (SCA8) an expanded CTG repeat in a gene expressing a non-coding RNA can lead to progressive cerebellar dysfunction (42). As in DM1, a CUG expansion RNA is presumably expressed in SCA8, but the CNS symptoms of these disorders show little overlap. The lack of RNA foci in DM1 cerebellar cortex, except for small inclusions in Purkinje cells, might account for this difference, but other factors, such as length of the CUG expansion or flanking sequence in the mutant RNA, may also influence the pattern of neuronal vulnerability.

Initial studies of DMPK immunolocalization showed expression in cortical neurons or ependymal cells (43,44), but questions about antibody specificity were raised (45). A more recent study used a panel of 16 different monoclonal antibodies to rigorously establish antibody specificity (46). DMPK protein was found exclusively in skeletal, cardiac and smooth muscles. Owing to lack of evidence for DMPK expression in brain, the authors postulated that CNS symptoms of DM1 result from haploinsufficiency for genes at the DM1 locus. The present studies, however, establish that the mutant DMPK mRNA is widely expressed in cortical and subcortical neurons. Expression in specific neuronal populations is consistent with northern blots showing that levels of DMPK mRNA are lower in brain than in skeletal or cardiac muscle (4) and a recent paper showing wide expression of DMPK in cortical neurons, assessed by in situ hybridization in mice (47). The failure to detect DMPK immunologically likely reflects its relatively low concentration in brain homogenates.

The most conspicuous effect on alternative splicing that we observed in DM1 brain was a 3-fold increase in the fraction of NMDA receptor 1 (NMDAR1) mRNA that includes exon 5. Inclusion of this exon influences the pharmacologic behavior, gating and cellular distribution (somatic rather than somato-dendritic expression) of NMDAR1 (48–50). NMDAR1 function is required for normal long term potentiation in the hippocampus and learning (51,52). It seems possible, therefore, that altered splicing of exon 5 may contribute to the memory impairment observed in DM1 (53,54).

Microtubule-associated protein tau (MAPT) pre-mRNA is alternatively spliced at exons 2, 3 and 10 (55). Tau transcripts in fetal brain do not include exon 10, whereas 50% of transcripts in adult brain include this exon which encodes an additional microtubule binding domain (55–57). Alternative splicing of exons 2 and 3 also is developmentally regulated (neither exon is included in the fetus, adults mainly include exon 2). The relative proportion of tau splice products is tightly regulated, as shown by kindreds with frontotemporal dementia and parkinsonism (FTDP-17) due to mutations in MAPT. Silent mutations in MAPT exon 10, or in the flanking intron, lead to FTDP-17 by disrupting cis elements that regulate splicing of tau pre-mRNA (58–61). Usually, these mutations lead to increased inclusion of exon 10 (62). However, some MAPT mutations that segregate with FTDP-17 have the opposite effect of reducing exon 10 inclusion (61,63). Notably, DM1 is associated with reduced exon 10 inclusion (Fig. 4), and FTDP-17 and DM1 are both associated with neurofibrillary tangles and neuronal aggregates of hyperphosphorylated tau (64–67). We also found that inclusion of tau exon 2 is reduced in DM1, confirming previous observations (68). Our results predict that fetal isoforms of tau (excluding exons 2, 3 and 10) are inappropriately expressed in adult DM1 brain, findings that correlate well with previous studies of tau protein in DM1 brain (68). Expression of human fetal tau in transgenic mice leads to formation of neurofibrillary tangles and axonopathy (69,70). It is unclear, however, whether the extent of the tau missplicing in DM1 is sufficient to cause neuronal dysfunction.

We have also found that DM1 is associated with increased expression of fetal splice isoforms for APP (exon 7 exclusion products). Taken together, these observations suggest that accumulation of mutant DMPK mRNA in the neuronal nucleus compromises a specific developmental program of alternative splicing.

Among the RNA binding proteins that we examined, only MBNL1 and MBNL2 are strongly recruited to nuclear foci of mutant RNA. In Drosophila, muscleblind is required for terminal differentiation of muscle and photoreceptor cells (71). Human orthologs of muscleblind were initially isolated as the major poly(CUG) binding proteins in HeLa nuclear extracts (17). The three mammalian muscleblind genes, MBNL1, MBNL2 and MBNL3, are closely related (20,30). When expressed as GFP fusion proteins, each member of this family can localize to RNA foci in DM1 cells (20). Our results are the first to show depletion of MBNL1 in the nucleoplasm of DM1 cells, supporting a model where CUG expansion RNA accumulates to levels sufficient to sequester and compromise the nuclear functions of MBNL1.

Loss of Mbnl1 is sufficient to induce DM1-like defects of alternative splicing in murine striated muscle (21). A recent paper has shown that human MBNL1 is a direct regulator of alternative splicing (72). RNA-binding proteins in the CELF family (CUG binding protein and ETR3-like factors) are also implicated in DM1-related defects of RNA processing (10,11,73). The initial observations linking CELF proteins with DM1 pathogenesis indicated that CUG binding protein 1 (CUGBP1) and ETR3/CUGBP2 bind to short oligo(CUG) RNAs in vitro (15,74). Subsequent studies, however, failed to show interaction of CELF proteins with expanded poly(CUG) in vitro or in DM1 cells (19,75,76). Proteins in this family nevertheless regulate splicing of several exons that show defective regulation in DM1 muscle (10,11,13). Notably, CELF proteins may also regulate alternative splicing for tau exon 10 and NMDAR1 exon 5 (77,78). However, it is unlikely that sequestration of CELF proteins on CUG expansion RNA is responsible for splicing defects in neurons because monoclonal antibody 3B1, which recognizes an epitope shared by several CELF proteins (79), does not colocalize with RNA inclusions. Moreover, the splicing defects that we observed would predict opposite effects on CELF protein activity [decreased activity of CELF3 or CELF4 in the case of tau exon 10 (78), or increased activity of ETR3/CUGBP2 in the case of NMDAR1 exon 5 (77)]. Our results do not suggest a unifying mechanism whereby CUG expansion RNA directly alters the activity of CELF proteins. It remains possible, however, that nuclear accumulation of mutant RNA or sequestration of muscleblind proteins may indirectly affect the activity of specific CELF proteins in the neuronal nucleus.

