Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of...

Myotonic dystrophy type 1 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. Thornton1

1Department of Neurology, University of Rochester School of Medicine and Dentistry, Box 673, 601

Elmwood Avenue, Rochester, NY 14642.

2Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, University of

Florida College of Medicine, Gainesville, FL 32610.

Corresponding Author:

Charles A. Thornton, MD

Department of Neurology Box 673 Room 5-5207

601 Elmwood Avenue

University of Rochester Medical Center

Rochester, NY 14642

Tel 585 275 2542

Fax 585 273 1255

Email [email protected]

Copyright © 2004 Oxford University Press

HMG Advance Access published October 20, 2004 by guest on M

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ABSTRACT

Myotonic dystrophy type 1 (DM1) is caused by expansion of a CTG repeat in the DMPK gene. In

skeletal muscle, 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.

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Introduction

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 core 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

by Harper (1) and Ashizawa (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 1,000 –

4,000 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



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(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 γ

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 the 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 if 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.



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Results

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.

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 Fig. 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) for neuronal markers NeuN (Fig. 1B) or MAP2 (Fig. 1C).

Counts of 100 NeuN-positive cells from temporal and frontal cortex of 4 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



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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 (each of 4

patients examined) (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 (area × intensity) than in Purkinje cells (p<10-10) and 18-fold greater than in

oligodendrocytes (p<10-10) 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 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, area × intensity) in frontal cortical neurons than in skeletal muscle from the same individual

(p<10-10, Fig. S2).

To determine if 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 4 DM1 and 3 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



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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 if 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 (20Sα, 11Sγ and 11Sα subunits) were recruited to RNA foci in

cortical neurons (Fig. 2A; Fig. S3A). We did not, however, find evidence for ubiquitination or

sumoylation of the foci (not shown). In contrast, antibodies to the PM/Scl75 or PM/Scl100 components

of the exosome did not colocalize with RNA foci (Fig. 2B)

Monoclonal antibody 3B1 showed 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 (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 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 Fig S3D). In contrast,

hnRNPs H and F colocalized with foci in cortical neurons to a limited extent (Fig. 2F, 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.



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Mutant DMPK mRNA is reported to interact with transcription factors retinoic acid receptor

gamma (RARγ) and Sp1 (14). In cortical neurons, these transcription factors were readily detected by

immunofluorescence but they did not colocalize with RNA foci (Figs. 2I and S3E) and their distribution

was similar in DM1 patients and controls (Fig. 2I and 2J).

Polyclonal antisera recognizing all members of the muscleblind family (MBNL1, MBNL2, and

MBNL3) showed strong colocalization with RNA foci (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 3 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 area × intensity units in DM1 patients vs 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.

To determine if 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 (supplemental Table 2).

For each exon, the ratio of inclusion versus exclusion isoforms was determined by reverse transcriptase-

PCR (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



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DM1 patients. Among 45 exons screened, 4 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 7 patients with DM1 and 5 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<10-5), and

decreased inclusion of tau exon 10 (21 ± 1% in DM1, 41 ± 5% in controls, p<10-6).

Discussion

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, if 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),



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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 mis-regulation 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 (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 unpublished observations, C Thornton and X Lin), 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



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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 muscle. Lacking 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



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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 mis-

splicing 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 orthologues 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.



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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



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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 (not

shown). This fits with expectations that the number and density of binding sites on a single transcript,

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

RARγ, “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 RARγ 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

Tissue samples.

Autopsy materials were obtained from ten DM1 patients (mean age 56 years, range 44-78 years, 7

men and 3 women) and 13 controls (6 with no neurologic disease, 2 with Alzheimer disease, 4 with



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Huntington disease, and one with refractory epilepsy). The mean post-mortem interval for DM1 patients

was 6 hours (range 2 to 14 hours). 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 7, sudden

cardiac death in 2). The other DM1 patient had minimal symptoms of DM1 and died at age 78 yrs of

unrelated disease. Genetic confirmation was performed as previously described by PCR or Southern blot

on DNA isolated from postmortem brain tissue (7). Southern blots of cortical DNA samples showed a

broad range of expanded alleles ranging in size from 5 to 12 kb (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.