The mutant DMPK mRNA in the RNA inclusion is, at least in part, the full-length mRNA (8,9). Nuclear mRNAs exist as ribonucleoprotein (RNP) complexes rather than naked polynucleotides (80). Thus, RNA inclusions would be expected to contain other nuclear RNA binding proteins in addition to MBNL1 and MBNL2. We found that hnRNPs H and F (but not hnRNP C, hnRNP I, hnRNP M, KSRP or HuR) colocalized with nuclear foci of poly (CUG) RNA to a limited extent. However, the overall nuclear distribution of hnRNP H or F in DM1 neurons did not show an obvious change. Also, the splicing of neuron-specific exon N1 of c-src, which is promoted by hnRNPs H and F (81,82), was not reduced in DM1 cerebral cortex. Indeed, inclusion of the N1 exon showed a slight (1.3-fold, P<0.02) increase in DM1 with respect to controls, opposite to the predicted effect of hnRNP F or H depletion (data not shown). This fits with expectations that the number and density of binding sites on a single transcript, and hence the capacity for protein sequestration, is much greater for proteins that bind to expanded poly(CUG) than for proteins that bind DMPK mRNA outside of the repeat tract.

A recent study suggested that mutant DMPK transcripts interact with transcription factors Sp1 and RAR, ‘leaching’ these factors away from normal binding sites on chromatin (14). At present, however, there is no direct biochemical evidence for interaction between poly(CUG) and DNA binding proteins. We have not found that Sp1 or RAR colocalize with RNA inclusions in DM1 neurons. A caveat is that post-mortem autolysis, loss of architectural preservation in frozen tissue or masking of epitopes may have limited our ability to detect RNA–protein interactions.

Factors that control the accumulation and degradation of mutant DMPK mRNA in the nucleus are not understood. In the polyglutamine disorders, the proteasome is recruited to nuclear foci of mutant protein (83). We postulated that the exosome, a multisubunit complex responsible for mRNA degradation (84), would be recruited to nuclear foci of mutant RNA in DM1. Opposite to this prediction, we found that the proteasome is recruited to RNA foci whereas the exosome is not. This observation raises the possibility that the proteasome is recruited by conformational changes in MBNL1, MBNL2 or other poly(CUG) binding proteins. If this is the case, loss of muscleblind function in DM1 may result from the combined effects of sequestration and accelerated degradation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Tissue samples
Autopsy materials were obtained from 10 DM1 patients (mean age 56 years, range 44–78 years, seven men and three women) and 13 controls (six with no neurologic disease, two with Alzheimer disease, four with Huntington disease and one with refractory epilepsy). The mean post-mortem interval for DM1 patients was 6 h (range 2–14 h). At the time of autopsy, coronal sections of brain were prepared and placed on aluminum slabs cooled on dry ice. In addition, selected regions were dissected and flash frozen in liquid nitrogen. All samples were stored at –70°C. Nine of the DM1 patients had signs of classical DM1 before age 30 and died of complications related to the disease (respiratory failure in seven, sudden cardiac death in two). The other DM1 patient had minimal symptoms of DM1 and died at age of 78 years from an unrelated disease. Genetic confirmation was performed as previously described by PCR or Southern blot on DNA isolated from post-mortem brain tissue (7). Southern blots of cortical DNA samples showed a broad range of expanded alleles ranging in size from 5 to 12 kb (data not shown). The individual with the minimal DM phenotype had a CTG repeat expansion length of 77 repeats in DNA isolated from peripheral blood, brain and other tissues.

FISH
FISH was performed as described (18) with slight modifications. Frozen sections (12 µm) were fixed in 3% paraformaldehyde PBS for 30 min, permeabilized in 2% acetone PBS (pre-chilled at –20°C) for 5 min and then prehybridized in 30% formamide and 2xSSC at room temperature for 10 min. Next, sections were hybridized with probe (1 ng/µl) for 2 h at 37°C in buffer (30% formamide, 2xSSC, 0.02%BSA, 66 µg/ml yeast rRNA, 2 mM vanadyl complex) and then washed for 30 min in 30% formamide/2xSSC at 42°C followed by 1xSSC for 30 min at room temperature. Probes were HPLC-purified 2-O-methyl RNA 20-mers (IDT, Coralville, IA, USA) composed of CAG-, CUG- or GUC-repeats, and labeled with Texas Red at the 5' end. Images were obtained on an Olympus AX70 epifluorescence microscope at 1000x magnification. To compare the relative fluorescence intensities for RNA foci, sections were processed on the same slide, imaged under the same illumination and exposure settings and then analyzed using MCID version 6.0 software (Imaging Research Inc., St Catherines, Ontario, Canada).

IF combined with FISH
Following the 1xSSC post-hybridization wash of the FISH procedure, sections were incubated in primary antibodies (listed in Supplementary Material, Table S1) overnight at 4°C, washed five times with PBS for 2 min and then incubated in secondary antibody (Alexa 488-labeled goat anti-rabbit polyclonal or Alexa 488-labeled goat anti-mouse polyclonal, Molecular Probes) and 33 nM diamidino-2-phenylindole (DAPI) for 30 min at room temperature. Sections were washed five times in PBS prior to mounting. To estimate relative MBNL1 concentration in nucleoplasm in DM1 nuclei versus controls, sections of temporal cortex were processed on the same slide and imaged under the same exposure settings. Merged images for Texas Red (to visualize RNA foci), Alexa 488 (for MBNL1) and DAP1 (for nuclear DNA) were obtained. Regions of interest were manually defined as nuclear area excluding nucleolus, RNA foci and overlapping lipofuscin. MBNL1 fluorescence intensity (mean optical density in monochrome mode in arbitrary units) in the region of interest was determined for 20 cortical neuronal nuclei per subject. Because of the difficulty in estimating background fluorescence from brain sections, the results are not corrected for background. This approach provides a conservative estimate of the fold-reduction for MBNL1 in DM1 nucleoplasm.

Splicing assays
Alternative splicing was assessed by RT–PCR. Total RNA was isolated from temporal cortex gray matter of seven DM1 patients and five non-neurologic disease controls using TriReagent (Molecular Research Center, Cincinnati, OH, USA). cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) with a mixture of oligo(dT)_12–18 and random hexamer primers. The cDNA was digested with RNase H and then amplified using PCR primers flanking alternatively spliced exons (Supplementary Material, Table S2). PCR products were resolved on agarose gels, stained with SybrGreenII (Molecular Probes) and analyzed on a fluorimager. An initial screen was performed on a subset of samples (four DM1 and two controls). Four exons appeared to show deregulated splicing in DM1. These differences were quantified in a second experiment including the full panel of seven DM1 and five control samples. The fraction of exon inclusion was determined on triplicate reactions using ImageQuant software (Amersham, Piscataway NJ, USA).

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
The authors thank Don Henderson for excellent technical assistance. The authors thank the University of Rochester Alzheimer Disease Center for tissue samples and Drs Thomas Cooper, Jeffrey Wilusz, Doug Black, Ganes Sen, C. Lee, D. Cho, K.L. Chan and E. Wagner for gifts of antibodies. This work comes from the University of Rochester Paul D. Wellstone Muscular Dystrophy Cooperative Research Center (AR050762). This work was supported by NIH/NIAMS AR49077, AR48143 (C.T.) and AR46799 (M.S.S.), the Muscular Dystrophy Association, and the Saunders Family Neuromuscular Research Fund.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +1 5852752542; Fax: +1 5852731255; E-mail: charles_thornton{at}urmc.rochester.edu

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 

1. Harper, P.S. (2001) Myotonic Dystrophy. Saunders, London, UK.

2. Ashizawa, T. (1998) Myotonic dystrophy as a brain disorder. Arch. Neurol., 55, 291–293.[Free Full Text]

3. Dyken, P.R. and Harper, P.S. (1973) Congenital dystrophia myotonica. Neurology, 23, 465–473.[Free Full Text]

4. Brook, J.D., McCurrach, M.E., Harley, H.G., Buckler, A.J., Church, D., Aburatani, H., Hunter, K., Stanton, V.P., Thirion, J.P., Hudson, T. et al. (1992) 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.[CrossRef][Web of Science][Medline]

5. Harley, H.G., Rundle, S.A., MacMillan, J.C., Myring, J., Brook, J.D., Crow, S., Reardon, W., Fenton, I., Shaw, D.J. and Harper, P.S. (1993) Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy. Am. J. Hum. Genet., 52, 1164–1174.[Web of Science][Medline]

6. Ashizawa, T., Dubel, J.R. and Harati, Y. (1993) Somatic instability of CTG repeat in myotonic dystrophy. Neurology, 43, 2674–2678.[Abstract/Free Full Text]

7. Thornton, C.A., Johnson, K. and Moxley, R.T. (1994) Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes. Ann. Neurol., 35, 104–107.[CrossRef][Web of Science][Medline]

8. Taneja, K.L., McCurrach, M., Schalling, M., Housman, D. and Singer, R.H. (1995) Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues. J. Cell. Biol., 128, 995–1002.[Abstract/Free Full Text]

9. Davis, B.M., McCurrach, M.E., Taneja, K.L., Singer, R.H. and Housman, D.E. (1997) 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, 7388–7393.[Abstract/Free Full Text]

10. Philips, A.V., Timchenko, L.T. and Cooper, T.A. (1998) Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science, 280, 737–741.[Abstract/Free Full Text]

11. Savkur, R.S., Philips, A.V. and Cooper, T.A. (2001) Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat. Genet., 29, 40–47.[CrossRef][Web of Science][Medline]

12. Mankodi, A., Takahashi, M.P., Jiang, H., Beck, C.L., Bowers, W.J., Moxley, R.T., Cannon, S.C. and Thornton, C.A. (2002) Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy. Mol. Cell, 10, 35–44.[CrossRef][Web of Science][Medline]

13. Charlet, B., Savkur, R.S., Singh, G., Philips, A.V., Grice, E.A. and Cooper, T.A. (2002) Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing. Mol. Cell, 10, 45–53.[CrossRef][Web of Science][Medline]

14. Ebralidze, A., Wang, Y., Petkova, V., Ebralidse, K. and Junghans, R.P. (2004) RNA leaching of transcription factors disrupts transcription in myotonic dystrophy. Science, 303, 383–387.[Abstract/Free Full Text]