Fluorescence in situ hybridization (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 2 X SSC 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, 2 X SSC, 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 1X SSC for 30 min at room temperature. Probes were HPLC-

purified 2-O-methyl RNA 20-mers (IDT, Coralville, IA) 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 1,000-fold 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 v6.0 software (Imaging Research Inc., St. Catherines, Ontario).



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Immunofluorescence (IF) combined with FISH

Following the 1 X SSC post-hybridization wash of the FISH procedure, sections were incubated in

primary antibodies (listed in supplemental Table 1) 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 vs 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 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 7 DM1 patients and 5 non-neurologic disease controls using TriReagent (Molecular Research

Center, Cincinnati). 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 (Table 2). PCR products were resolved

on agarose gels, stained with SybrGreenII (Molecular Probes), and analyzed on a fluorimager. An initial



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screen was performed on a subset of samples (4 DM1 and 2 control). Four exons appeared to show

deregulated splicing in DM1. These differences were quantified in a second experiment including the full

panel of 7 DM1 and 5 control samples. The fraction of exon inclusion was determined on triplicate

reactions using ImageQuant software (Amersham, Piscataway).



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ACKNOWLEDGEMENTS

The authors thank Don Henderson for excellent technical assistance. 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. 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, KL Chan, E. Wagner for gifts of antibodies. This work comes from the

University of Rochester Paul D. Wellstone Muscular Dystrophy Cooperative Research Center

(AR050762).



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FIGURE LEGENDS

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 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.

Figure 2. FISH and IF on sections of temporal or frontal cortical neurons show colocalization of mutant

DMPK mRNA [(CUG)n] with 20Sα 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 to 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). RARγ does not colocalize with RNA foci in DM1 cortical neurons (I). The distribution of

RARγ in the DM1 (I) and non-neurologic-disease (J) neuronal nucleus is similar. Bar, 5 µm, applies to

all panels.



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Figure 3. MBNL1 is decreased in nucleoplasm of DM1 cortical neurons. Immunofluorescence (area ×

intensity) for MBNL1 in the nucleus, excluding nucleolus and RNA foci, was determined for 20 neurons

in sections of temporal cortex from 3 individuals with DM1 and 3 controls without neurologic disease

(C).

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.

Figure S1. RNA foci in dentate gyrus and subcortical neurons in DM1. FISH (CAG repeat probe, red)

merged with IF (anti-NeuN antibody, green) and nuclear stain (DAPI, blue). SN, substantia nigra. Bar, 5

µm, applies to all panels.

Figure S2. Foci of mutant RNA in neuronal and muscle nuclei. A. When processed on the same slide

and imaged under the same exposure settings, RNA foci in frontal cortex were larger and more intensely

fluorescent than in biceps muscle from the same patient. B. In paired samples of frontal cortex and

skeletal muscle from the same patient, fluorescence area × intensity of RNA foci was 3.1-fold greater in

nuclei of cortical neurons than in skeletal muscle, n=3 patients, 20 nuclei per sample (p<10-10).

Figure S3. FISH combined with IF on sections of DM1 temporal or frontal cortex. FISH (CAG repeat

probe, red, left panels) and IF (middle panels, antibody to indicated protein, green) are merged with

nuclear stain (DAPI, blue) in right panels. CUG expansion RNA colocalizes with proteasome 11Sγ



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subunit (A) and hnRNP H (C) but not with double-stranded RNA binding protein ADAR1 (B), hnRNP M

(D), or Sp1 (E). Bar, 5 µm, applies to all panels.