15. Timchenko, L.T., Miller, J.W., Timchenko, N.A., DeVore, D.R., Datar, K.V., Lin, L., Roberts, R., Caskey, C.T. and Swanson, M.S. (1996) Identification of a (CUG)n triplet repeat RNA-binding protein and its expression in myotonic dystrophy. Nucl. Acids Res., 24, 4407–4414.[Abstract/Free Full Text]

16. Tian, B., White, R.J., Xia, T., Welle, S., Turner, D.H., Mathews, M.B. and Thornton, C.A. (2000) Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR. RNA 6, 79–87.[Abstract]

17. Miller, J.W., Urbinati, C.R., Teng-Umnuay, P., Stenberg, M.G., Byrne, B.J., Thornton, C.A. and Swanson, M.S. (2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J., 19, 4439–4448.[CrossRef][Web of Science][Medline]

18. Mankodi, A., Urbinati, C.R., Yuan, Q.P., Moxley, R.T., Sansone, V., Krym, M., Henderson, D., Schalling, M., Swanson, M.S. and Thornton, C.A. (2001) Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum. Mol. Genet., 10, 2165–2170.[Abstract/Free Full Text]

19. Fardaei, M., Larkin, K., Brook, J.D. and Hamshere, M.G. (2001) In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts. Nucl. Acids Res., 29, 2766–2771.[Abstract/Free Full Text]

20. Fardaei, M., Rogers, M.T., Thorpe, H.M., Larkin, K., Hamshere, M.G., Harper, P.S. and Brook, J.D. (2002) Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum. Mol. Genet., 11, 805–814.[Abstract/Free Full Text]

21. Kanadia, R.N., Johnstone, K.A., Mankodi, A., Lungu, C., Thornton, C.A., Esson, D., Timmers, A.M., Hauswirth, W.W. and Swanson, M.S. (2003) A muscleblind knockout model for myotonic dystrophy. Science, 302, 1978–1980.[Abstract/Free Full Text]

22. Otten, A.D. and Tapscott, S.J. (1995) Triplet repeat expansion in myotonic dystrophy alters the adjacent chromatin structure. Proc. Natl Acad. Sci. USA, 92, 5465–5469.[Abstract/Free Full Text]

23. Klesert, T.R., Otten, A.D., Bird, T.D. and Tapscott, S.J. (1997) Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. Nat. Genet., 16, 402–406.[CrossRef][Web of Science][Medline]

24. Thornton, C.A., Wymer, J.P., Simmons, Z., McClain, C. and Moxley, R.T. (1997) Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene. Nat. Genet., 16, 407–409.[CrossRef][Web of Science][Medline]

25. Berul, C.I., Maguire, C.T., Aronovitz, M.J., Greenwood, J., Miller, C., Gehrmann, J., Housman, D., Mendelsohn, M.E. and Reddy, S. (1999) DMPK dosage alterations result in atrioventricular conduction abnormalities in a mouse myotonic dystrophy model. J. Clin. Invest, 103, R1–R7.[Medline]

26. Klesert, T.R., Cho, D.H., Clark, J.I., Maylie, J., Adelman, J., Snider, L., Yuen, E.C., Soriano, P. and Tapscott, S.J. (2000) Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nat. Genet., 25, 105–109.[CrossRef][Web of Science][Medline]

27. Sarkar, P.S., Appukuttan, B., Han, J., Ito, Y., Ai, C., Tsai, W., Chai, Y., Stout, J.T. and Reddy, S. (2000) Heterozygous loss of Six5 in mice is sufficient to cause ocular cataracts. Nat. Genet., 25, 110–114.[CrossRef][Web of Science][Medline]

28. Lam, L.T., Pham, Y.C., Nguyen, T.M. and Morris, G.E. (2000) Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart. Hum. Mol. Genet., 9, 2167–2173.[Abstract/Free Full Text]

29. Skinner, P.J., Koshy, B.T., Cummings, C.J., Klement, I.A., Helin, K., Servadio, A., Zoghbi, H.Y. and Orr, H.T. (1997) Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures. Nature, 389, 971–974.[CrossRef][Medline]

30. Kanadia, R.N., Urbinati, C.R., Crusselle, V.J., Luo, D., Lee, Y.J., Harrison, J.K., Oh, S.P. and Swanson, M.S. (2003) Developmental expression of mouse muscleblind genes Mbnl1, Mbnl2 and Mbnl3. Gene Expression Patterns, 3, 459–462.[Medline]

31. Jansen, G., Groenen, P.J., Bachner, D., Jap, P.H., Coerwinkel, M., Oerlemans, F., van den, B.W., Gohlsch, B., Pette, D., Plomp, J.J. et al. (1996) Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice. Nat. Genet., 13, 316–324.[CrossRef][Web of Science][Medline]

32. Reddy, S., Smith, D.B., Rich, M.M., Leferovich, J.M., Reilly, P., Davis, B.M., Tran, K., Rayburn, H., Bronson, R., Cros, D. et al. (1996) Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy. Nat. Genet., 13, 325–335.[CrossRef][Web of Science][Medline]

33. Mankodi, A., Logigian, E., Callahan, L., McClain, C., White, R., Henderson, D., Krym, M. and Thornton, C.A. (2000) Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science, 289, 1769–1773.[Abstract/Free Full Text]

34. Seznec, H., Agbulut, O., Sergeant, N., Savouret, C., Ghestem, A., Tabti, N., Willer, J.C., Ourth, L., Duros, C., Brisson, E. et al. (2001) Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities. Hum. Mol. Genet., 10, 2717–2726.[Abstract/Free Full Text]

35. Schulz, P.E., McIntosh, A.D., Kasten, M.R., Wieringa, B. and Epstein, H.F. (2003) A role for myotonic dystrophy protein kinase in synaptic plasticity. J. Neurophysiol., 89, 1177–1186.[Abstract/Free Full Text]

36. Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L. and Bates, G.P. (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell, 90, 537–548.[CrossRef][Web of Science][Medline]

37. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P. and Aronin, N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science, 277, 1990–1993.[Abstract/Free Full Text]

38. Haltia, M., Somer, H., Palo, J. and Johnson, W.G. (1984) Neuronal intranuclear inclusion disease in identical twins. Ann. Neurol., 15, 316–321.[CrossRef][Web of Science][Medline]

39. Greco, C.M., Hagerman, R.J., Tassone, F., Chudley, A.E., Del Bigio, M.R., Jacquemont, S., Leehey, M. and Hagerman, P.J. (2002) Neuronal intranuclear inclusions in a new cerebellar tremor/ataxia syndrome among fragile X carriers. Brain, 125, 1760–1771.[Abstract/Free Full Text]

40. Klement, I.A., Skinner, P.J., Kaytor, M.D., Yi, H., Hersch, S.M., Clark, H.B., Zoghbi, H.Y. and Orr, H.T. (1998) Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell, 95, 41–53.[CrossRef][Web of Science][Medline]

41. Sisodia, S.S. (1998) Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial? Cell, 95, 1–4.[CrossRef][Web of Science][Medline]

42. Liquori, C.L., Ricker, K., Moseley, M.L., Jacobsen, J.F., Kress, W., Naylor, S.L., Day, J.W. and Ranum, L.P. (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science, 293, 864–867.[Abstract/Free Full Text]

43. van der Ven, P.F., Jansen, G., van Kuppevelt, T.H., Perryman, M.B., Lupa, M., Dunne, P.W., ter Laak, H.J., Jap, P.H., Veerkamp, J.H., Epstein, H.F. et al. (1993) Myotonic dystrophy kinase is a component of neuromuscular junctions. Hum. Mol. Genet., 2, 1889–1894.[Abstract/Free Full Text]

44. Whiting, E.J., Waring, J.D., Tamai, K., Somerville, M.J., Hincke, M., Staines, W.A., Ikeda, J.E. and Korneluk, R.G. (1995) Characterization of myotonic dystrophy kinase (DMK) protein in human and rodent muscle and central nervous tissue. Hum. Mol. Genet., 4, 1063–1072.[Abstract/Free Full Text]

45. Pham, Y.C., Man, N., Lam, L.T. and Morris, G.E. (1998) Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies. Hum. Mol. Genet., 7, 1957–1965.[Abstract/Free Full Text]

46. Lam, L.T., Pham, Y.C., Nguyen, T.M. and Morris, G.E. (2000) Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart. Hum. Mol. Genet., 9, 2167–2173.[Abstract/Free Full Text]

47. Sarkar, P.S., Han, J. and Reddy, S. (2004) In situ hybridization analysis of Dmpk mRNA in adult mouse tissues. Neuromuscul. Disord., 14, 497–506.[CrossRef][Web of Science][Medline]

48. Pal, R., Agbas, A., Bao, X., Hui, D., Leary, C., Hunt, J., Naniwadekar, A., Michaelis, M.L., Kumar, K.N. and Michaelis, E.K. (2003) Selective dendrite-targeting of mRNAs of NR1 splice variants without exon 5: identification of a cis-acting sequence and isolation of sequence-binding proteins. Brain Res., 994, 1–18.[CrossRef][Web of Science][Medline]

49. Durand, G.M., Bennett, M.V. and Zukin, R.S. (1993) Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C. Proc. Natl Acad. Sci. USA, 90, 6731–6735.[Abstract/Free Full Text]

50. Traynelis, S.F., Hartley, M. and Heinemann, S.F. (1995) Control of proton sensitivity of the NMDA receptor by RNA splicing and polyamines. Science, 268, 873–876.[Abstract/Free Full Text]

51. Tsien, J.Z., Huerta, P.T. and Tonegawa, S. (1996) The essential role of hippocampal CA1 NMDA receptor-dependent synaptic plasticity in spatial memory. Cell, 87, 1327–1338.[CrossRef][Web of Science][Medline]

52. Tonegawa, S., Tsien, J.Z., McHugh, T.J., Huerta, P., Blum, K.I. and Wilson, M.A. (1996) Hippocampal CA1-region-restricted knockout of NMDAR1 gene disrupts synaptic plasticity, place fields, and spatial learning. Cold Spring Harb. Symp. Quant. Biol., 61, 225–238.[Abstract/Free Full Text]

53. Portwood, M.M., Wicks, J.J., Lieberman, J.S. and Duveneck, M.J. (1986) Intellectual and cognitive function in adults with myotonic muscular dystrophy. Arch. Phys. Med. Rehabil., 67, 299–303.[Web of Science][Medline]

54. Rubinsztein, J.S., Rubinsztein, D.C., McKenna, P.J., Goodburn, S. and Holland, A.J. (1997) Mild myotonic dystrophy is associated with memory impairment in the context of normal general intelligence. J. Med. Genet., 34, 229–233.[Abstract/Free Full Text]

55. Goedert, M., Spillantini, M.G., Jakes, R., Rutherford, D. and Crowther, R.A. (1989) Multiple isoforms of human microtubule-associated protein tau: sequences and localization in neurofibrillary tangles of Alzheimer's disease. Neuron, 3, 519–526.[CrossRef][Web of Science][Medline]