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Supplemental table 1

Target antigen Final Dilution Source

muscleblind

(MBNL1)

mAb (3B10): 1:1500

pAb (EXP 42) : 1:1500

M. Swanson, FL

muscleblind

(MBNL2)

mAb (2D9): 1:10,000

M. Swanson, FL

CUGBP1 mAb (3B1): 1:500 M. Swanson, FL

CELF4 pAb (#440): 1:500

T. Cooper, TX

ETR3 pAb ( #163): 1:500

T. Cooper, TX

PKR pAb (pT451): 1:500

pAb (M515):1:500

pAb (D20): 1:500

mAb (B10): 1:500

Biosource International,CA

Santa Cruz, CA

Santa Cruz, CA

Santa Cruz, CA

RNA helicase A pAb: 1:500

C. Lee, NJ

ADAR1 mAb: 1:500 D. Cho, PA

HRBP pAb (#1683):1:500

C. Thornton

NF90 (DRBP76) pAb (p90 AB4): 1:500

G. Sen, OH

Staufen pAb 1:500 (AB 5819)

Chemicon, Temecula, CA

Proteosome

19S S10a

11S α

11S γ

20S β3 (HC10)

20S α

pAb: 1:500

pAb: 1:1000

pAb: 1:1000

mAb: 1:500

pAb: 1:1000

Affiniti, Exeter, UK



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Ubiquitin pAb: 1:1000 DAKO, Glostrup, Denmark

p80 coilin pAb (R288):1:500 KL Chan, CA

C23 nucleolin mAb: 1:500

Santa Cruz, CA

PML pAb: 1:500

Santa Cruz, CA

PTB pAb: 1:500

E. Wagner, NC

PM-Scl 75 pAb: 1:500

J. Wilusz, Newark

hnRNP H

C-terminal

N-terminal

pAb: 1:1000

pAb: 1: 500

J. Wilusz, Newark

hnRNP F pAb: 1:1000

J. Wilusz, Newark

hnRNP H

hnRNP F

hnRNPI /PTB

KSRP

4F4 (hnRNP C)

1D8 (hnRNP M)

pAb: 1:1000

pAb: 1:1000

pAb: 1:1000

pAb: 1:1000

mAb: 1:500

mAb: 1:500

Doug Black, CA

M. Swanson, FL

CNPase

mAb: 1:1000 Sigma, St. Louis, MO

MAP2 mAb: 1:500

Sigma, St. Louis, MO

NeuN mAb: 1:500

Chemicon, Temecula, CA

Sp1 (sc-59) pAb: 1:500

Santa Cruz Biotechnology, Santa Cruz,

CA

RARγ(sc-550) pAb: 1:500

Santa Cruz Biotechnology, Santa Cruz,

CA



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Staufen (AB5819) pAb: 1:500

Chemicon, Temecula, CA92590

SUMO-1 mAb

Zymed Laboratories Inc., South San

Francisco. CA94080

Supplemental Table 1. Sources for primary antibodies. mAb, monoclonal antibody; pAb, polyclonal

antibody.



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Supplemental table 2

Gene Name Unigene

Alternatively

spliced exon Acc. No. Nucleotides

Amyloid beta (A4) precursor protein APP ex2 NM_000484 205-372

ex7 NM_000484 1013-1180

ex15 NM_000484 1181-1237

Actin-related protein 3-beta ARP3BETA ex2 BC008682.1 134-189

Beta-site APP-cleaving enzyme 2 BACE2 ex9 NM_012105 1448-1597

ex10 NM_012105 1598-1766

Neuronal apoptosis inhibitory protein BIRC1 ex10-11 NM_004536 1314-1453

Calcium channel, voltage-dependent, P/Q

type, alpha 1A subunit

CACNA1A ex38 AF004883 5783-5879

Calcium/calmodulin protein kinase II

dependent delta

CAMK2D alt splic donor in

ex21(44bp)