56. Hong, M., Zhukareva, V., Vogelsberg-Ragaglia, V., Wszolek, Z., Reed, L., Miller, B.I., Geschwind, D.H., Bird, T.D., McKeel, D., Goate, A. et al. (1998) Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science, 282, 1914–1917.[Abstract/Free Full Text]

57. Goedert, M. and Jakes, R. (1990) Expression of separate isoforms of human tau protein: correlation with the tau pattern in brain and effects on tubulin polymerization. EMBO J., 9, 4225–4230.[Web of Science][Medline]

58. Hutton, M., Lendon, C.L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., Grover, A. et al. (1998) Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature, 393, 702–705.[CrossRef][Medline]

59. Spillantini, M.G., Murrell, J.R., Goedert, M., Farlow, M.R., Klug, A. and Ghetti, B. (1998) Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl Acad. Sci. USA, 95, 7737–7741.[Abstract/Free Full Text]

60. Poorkaj, P., Bird, T.D., Wijsman, E., Nemens, E., Garruto, R.M., Anderson, L., Andreadis, A., Wiederholt, W.C., Raskind, M. and Schellenberg, G.D. (1998) Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol., 43, 815–825.[CrossRef][Web of Science][Medline]

61. D'Souza, I., Poorkaj, P., Hong, M., Nochlin, D., Lee, V.M., Bird, T.D. and Schellenberg, G.D. (1999) Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements. Proc. Natl Acad. Sci. USA, 96, 5598–5603.[Abstract/Free Full Text]

62. Lee, V.M., Goedert, M. and Trojanowski, J.Q. (2001) Neurodegenerative tauopathies. Annu. Rev. Neurosci., 24, 1121–1159.[CrossRef][Web of Science][Medline]

63. Stanford, P.M., Shepherd, C.E., Halliday, G.M., Brooks, W.S., Schofield, P.W., Brodaty, H., Martins, R.N., Kwok, J.B. and Schofield, P.R. (2003) Mutations in the tau gene that cause an increase in three repeat tau and frontotemporal dementia. Brain, 126, 814–826.[Abstract/Free Full Text]

64. Foster, N.L., Wilhelmsen, K., Sima, A.A., Jones, M.Z., D'Amato, C.J. and Gilman, S. (1997) Frontotemporal dementia and parkinsonism linked to chromosome 17: a consensus conference. Ann. Neurol., 41, 706–715.[CrossRef][Web of Science][Medline]

65. Kiuchi, A., Otsuka, N., Namba, Y., Nakano, I. and Tomonaga, M. (1991) Presenile appearance of abundant Alzheimer's neurofibrillary tangles without senile plaques in the brain in myotonic dystrophy. Acta Neuropathol., 82, 1–5.[CrossRef][Medline]

66. Yoshimura, N., Otake, M., Igarashi, K., Matsunaga, M., Takebe, K. and Kudo, H. (1990) Topography of Alzheimer's neurofibrillary change distribution in myotonic dystrophy. Clin. Neuropathol., 9, 234–239.[Web of Science][Medline]

67. Vermersch, P., Sergeant, N., Ruchoux, M.M., Hofmann-Radvanyi, H., Wattez, A., Petit, H., Dwailly, P. and Delacourte, A. (1996) Specific tau variants in the brains of patients with myotonic dystrophy. Neurology, 47, 711–717.[Abstract/Free Full Text]

68. Sergeant, N., Sablonniere, B., Schraen-Maschke, S., Ghestem, A., Maurage, C.A., Wattez, A., Vermersch, P. and Delacourte, A. (2001) Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1. Hum. Mol. Genet., 10, 2143–2155.[Abstract/Free Full Text]

69. Ishihara, T., Zhang, B., Higuchi, M., Yoshiyama, Y., Trojanowski, J.Q. and Lee, V.M. (2001) Age-dependent induction of congophilic neurofibrillary tau inclusions in tau transgenic mice. Am. J. Pathol., 158, 555–562.[Abstract/Free Full Text]

70. Probst, A., Gotz, J., Wiederhold, K.H., Tolnay, M., Mistl, C., Jaton, A.L., Hong, M., Ishihara, T., Lee, V.M., Trojanowski, J.Q. et al. (2000) Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein. Acta Neuropathol., 99, 469–481.[CrossRef][Medline]

71. Artero, R., Prokop, A., Paricio, N., Begemann, G., Pueyo, I., Mlodzik, M., Perez-Alonso, M. and Baylies, M.K. (1998) The muscleblind gene participates in the organization of Z-bands and epidermal attachments of Drosophila muscles and is regulated by Dmef2. Dev. Biol., 195, 131–143.[CrossRef][Web of Science][Medline]

72. Ho, T.H., Charlet, B., Poulos, M.G., Singh, G., Swanson, M.S. and Cooper, T.A. (2004) Muscleblind proteins regulate alternative splicing. EMBO J., 23, 3103–3112.[CrossRef][Web of Science][Medline]

73. Ladd, A.N., Charlet, N. and Cooper, T.A. (2001) The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol. Cell. Biol., 21, 1285–1296.[Abstract/Free Full Text]

74. Lu, X., Timchenko, N.A. and Timchenko, L.T. (1999) Cardiac elav-type RNA-binding protein (ETR-3) binds to RNA CUG repeats expanded in myotonic dystrophy. Hum. Mol. Genet., 8, 53–60.[Abstract/Free Full Text]

75. Michalowski, S., Miller, J.W., Urbinati, C.R., Paliouras, M., Swanson, M.S. and Griffith, J. (1999) Visualization of double-stranded RNAs from the myotonic dystrophy protein kinase gene and interactions with CUG-binding protein. Nucl. Acids Res., 27, 3534–3542.[Abstract/Free Full Text]

76. Mankodi, A., Teng-Umnuay, P., Krym, M., Henderson, D., Swanson, M. and Thornton, C.A. (2003) Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann. Neurol., 54, 760–768.[CrossRef][Web of Science][Medline]

77. Zhang, W., Liu, H., Han, K. and Grabowski, P.J. (2002) Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR. RNA, 8, 671–685.[Abstract]

78. Wang, J., Gao, Q.S., Wang, Y., Lafyatis, R., Stamm, S. and Andreadis, A. (2004) Tau exon 10, whose missplicing causes frontotemporal dementia, is regulated by an intricate interplay of cis elements and trans factors. J. Neurochem., 88, 1078–1090.[CrossRef][Web of Science][Medline]

79. Good, P.J., Chen, Q., Warner, S.J. and Herring, D.C. (2000) A family of human RNA-binding proteins related to the Drosophila Bruno translational regulator. J. Biol. Chem., 275, 28583–28592.[Abstract/Free Full Text]

80. Dreyfuss, G., Kim, V.N. and Kataoka, N. (2002) Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell. Biol., 3, 195–205.[CrossRef][Web of Science][Medline]

81. Min, H., Chan, R.C. and Black, D.L. (1995) The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event. Genes Dev., 9, 2659–2671.[Abstract/Free Full Text]

82. Chou, M.Y., Rooke, N., Turck, C.W. and Black, D.L. (1999) hnRNP H is a component of a splicing enhancer complex that activates a c-src alternative exon in neuronal cells. Mol. Cell. Biol., 19, 69–77.[Abstract/Free Full Text]

83. Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T. and Zoghbi, H.Y. (1998) Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet., 19, 148–154.[CrossRef][Web of Science][Medline]

84. Mitchell, P., Petfalski, E., Shevchenko, A., Mann, M. and Tollervey, D. (1997) The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'5' exoribonucleases. Cell, 91, 457–466.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				T. M. Wheeler, K. Sobczak, J. D. Lueck, R. J. Osborne, X. Lin, R. T. Dirksen, and C. A. Thornton Reversal of RNA Dominance by Displacement of Protein Sequestered on Triplet Repeat RNA Science, July 17, 2009; 325(5938): 336 - 339. [Abstract] [Full Text] [PDF]

				I. Holt, V. Jacquemin, M. Fardaei, C. A. Sewry, G. S. Butler-Browne, D. Furling, J. D. Brook, and G. E. Morris Muscleblind-Like Proteins: Similarities and Differences in Normal and Myotonic Dystrophy Muscle Am. J. Pathol., January 1, 2009; 174(1): 216 - 227. [Abstract] [Full Text] [PDF]

				J. P. Orengo, P. Chambon, D. Metzger, D. R. Mosier, G. J. Snipes, and T. A. Cooper Expanded CTG repeats within the DMPK 3' UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy PNAS, February 19, 2008; 105(7): 2646 - 2651. [Abstract] [Full Text] [PDF]

				E. Ciafaloni, E. Mignot, V. Sansone, J. E. Hilbert, L. Lin, X. Lin, L. C. Liu, W. R. Pigeon, M. L. Perlis, and C. A. Thornton The hypocretin neurotransmission system in myotonic dystrophy type 1 Neurology, January 15, 2008; 70(3): 226 - 230. [Abstract] [Full Text] [PDF]

				M. B. Warf and J. A. Berglund MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T RNA, December 1, 2007; 13(12): 2238 - 2251. [Abstract] [Full Text] [PDF]

				J. P. Chapple, K. Anthony, T. R. Martin, A. Dev, T. A. Cooper, and J.-M. Gallo Expression, localization and tau exon 10 splicing activity of the brain RNA-binding protein TNRC4 Hum. Mol. Genet., November 15, 2007; 16(22): 2760 - 2769. [Abstract] [Full Text] [PDF]

				K. P. Smith, M. Byron, C. Johnson, Y. Xing, and J. B. Lawrence Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains J. Cell Biol., September 7, 2007; 178(6): 951 - 964. [Abstract] [Full Text] [PDF]

				I. Holt, S. Mittal, D. Furling, G. S Butler-Browne, J. D. Brook, and G. E. Morris Defective mRNA in myotonic dystrophy accumulates at the periphery of nuclear splicing speckles Genes Cells, September 1, 2007; 12(9): 1035 - 1048. [Abstract] [Full Text] [PDF]

				Y. Yuan, S. A. Compton, K. Sobczak, M. G. Stenberg, C. A. Thornton, J. D. Griffith, and M. S. Swanson Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs Nucleic Acids Res., August 15, 2007; (2007) gkm601v1. [Abstract] [Full Text] [PDF]

				M. F. Mehler and J. S. Mattick Noncoding RNAs and RNA Editing in Brain Development, Functional Diversification, and Neurological Disease Physiol Rev, July 1, 2007; 87(3): 799 - 823. [Abstract] [Full Text] [PDF]

				J. D. Lueck, C. Lungu, A. Mankodi, R. J. Osborne, S. L. Welle, R. T. Dirksen, and C. A. Thornton Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1 Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1291 - C1297. [Abstract] [Full Text] [PDF]