NM_172127 1709-1981

Clathrin light chain B CLTB exon casette NM_007097 641-694

Homo sapiens discs, large homolog 1 DLG1 ex8 NM_004087 771-824

Dopamine receptor type 2 DRD2 ex6 NM_000795 889-975

Erythrocyte membrane protein band 4.1-like 1 EPB41L1 ex5 NM_012156 514-618

ex21 NM_012156 2356-2439

GABA receptor alpha 2 GABRA4 ex3-8 NM_000809 345-1274

Gephyrin GPHN ex9 NM_020806 742-840

ex12 NM_020806 976-1018

LIM domain binding 3 LDB3 ex10 AB 014513 918-1106

NMDA receptor NR1 GRIN1 ex5 AF015730 644-706

ex20 NM_007327 3683-3793

ex21 NM_007327 3794-3910

Neurotrophic tyrosine kinase, receptor, type 2 NTRK2 exon casette AF410901 1878-1926

C-Jun N-terminal kinase 2 JNK2 E6b, E6a NM_002752 666-737

Netrin G1 Ntng2 E5 NM_032536 1115-1138

Neogenin NEO1 ex26 NM_002499 3879-4037

Neurofibromin 1 NF1 ex9a NT010799 108004-108033

Neuronatin NNAT ex2 NM_005386 200-280



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Neurorexin 1 NRXN1 ex3a NM_004801 965-1024

ex4

ex5

ex7a NM_004801 1327-1250

ex12 NM_004801 2540-2566

Neurorexin 2 NRXN2 ex12 NM_015080 2829-2855

ex20 NM_015080 4197-4286

Neurorexin 3 NRXN3 ex12 NM_004796 1621-1647

NUMB NUMB ex8 AF015040 480-512

ex15 AF015040 1367-1510

Presynaptic cytomatrix protein PICO ex10 AB011131 3056-3082

Peanut-like 2 PNUTL2 ex2 NM_004574 189-579

Protein phosphatase 2, regulatory subunit B

(PR 52), beta isoform

PPP2R2B ex6 MN_181674 364-557

REST/NRSF/SBR REST ex5 AF228045 410-459

Microtubule-associated protein tau MAPT ex2 NM_005910 370-456

ex10 NM_005910 1059-1151

Sarcolemma associated protein SLMAP ex4 NM_007159 273-395

v-src sarcoma (Schmidt-Ruppin A-2) viral

oncogene homolog (avian)

SRC exon casette NM_005417 additional exon

cassette (18bp or

50bp)

Supplemental table 2. List of exons screened for abnormal regulation of alternative splicing in DM1

compared to controls without neurologic disease. “Nucleotides” indicates which portion of the specified

cDNA (GenBank accession number) was amplified by RT-PCR.



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Figure 1



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Figure 2



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Figure 3

C C C DM1 DM1 DM1

MB

NL1

in n

ucle

opla

sm(fl

uore

scen

ce in

tens

ity x

are

a)

0

20

40

60

80

100

120



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Figure 4

4 5 64 6

6 7 96 9

1 2 4

1 4

9 10 119 11

APP

NMDAR1

MAPT

MAPT

4 5 64 64 5 64 5 64 64 6

6 7 96 7 96 96 9

1 2 41 2 4

1 41 4

9 10 119 10 119 119 11

APP

NMDAR1

MAPT

MAPT

A

C DM1 C DM1 C DM1 C DM1 APP NMDAR1 MAPT MAPT ex7 ex5 ex2 ex10

% in

clus

ion

0

10

20

30

40

50

60B

C DM1 C DM1 C DM1 C DM1 APP NMDAR1 MAPT MAPT ex7 ex5 ex2 ex10

% in

clus

ion

0

10

20

30

40

50

60Bcontrol DM1



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Supplemental figure 1



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0

10000

20000

Cortical Neurons Sk. Muscle

Cortical neuron Biceps muscle

Fluo

resc

ence

(are

a ×

inte

nsity

)



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Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of...

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