				R. J. Osborne and C. A. Thornton RNA-dominant diseases Hum. Mol. Genet., October 15, 2006; 15(suppl_2): R162 - R169. [Abstract] [Full Text] [PDF]

				M. F. Mehler and J. S. Mattick Non-coding RNAs in the nervous system J. Physiol., September 1, 2006; 575(2): 333 - 341. [Abstract] [Full Text] [PDF]

				R. N. Kanadia, J. Shin, Y. Yuan, S. G. Beattie, T. M. Wheeler, C. A. Thornton, and M. S. Swanson Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy PNAS, August 1, 2006; 103(31): 11748 - 11753. [Abstract] [Full Text] [PDF]

				M. de Haro, I. Al-Ramahi, B. De Gouyon, L. Ukani, A. Rosa, N. A. Faustino, T. Ashizawa, T. A. Cooper, and J. Botas MBNL1 and CUGBP1 modify expanded CUG-induced toxicity in a Drosophila model of myotonic dystrophy type 1 Hum. Mol. Genet., July 1, 2006; 15(13): 2138 - 2145. [Abstract] [Full Text] [PDF]

				P. Garcia, Z. Ucurum, R. Bucher, D. I. Svergun, T. Huber, A. Lustig, P. V. Konarev, M. Marino, and O. Mayans Molecular insights into the self-assembly mechanism of dystrophia myotonica kinase FASEB J, June 1, 2006; 20(8): 1142 - 1151. [Abstract] [Full Text] [PDF]

				E. Bonifazi, F. Gullotta, L. Vallo, R. Iraci, A. M. Nardone, E. Brunetti, A. Botta, and G. Novelli Use of RNA Fluorescence In Situ Hybridization in the Prenatal Molecular Diagnosis of Myotonic Dystrophy Type I Clin. Chem., February 1, 2006; 52(2): 319 - 322. [Abstract] [Full Text] [PDF]

				C. L. ZHENG, Y.-S. KWON, H.-R. LI, K. ZHANG, G. COUTINHO-MANSFIELD, C. YANG, T. M. NAIR, M. GRIBSKOV, and X.-D. FU MAASE: An alternative splicing database designed for supporting splicing microarray applications RNA, December 1, 2005; 11(12): 1767 - 1776. [Abstract] [Full Text] [PDF]

				A. Mankodi, X. Lin, B. C. Blaxall, M. S. Swanson, and C. A. Thornton Nuclear RNA Foci in the Heart in Myotonic Dystrophy Circ. Res., November 25, 2005; 97(11): 1152 - 1155. [Abstract] [Full Text] [PDF]

				C. A. Maurage, B. Udd, M. M. Ruchoux, P. Vermersch, H. Kalimo, R. Krahe, A. Delacourte, and N. Sergeant Similar brain tau pathology in DM2/PROMM and DM1/Steinert disease Neurology, November 22, 2005; 65(10): 1636 - 1638. [Abstract] [Full Text] [PDF]

				J.-M. Gallo, P. Jin, C. A. Thornton, H. Lin, J. Robertson, I. D'Souza, and W. W. Schlaepfer The Role of RNA and RNA Processing in Neurodegeneration J. Neurosci., November 9, 2005; 25(45): 10372 - 10375. [Full Text] [PDF]

				T. Kimura, M. Nakamori, J. D. Lueck, P. Pouliquin, F. Aoike, H. Fujimura, R. T. Dirksen, M. P. Takahashi, A. F. Dulhunty, and S. Sakoda Altered mRNA splicing of the skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in myotonic dystrophy type 1 Hum. Mol. Genet., August 1, 2005; 14(15): 2189 - 2200. [Abstract] [Full Text] [PDF]

				D.-H. Kim, M.-A. Langlois, K.-B. Lee, A. D. Riggs, J. Puymirat, and J. J. Rossi HnRNP H inhibits nuclear export of mRNA containing expanded CUG repeats and a distal branch point sequence Nucleic Acids Res., July 15, 2005; 33(12): 3866 - 3874. [Abstract] [Full Text] [PDF]

				T. H. Ho, R. S. Savkur, M. G. Poulos, M. A. Mancini, M. S. Swanson, and T. A. Cooper Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy J. Cell Sci., July 1, 2005; 118(13): 2923 - 2933. [Abstract] [Full Text] [PDF]

				T. H. Ho, D. Bundman, D. L. Armstrong, and T. A. Cooper Transgenic mice expressing CUG-BP1 reproduce splicing mis-regulation observed in myotonic dystrophy Hum. Mol. Genet., June 1, 2005; 14(11): 1539 - 1547. [Abstract] [Full Text] [PDF]

				C. J. McLeod, L. V. O'Keefe, and R. I. Richards The pathogenic agent in Drosophila models of 'polyglutamine' diseases Hum. Mol. Genet., April 15, 2005; 14(8): 1041 - 1048. [Abstract] [Full Text] [PDF]

				Y. Wang, J. Wang, L. Gao, R. Lafyatis, S. Stamm, and A. Andreadis Tau Exons 2 and 10, Which Are Misregulated in Neurodegenerative Diseases, Are Partly Regulated by Silencers Which Bind a SRp30c{middle dot}SRp55 Complex That Either Recruits or Antagonizes htra2{beta}1 J. Biol. Chem., April 8, 2005; 280(14): 14230 - 14239. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Supplementary Data All Versions of this Article: 13/24/3079    most recent ddh327v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by Jiang, H. Articles by Thornton, C. A. Search for Related Content PubMed PubMed Citation Articles by Jiang, H. Articles by Thornton, C. A. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics