Reduced cytoplasmic MBNL1 is an early event in a brain-specific mouse model of myotonic dystrophy

Abstract

Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats in the 3’ untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Cognitive impairment associated with structural change in the brain is prevalent in DM1. How this histopathological abnormality during disease progression develops remains elusive. Nuclear accumulation of mutant DMPK mRNA containing expanded CUG RNA disrupting the cytoplasmic and nuclear activities of muscleblind-like (MBNL) protein has been implicated in DM1 neural pathogenesis. The association between MBNL dysfunction and morphological changes has not been investigated. We generated a mouse model for postnatal expression of expanded CUG RNA in the brain that recapitulates the features of the DM1 brain, including the formation of nuclear RNA and MBNL foci, learning disability, brain atrophy and misregulated alternative splicing. Characterization of the pathological abnormalities by a time-course study revealed that hippocampus-related learning and synaptic potentiation were impaired before structural changes in the brain, followed by brain atrophy associated with progressive reduction of axon and dendrite integrity. Moreover, cytoplasmic MBNL1 distribution on dendrites decreased before dendrite degeneration, whereas reduced MBNL2 expression and altered MBNL-regulated alternative splicing was evident after degeneration. These results suggest that the expression of expanded CUG RNA in the DM1 brain results in neurodegenerative processes, with reduced cytoplasmic MBNL1 as an early event response to expanded CUG RNA.

Introduction

Cognitive and behavioral abnormalities vary among people with myotonic dystrophy type 1 (DM1) and include apathy, excessive daytime sleepiness, mental retardation, attention deficit, hyperactivity, and psychiatric disorders (1,2). MRI evidence has shown the association of cognitive impairment and presence of white matter lesions (3,4), atrophy in corpus callosum and cerebral cortex, and reduced grey and white matter (5–7) in the DM1 brain. Thus, structural change is a major feature of the DM1 brain. Altered brain structure, such as neuronal morphologic features and connections, adversely affects neuronal functions such as synaptic transmission, thus resulting in cognitive impairment. However, the development of such histopathological abnormalities in the DM1 brain is poorly understood.

The genetic basis of DM1 is expanded CTG repeats in the 3’ untranslated region (UTR) of dystrophia myotonia protein kinase (DMPK). DMPK mRNA containing CUG repeats accumulates in nuclear foci and affects the cytoplasmic and nuclear functions of muscleblind-like (MBNL) and CUGBP and ETR3-like factor (CELF) (8). The pathogenic RNA binds and sequesters MBNL and induces protein kinase C (PKC)-mediated CELF1 hyperphosphorylation and upregulation, thereby leading to misregulated alternative splicing of specific pre-mRNAs in DM1 tissues (9–11). The DMSXL mouse model, expressing a large CTG expansion in many tissues including the brain, has demonstrated that expanded CUG RNA in the brain causes misregulation of alternative splicing, growth retardation, and neurotransmission dysfunctions (12–15). However, because of the severe defects occurring in other tissues, it is unclear whether expanded CUG RNA contributes to impaired neurodevelopment or neurodegeneration or both that account for the abnormalities. Mbnl knockout models, including Mbnl1^ΔE3/ΔE3 and Mbnl2^ΔE2/ΔE2, show different features of the DM1 brain, including misregulated RNA processing, motivation and spatial learning deficits, and abnormal REM sleep propensity (16,17), which suggests a role for MBNL dysfunction in DM1 neural pathogenesis. However, the basis of the behavioral abnormalities caused by expanded CUG RNA or MBNL dysfunction is largely unclear. How expanded CUG RNA affects the brain structure or whether MBNL dysfunction contributes to abnormal brain connectivity resulting in behavioral defects remains unclear.

To study the histopathological abnormalities caused by expanded CUG RNA after the development of the nervous system, we generated a mouse model expressing 960 CUG RNA repeats in the postnatal brain (18). Adult mice expressing expanded CUG RNA in the brain exhibited several features of DM1, including learning disability, brain atrophy-associated morphological defects in axons and dendrites and misregulation of alternative splicing. Analysis of morphological changes during phenotype progression by a time-course study demonstrated reduced cytoplasmic MBNL1 expression in dendrites at 6 months, followed by reduced axon and dendrite integrity at 9 months and reduced MBNL2 expression and splicing changes at 12 months. These results suggest that reduced MBNL1 cytoplasmic content may be an early event in the neural pathogenesis of DM1.

Results

The EpA960/CaMKII-Cre mouse model recapitulates DM1 molecular features

To establish a brain-specific mouse model for DM1, we crossed the EpA960 line, which carries a transgene expressing an inducible human DMPK 3’ UTR containing 960 copies of CTG repeats (18), with a CaMKII-Cre line (19). The CaMKII promoter-driven Cre expression is activated in the forebrain region, including the cortex and hippocampus, after postnatal 2 weeks and throughout adulthood; therefore brain development during the embryonic stage is preserved (19). We first determined the expression of recombined EpA960 [EpA960(R)] mRNA containing expanded CUG repeats in the forebrain regions, including the cortex and hippocampus, of EpA960 and EpA960/CaMKII-Cre animals. EpA960(R) mRNA was detected in the forebrains of EpA960/CaMKII-Cre mice but not EpA960 mice, which indicates no leakage of EpA960(R) RNA expression from EpA960 animals (Fig. 1A). As well, the level was comparable in EpA960/CaMKII-Cre animals at 6, 9 and 12 months of age (Fig. 1B). In situ hybridization with a Cy3-labeled-(CAG)₇ LNA probe combined with immunofluorescence staining revealed that the EpA960(R) mRNA formed nuclear foci and co-localized with MBNL1 and 2 foci in EpA960/CaMKII-Cre brains, but the same probe did not detect RNA foci in the EpA960 brain (Fig. 1C and D). Therefore, the results suggest that the EpA960/CaMKII-Cre brain develops MBNL-co-localized nuclear RNA foci similar to the pattern seen in the human brain with DM1 (20).

Figure 1

Expression of EpA960(R) mRNA and formation of nuclear RNA and MBNL foci in EpA960/CaMKII-Cre brain. (A) Representative RT-PCR analysis of mRNA expression of EpA960(R) RNA in cortex and hippocampus of EpA960/CaMKII-Cre mice after Cre-mediated recombination. Forebrain regions from the brains of EpA960 mice were used as controls to demonstrate the lack of leakage of EpA960(R) RNA expression from EpA960 animals. (B) RT-qPCR of EpA960(R) mRNA after Cre-mediated recombination at different ages. (n = 5 animals from each group). n.s., not significant. (C) Detection of nuclear RNA foci by in situ hybridization with a Cy3-labeled-(CAG)₇ LNA probe. EpA960 brain was a control. (D) Representative images of combined in situ hybridization with a Cy3-labeled-(CAG)₇ LNA probe and immunostaining to detect nuclear RNA, MBNL1 and MBNL2 foci. CA1 region of hippocampus was imaged by z-stacking and two-dimensionally deconvoluted. Images were computed from a collapsed series of images collected within a 1-μm focus interval optimized to contain as many foci-expressing cells as possible. (n = 3 mice). Arrows indicate cells detected with RNA foci and MBNL foci. Scale: C, D, 15 μm.

EpA960(R) mRNA causes axon and dendrite degeneration in the EpA960/CaMKII-Cre brain

EpA960/CaMKII-Cre mice exhibited a normal lifespan relative to their littermate controls, including EpA960, CaMKII-Cre and wild-type animals. To investigate how expanded CUG RNA causes histopathological abnormalities, we first examined the brain structure of EpA960/CaMKII-Cre animals at age 12 months by immunohistochemistry with anti-NeuN antibody to detect neurons. Compared to all genotype control brains, EpA960/CaMKII-Cre brains showed smaller cortical layers (Fig. 2A-1) and the corpus callosum was thinner (Fig. 2A-2). Atrophy in the cortex and corpus callosum is commonly seen in the human DM1 brain (5,6). With the reduced thickness of the cortex and corpus callosum in the EpA960/CaMKII-Cre brain at 12 months, we wondered whether EpA960(R) mRNA affected axon and dendrite integrity. We examined the integrity of axons and dendrites in the cortex by immunohistochemistry with anti-neurofilament (NF-160) and -microtubule–associated protein 2 (MAP2) antibodies, respectively. The neurofilament immunoreactivity presented as NF160-positive lengths revealed similar axonal projections in cortical layer II/III of genotype control brains (Fig. 2A-3). However, EpA960/CaMKII-Cre brains showed axon shortening as revealed by reduced NF160-positive length. Similarly, as revealed by MAP2-positive length, dendritic processes were less intact and shorter in cortical layer II/III of EpA960/CaMKII-Cre brains than genotype control brains (Fig. 2A-4). Thus, EpA960(R) mRNA expression in the brain affected axon and dendrite integrity.

Figure 2

Expression of EpA960(R) mRNA in brain causes neurodegeneration. (A) Representative immunohistochemical images of atrophy in the cortex (CTX) (1) and corpus callosum (2) of EpA960/CaMKII-Cre mice at age 12 months. Anti-NeuN antibody was used for immunohistochemistry. (3,4) Representative images of EpA960(R) mRNA affecting the integrity of axons (3) and dendrites (4) of cortical-layer II/III neurons in EpA960/CaMKII-Cre brains at age 12 months. Anti-neurofilament-160 (NF-160) and microtubule-associated protein 2 (MAP2) antibodies were used as axon and dendrite markers, respectively. Quantification of thickness of cortex and corpus callosum and length of NF-160-positive and MAP2-positive immunoreactivity. (B, C) Representative images of the integrity of axons (B) and dendrites (C) of cortical-layer II/III neurons in EpA960/CaMKII-Cre brains at 6 and 9 months. (D) Representative images of fluorescence Nissl staining revealed degenerating cells in cortex of EpA960/CaMKII-Cre brain at 12 months. Number of animals (N) in each group is indicated. Comparisons (in A, C, D) are shown as percentage relative to the wildtype. Data are mean ± SEM.*P< 0.05, **P< 0.01, ***P< 0.001, by one-way ANOVA. Scale: A-1, 2 mm; A-3, 4, 100 μm; B, 500 μm; C, 100 μm; D, 30 μm.

Figure 3

Learning disability and neurotransmission dysfunction in the EpA960/CaMKII-Cre mice. (A–C) EpA960/CaMKII-Cre animals showed learning disability. (A) Training for Morris water maze test. (B) Probe test. (C) Swimming speed. (D, E) EpA960/CaMKII-Cre animals showed impaired hippocampal long-term potentiation (LTP). High-frequency stimulation-induced LTP recorded in the hippocampal CA1 region. Field excitatory postsynaptic potentials were plotted by time. Representative traces shown on top are the mean of 40 fEPSPs from baseline (1) and the last 10 min of recording (2). The last 10 min of recording was analyzed quantitatively. Calibration: 0.5 mV, 20 msec. Animals from four genotypes at 6 months old were included in experiments. Number of animals (N) and hippocampal slices (n) in each group is indicated. Data are mean ± SEM.*P< 0.05, by one-way ANOVA.

Figure 4

Reduced expression of cytoplasmic MBNL1 in the EpA960/CaMKII-Cre brain. (A) Representative immunofluorescence staining of co-localization of MBNL1 and MAP2, indicated by arrowheads, in the cortex of the adult mouse brain. (4 images/animal, 2 mice) (B) Representative immunofluorescence staining of MBNL1 expression in apical dendrites of cortical-layer V neurons in mice at age 6, 9 and 12 months. Number of animals (N) in each group is indicated. Data are mean ± SEM. *P<0.05; **P<0.01; ***P< 0.001 by one-way ANOVA. Scale: A, B, 50 μm.

We next determined whether the histopathological abnormalities caused by EpA960(R) mRNA was progressive, thereby resulting in neurodegeneration. In EpA960/CaMKII-Cre brains at age 6 months, axon and dendrite morphologic features were relatively normal as compared with genotype control brains (Fig. 2B and C). However, by 9 months, EpA960/CaMKII-Cre brains featured prominent axon and dendrite shortening; axon and dendrite morphologic features were still intact in age-matched control brains. The reduction in axon and dendrite length compared to control brains was greater at 12 than 9 months, which suggests degeneration (Fig. 2B and C). We further used Nissl staining to examine whether the reduced axon and dendrite integrity was linked to degeneration (21). The volume of the Nissl-stained cortical neurons was smaller in EpA960/CaMKII-Cre brains at 12 months than in other genotype control brains (Fig. 2D), which also suggests degeneration. Thus, EpA960(R) mRNA caused a progressive defect in the integrity of neuronal morphologic features resulting in neurodegeneration in EpA960/CaMKII-Cre brains.

EpA960/CaMKII-Cre animals exhibit learning disability and neurotransmission dysfunction

Learning disability is prevalent among people with DM1 (22), so we examined the learning ability of EpA960/CaMKII-Cre animals at age 6 months by the Morris water maze test, which tests hippocampus-dependent spatial learning and memory (23). Although axon and dendrite shortening became prominent by 9 months, we wondered whether behavioral abnormalities could be detected at an earlier stage such as at 6 months. During the 5 days of training, swimming speed was comparable among all animals, and at completion of the training, all animals learned to locate the hidden platform by using visual cues, as shown by the probe test (Fig. 3A and C). However, on training days 2 and 3, the escape latency was greater for EpA960/CaMKII-Cre animals than other genotype controls (Fig. 3A), which suggests a slower learning process in EpA960/CaMKII-Cre animals. We next performed an electrophysiology study of hippocampal slices to examine NMDA receptor-dependent long-term potentiation (LTP) induced by high-frequency stimulation (Fig. 3D and E). Field excitatory postsynaptic potential (fEPSP) was lower in EpA960/CaMKII-Cre hippocampal slices than slices from other genotype controls, which suggests decreased synaptic activity in EpA960/CaMKII-Cre brains. Therefore, EpA960/CaMKII-Cre animals showed learning disabilities and decreased NMDA receptor-dependent synaptic activity.

Reduced expression of MBNL1 and 2 in the EpA960/CaMKII-Cre brain

Loss of MBNL1 and 2 function has been implicated in DM1 neural pathogenesis (24,25), but whether and how expanded CUG RNA affects their expression pattern in the brain has not been investigated. We first examined the expression pattern of MBNL1 in the adult mouse brain by using the anti-MBNL1 polyclonal antibody (18,26,27). MBNL1 was expressed in the forebrain, including the cortex and hippocampus, and localized in the nucleus and cytoplasm of the cell body as well as dendrites on co-localization with MAP2 (Fig. 4A). The staining pattern was specific because pre-incubation of MBNL1 antibody with recombinant protein blocked the immunofluorescence signals (Supplementary Material, Fig. S1).

We next determined the effect of EpA960(R) mRNA on MBNL1 expression during the progression of morphological alteration. In cortical layer V neurons, MBNL1 was localized in the cell body and apical dendrites (Fig. 4B, wildtype). EpA960/CaMKII-Cre brains at age 6 months showed reduced MBNL1 immunoreactivity in apical dendrites of cortical layer V neurons as compared with other genotype control brains (Fig. 4B). To compare the difference, we measured the intensity of cytoplasmic MBNL1 by quantifying the total MBNL1 immunoreactivity in both the cell body and dendrites but excluding that in the nucleus to represent the cytoplasmic fraction of MBNL1. The cytoplasmic-to-nuclear ratio of MBNL1 immunoreactive intensity was used for comparison. With increasing age and as compared with control brains, EpA960/CaMKII-Cre brains showed reduced MBNL1 cytoplasmic content at age 9 and 12 months, when altered dendrite integrity was also detected in cortical layer II/III (Fig. 2C). At age 12 months, the cytoplasmic content including dendrites was reduced to ∼50% that of control brains.

We next examined MBNL2 expression in EpA960/CaMKII-Cre brains using anti-MBNL2 monoclonal antibody; the specificity has been shown in the Mbnl2^ΔE2/ΔE2brain (16). In the cortex, MBNL2 is localized both in the nucleus and cytoplasm (16). We wondered whether cortical layer V neurons showed a similar alteration in the subcellular distribution of MBNL2 as for MBNL1. The cytoplasmic-to-nuclear ratio of MBNL2 immunoreactivity showed a similar pattern among the four genotype brains at 6 months and no change up to 12 months (Fig. 5A). However, MBNL2-expressing cells were smaller in the EpA960/CaMKII-Cre than control brain at age 12 months (Fig. 5B), which was reminiscent of the degeneration shown by Nissl staining. We further determined the expression of MBNL2 in the cytoplasmic and nuclear fractions of the EpA960/CaMKII-Cre brain. Compared to control brains, EpA960/CaMKII-Cre brains showed reduced MBNL2 levels in both the cytoplasmic and nuclear fraction at 12 but not 6 or 9 months (Fig. 5C). Thus, EpA960(R) mRNA expression in the EpA960/CaMKII-Cre brain affected MBNL1 subcellular distribution, which occurred before reduced MBNL2 level at a later stage of phenotype progression.

Figure 5

Reduced expression of MBNL2 in the EpA960/CaMKII-Cre brain. (A, B) (A) Quantifications of subcellular distribution of MBNL2 in EpA960/CaMKII-Cre and control brains at 6 and 12 months. (B) Representative images showing subcellular distribution of MBNL2 in EpA960/CaMKII-Cre and control brains at 12 months. Number of animals (N) in each group is indicated. (C) Western blot quantification of cytoplasmic and nuclear expression of MBNL2 at 6, 9 and 12 months. Data are mean ± SEM. **P< 0.01, ***P < 0.001, by one-way ANOVA. Scale: B, 20 μm.

EpA960/CaMKII-Cre brain exhibits aberrant MBNL-regulated alternative splicing

One of the characteristic molecular features of DM1 is the misregulation of developmental alternative splicing transitions (9). We examined the alternative splicing events regulated by MBNL by using RNAs from the cortex and hippocampus of 12- to 16-month-old mice. The inclusion of Mbnl1 exon 5 and Mbnl2 exon 7 was increased in EpA960/CaMKII-Cre mice as compared with the genotype controls, which revealed the developmental splicing pattern in EpA960/CaMKII-Cre animals (Fig. 6A). In the postnatal day 2 (P2) brain, the percentage of Cacna1d exon 12a inclusion was low but was increased in adulthood. The inclusion of Cacna1d exon 12a in the EpA960/CaMKII-Cre brain was reduced (Fig. 6A). Therefore, the EpA960/CaMKII-Cre brain showed the abnormal MBNL-regulated splicing events observed in the DM1 brain.

Figure 6

Misregulated alternative splicing in the EpA960/CaMKII-Cre brain. (A) Quantitative analyses of the inclusion of Mbnl1 exon 5, Mbnl2 exon 7, and Cacna1d exon 12a with RNA in 12- to 16-month-old animals. Control animals were CaMKII-Cre or EpA960 animals. RNA collected from brains at postnatal day 2 (P2) or day 7 (P7) and adult brains (Ad, 4 months) was used to show the pattern of developmental transition. (B, C) MBNL-regulated alternative splicing in 6- (B) and 9-month-old (C) EpA960/CaMKII-Cre brains as compared with control brains. Number of animals (N) in each group is indicated. Data are mean ± SEM.*P< 0.05, **P< 0.01, ***P< 0.001 by Student’s t test. n.s., not significant.

We also determined whether altered alternative splicing events could be detected at early stages of phenotype progression. However, we found neither increased inclusion of Mbnl1 exon 5 and Mbnl2 exon 7 nor increased exclusion of Cacna1d exon 12a in 6- and 9-month-old EpA960/CaMKII-Cre brains as compared with control brains (Fig. 6B and C), so changes in alternative splicing may occur at later stages of phenotype progression.

Reduced MBNL1 cytoplasmic content in dendrites is an early event in the EpA960/CaMKII-Cre brain

Reduced MBNL2 expression at 12 months was associated with axon and dendrite degeneration, so we wondered whether the reduced MBNL1 expression in dendrites at 6 months was also due to dendrite degeneration in cortical layer V neurons. We examined MBNL1 distribution on dendrites and dendrite integrity by MAP2 immunofluorescence. The z-stacking immunofluorescence images of individual cortical layer V neurons labeled with MAP2 and MBNL1 were used to compare dendrite integrity and the length of MBNL1-positive immunoreactivity on dendrites. Dendrite integrity was intact and comparable among all genotype brains including EpA960/CaMKII-Cre brains; however, MBNL1-positive length was shorter in the EpA960/CaMKII-Cre brain than other control brains (Fig. 7A and B). Thus, our results suggest that reduced cytoplasmic MBNL1 expression in the EpA960/CaMKII-Cre brain at 6 months likely occurred before the reduced dendrite integrity and therefore was an early event following expanded CUG RNA expression.

Figure 7

Reduced cytoplasmic MBNL1 expression on dendrites is an early event in EpA960/CaMKII-Cre brain. (A) Representative images of length of MAP2 and MBNL1 immunoreactivity in cortical-layer V neurons. Layer V neurons expressing MAP2 and MBNL1 were imaged by z-stacking. Each image was computed from a collapsed series of images optimized to contain the entire apical dendrite extending from the cell body. (B) Quantification of length of MAP2 (left) and MBNL1 (right) in cortical-layer V neurons. Number of neurons (n) and animals (N) in each group is indicated. Data are mean ± SEM. *P<0.05; **P<0.01; ***P< 0.001 by one-way ANOVA. Scale: 15 μm.

Discussion

Structural changes in the brain associated with cognitive impairment are prevalent in people with DM1 (1). How CUG-repeat RNA causes such histopathological abnormalities in the brain has not been investigated. In the present study, we found that the EpA960/CaMKII-Cre mouse model recapitulated features of the DM1 brain, including the formation of nuclear RNA and MBNL foci, misregulation of alternative splicing, neurodegeneration and learning disability. Importantly, we observed the sequential phenotypic progression in EpA960/CaMKII-Cre brains: reduced cytoplasmic MBNL1 expression on dendrites at 6 months, followed by axon and dendrite degeneration at 9 months, and reduced MBNL2 expression and aberration in MBNL-regulated alternative splicing at 12 months. Comparison of Dmpk mRNA and CaMKII-Cre expression in the adult mouse brain revealed a similar distribution: both are abundant in the cortex and hippocampus and at low level in the thalamus and hypothalamus (19,28). Thus, the expression of expanded CUG RNA driven by the CaMKII promoter may provide an eligible tool for examining the effect of expanded CUG RNA in the DM1 brain after birth.

Several mouse models have been established to understand the molecular pathogenesis caused by expanded CUG [(CUG)n] RNA (12,18,26,29,30) or the contribution of MBNL loss of function (16,24) and elevated expression of CELF1 to disease features (31,32). The length of (CUG)n RNA or copy number of transgenes expressing (CUG)n RNA determines the MBNL1 and CELF1 responsiveness. Short (CUG)n RNA induces only MBNL1 sequestration, whereas long (CUG)n RNA concomitantly induces MBNL1 sequestration and CELF1 elevation, so MBNL1 is susceptible to (CUG)n RNA. Consistently, our findings of the early detection of reduced MBNL1 cytoplasmic content at 6 months in the EpA960/CaMKII-Cre brain support this notion. In contrast, reduced MBNL2 expression accompanying misregulated alternative splicing and neurodegeneration was a later event induced by EpA960(R) RNA in the EpA960/CaMKII-Cre brain. At the same time point, we observed aberrant splicing of MBNL2-regulated targets, which suggests that MBNL2 dysfunction likely contributed to misregulated alternative splicing in DM1. These findings, reduced cytoplasmic MBNL1 expression on dendrites and reduced MBNL2 expression in the cell body, reminisced that distinct regulatory mechanisms are involved in regulating the degeneration of axons and dendrites and the death of the cell body (33,34). Accordingly, we hypothesized that reduced MBNL1 cytoplasmic content and defective MBNL2-regulated RNA processing likely represent two transitions in DM1 neural pathogenesis or two proceeding pathways in different cellular compartments.

The occurrence of synaptic dysfunction before axon and dendrite degeneration in EpA960/CaMKII-Cre animals is reminiscent of models of neurodegenerative diseases showing dysfunction in synaptic activity and structure preceding axon and dendrite degeneration (35–37). Decline in cognitive function over time in DM1 has been reported and suggested as an early aging process (38). The axon and dendrite degeneration seen in the EpA960/CaMKII-Cre brain provides histopathological evidence for brain atrophy and aging. Thus, EpA960/CaMKII-Cre animals may be a model for studying the underlying mechanism of neurodegeneration in DM1. This model can also be used for understanding how defective axon and dendrite morphological features lead to a disrupted complex neuronal network, thereby affecting cognitive functions.

In the EpA960/CaMKII-Cre brain, reduced MBNL1 distribution in dendrites occurred before misregulation of splicing, which suggests that change in MBNL1 distribution was likely not due to misregulated splicing. Pathogenic RNA-induced splicing-independent events have also been reported in the DMSXL mouse brain (15,39) and in hearts expressing EpA960(R) RNA (40,41). In the DMSXL brain, aberrant expression of synaptic proteins including RAB3A and synapsin was not due to misregulated splicing. The EpA960(R) RNA-induced PKC activation was independent of splicing change, although PKC-mediated hyperphosphorylation of CELF1 results in misregulated splicing and plays an important role in DM1 cardiac pathogenesis (31,40). The molecular mechanism causing reduced cytoplasmic MBNL1 needs further investigation. Study of C. elegans has shown that the MBNL homolog mbl-1 regulates synapse formation in the neuromuscular junction (42). Dysregulation of synaptic proteins has been reported in DMSXL and Mbnl1^ΔE3/ΔE3 but not Mbnl2^ΔE2/ΔE2brains (15), which suggests a role for loss of MBNL1 function contributing to dysregulated synaptic transmission. In the EpA960/CaMKII-Cre brain at 6 months, reduced cytoplasmic MBNL1 level in dendrites and dysfunction in synaptic transmission with reduced hippocampal LTP suggests MBNL1 dysfunction contributing to dysregulated synaptic transmission in the DM1 brain.

Materials and Methods

Animals

The EpA960 mouse line was originally generated in an FVB background (18) and crossed with mice in a C57BL/6J background for more than 10 generations. The CaMKII-Cre mouse line (19) was maintained in an C57BL/6J background. EpA960/CaMKII-Cre mice were F1 offspring of EpA960 mice crossed with CaMKII-Cre mice. All animals were maintained on a standard 12-h light/dark cycle with light on at 8 AM. Food and water were available ad libitum. Brain samples used for RNA or protein extraction or immunohistochemistry were from littermate animals of both genders. All animal experiments were performed with the approval of the Academia Sinica Institutional Animal Care and Utilization Committee in strict accordance with its guidelines and those of the Council of Agriculture Guidebook for the Care and Use of Laboratory Animals.

RT-PCR splicing analysis

To determine the splicing of Mbnl1 exon 5, Mbnl2 exon 7 and Cacna1d exon 12a, 5 μg total RNA was used for synthesis of cDNA followed by PCR amplification as described (18). Primers used for amplifying Mbnl1 exon 5, Mbnl2 exon 7 and Cacna1d exon 12a were for Mbnl1-F: 5’-AAGATCAAGGCTGCCCAATA-3’; Mbnl1-R: 5’-GCATGTTGGCTAGAGCCTGT-3’; Mbnl2-F: 5’-AC CG TAACCGTTTGTATGGATTAC-3’; Mbnl2-R: 5’-CTTTGGT AA GGG AT GAAGAGCAC-3’; Cacna1d-F: 5’-CAAACGAAAC ACTAGCA TGCC-3’; Cacna1d-R: 5’-CTGAGTTTGGATTTCGAGATGG-3’. The PCR products were separated on 5% nondenaturing polyacrylamide gels. Quantification of percentage exon inclusion was as described (43).

Fluorescence in situ hybridization, immunofluorescence staining and immunohistochemistry

For immunofluorescence and immunohistochemistry of mouse brain sections, mice were anesthetized and perfused transcardially with phosphate buffered saline (PBS), then 4% paraformaldehyde (PFA), and brain sections were collected by vibratome sectioning. To detect RNA foci on mouse brain sections, sections first underwent UV cross-linking (150 mJ/cm²) in PBS, followed by permeabilization with 0.5% Triton-X 100 and 3% H₂O₂ in TBS for 10 min at room temperature. After DNase treatment for 1 h, sections were incubated with prehybridization solution (40% formamide, 2X SSC, 67 ng/μl yeast tRNA, 2 mM vanadyl ribonucleotide) at 42^˚C for 1 h, followed by hybridization with Cy3-labeled (CAG)₇ LNA probe (1 ng/μl) in hybridization solution (50% formamide, 4X SSC, 5X Denhardt’s solution, 67 ng/μl yeast tRNA, 2 mM vanadyl ribonucleotide) at 42^˚C for 2 h, and washed with hybridization buffer at 42^˚C for 15 min, with serial washes with 40% formamide, 2X SSC; 40% formamide, 1X SSC; 1X SSC; 0.5X SSC.

For immunofluorescence staining, brain sections were incubated in blocking solution (3% normal goat serum in 1X PBS containing 0.2% Triton X-100), then with specific antibodies in antibody solution (1% normal goat serum and 0.2% Triton X-100 in 1X PBS) for 1–2 days. After washing, sections were incubated with secondary antibody conjugated with Alexa Fluor 488 (Invitrogen), then washed with PBS, counterstained for DNA with DAPI and mounted with Fluoro-gel (EMS) for imaging.

Fluorescent Nissl staining (NeuroTrace, Molecular Probes) was performed following the manufacturer’s instructions. Briefly, brain sections were permeabilized in 0.1% Triton X-100/PBS for 10 min, washed with PBS, incubated with NeuroTrace (1:200) for 20 min, washed in 0.1% Triton X-100/PBS for 10 min and in PBS at 4^˚C overnight, then mounted with Fluoro-gel (EMS) for imaging. Images were acquired under a fluorescent microscope (AxioImager M2, Carl Zeiss) equipped with a 63×/1.4/oil (Plan-Apochromat; Carl Zeiss) objective lens.

Quantification of morphological features and MBNL1 immunoreactivity in mouse brain

The thickness of the somatosensory cortex (bregma −1.70 mm) and corpus callosum (bregma 0.26 mm) was measured from three coronal sections of each animal. To examine the axon integrity, the length of NF-160 immunoreactivity in the layer II/III neurons of the somatosensory cortex (Bregma −1.7 to −2.06) was measured by using ImageJ 1.45 (NIH). In each image frame, measurements were collected from 8–10 neurons in which the longest NF-160 immunoreactivity from each neuron was included. To examine dendrite integrity, we measured the length of MAP2-positive immunoreactivity > 10 μm within the layer II/III region. In each image frame, measurements were collected from 12 to 16 MAP2-positive dendrites. For each animal, eight image frames were collected.

For quantification of fluorescence Nissl staining, Nissl-stained areas were measured by using Metamorph 7.7.5.0 (Molecular Devices). Under “cell scoring”, the range of the diameter of the Nissl-stained area was defined for measurement and DAPI was used for defining the nucleus. For quantification, 450–750 cells per animals were used. Images were acquired by using a Zeiss confocal laser scanning microscope (LSM700) (Carl Zeiss) and an objective lens 20x/0.8 (for NF-160 and MAP2 staining) or 40x/1.4/oil (for Nissl stain) (Plan-Apochromat, Carl Zeiss).

To determine the cytoplasmic fraction of MBNL1 in the cortical layer V neurons, we first measured the total and nuclear MBNL1 immunoreactive intensity as described above. The ratio of cytoplasmic to the nuclear fraction of MBNL1 immunoreactive intensity was used for comparing the four genotypes after normalizing the ratio to that of the wild-type in Figure 4B. Images were acquired under a Zeiss confocal laser scanning microscope (LSM700) equipped with a 20x/0.8 (Plan-Apochromat, Carl Zeiss) objective lens.

To determine the MAP2 and MBNL1 immunoreactivity in cortical layer V neurons shown in Figure 7, neurons expressing MAP2 and MBNL1 were imaged by z-stacking under a Zeiss confocal laser scanning microscope (LSM700) equipped with a 40x/1.4/oil (Plan-Apochromat, Carl Zeiss) objective lens. The images were obtained by computing from a collapsed series of images by using Metamorph software. The length of MAP2 and MBNL1 immunoreactivity was analyzed by using Imaris 8.0.1 (licensed from the Institute of Molecular Biology, Academia Sinica).

For immunofluorescence (IF) staining described above, brain sections collected from four genotypes were processed at the same time and considered the same batch of IF preparation. Subsequently, images were acquired under the same setting for the same batch of IF preparation. The IF intensity among different batches of IF preparation was variable or the autofluorescence from different sections was variable; therefore, we used the ratio of cytoplasm to nucleus, in which the immunoreactive intensity in the cytoplasm and nucleus from the same neurons was measured and used for calculating the ratio, or the length of axon or dendrites not affected by variation of absolute intensity from different batches of preparation was measured.

Antibodies

The antibodies anti-NeuN (A60, 1:1000, Millipore), anti-NF 160 (3H11, 1:500, Covance), and anti-MAP2 (AP20, 1:500, Millipore) were used to label neurons, axons and dendrites, respectively. The polyclonal anti-MBNL1 (ABE241, 1:1000, Millipore) (18) or MBNL2 antibody (3B4, 1:500, Santa Cruz) (16) was used for IF staining.

Morris Water Maze

The Morris water maze task was used to test hippocampus-dependent spatial learning and memory (23). Animals at 6 months old from all genotypes were used for behavioral testing. The apparatus was a 151-cm circular stainless steel tank filled with water (20°C, 51 cm deep) made opaque by the addition of ∼2000 ml whole milk. A clear Plexiglas adjustable platform was submerged 1 cm below the water surface or elevated 0.5 cm above the water level. The training consisted of daily training trials on the visible platform task for 2 days and on the hidden platform tasks for 5 days with a probe trial 24 hr after the last hidden-platform training session. In the daily trial, animals underwent four trials with a 30-min inter-trial interval. Each trial lasted for 1 min, latency to reach the platform was measured, and animals were tracked by using Smart tracker software (San Diego Instruments, San Diego, CA). After the platform was removed from the apparatus, the animal was given one 60-s probe trial starting from a novel location to determine memory for where the platform had been.

Electrophysiology

Animals were anesthetized with isoflurane, then decapitated. The brain was quickly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF, 119 mM NaCl, 2.5 mM KCl, 26.2 mM NaHCO₃, 1 mM NaH₂PO₄, 1.3 mM MgSO₄, 11 mM glucose, and 2.5 mM CaCl₂, with pH adjusted to 7.4 by gassing with 5% CO₂/95% O₂) before isolation of the caudal portion containing the hippocampus and entorhinal cortex. After isolation, hippocampal slices (450 μm) were cut transversely with the use of a vibrating tissue slicer (D.S.K. Microslicer DTK-1000; Dosaka EM, Kyoto, Japan) and transferred to the holding chamber at room temperature (24–25°C) for recovery for at least 90 min before recording. Electrophysiology was performed as described (44,45). Briefly, hippocampal slices were kept in the recording chamber on an upright microscope (BX51WI; Olympus Optical, Kyoto, Japan) equipped with an infrared-differential interference-contrast microscopic video. Oxygenated ACSF (5% CO₂/95% O₂) containing 0.1 mM picrotoxin (GABA_A receptor antagonist) was perfused into the recording chamber at 1–2 ml/min. Recordings were made at room temperature (24–25°C). To prevent epileptiform discharge of pyramidal neurons, the border between the CA1 and CA3 areas was cut. Extracellular stimuli were administered on the border of areas CA3 and CA1 along the Schaffer collaterals by using a constant current isolated stimulator (model DS3; Digitimer, England) and bipolar stainless steel stimulating electrodes (Frederick Haer Co., Bowdoinham, ME) (10 ΩM impedance). Field excitatory postsynaptic potentials (fEPSPs) were recorded by using a glass pipette filled with 3 M NaCl and positioned in the stratum radiatum, on opposite sides of the stimulating electrodes. Stable baseline fEPSP activity was recorded by applying a short-duration current pulse (∼40 μs) at the determined intensity every 15 s for at least 10 min. For long-term potentiation (LTP) experiments, LTP was induced by administering 3 trains of 100 stimuli at 100 Hz (1 s, high-frequency stimulation; HFS) with an intertetanus interval of 60 s. Electrophysiological traces were amplified by using an amplifier (Multiclamp 700 B; Axon Instruments, Union City, CA). All signals were low-pass–filtered at a corner frequency of 1 kHz and digitized at 10 kHz by using a CED Micro 1401 mKII interface (Cambridge Electronic Design). Data were collected by using Signal software (Cambridge Electronic Design, Cambridge, UK). The initial slopes of the fEPSP were measured for data analysis. Synaptic responses were normalized to the mean baseline response. The mean size of the slope of the fEPSPs recorded from the last 10 min after HFS was used for comparison.

Statistical Analysis

Data are presented as mean ± SEM and were analyzed by ANOVA with SigmaPlot 12.5 (Systat Software Inc.) and by Student’s t test with Microsoft Excel. Two groups were compared by unpaired two-tailed Student’s t tests and more than tow groups by one-way ANOVA, followed by Holm-Sidak multiple comparison tests. P < 0.05 was considered statistically significant. Data collection and analyses were not blinded. For data collected from animals, each N value corresponds to a single mouse.

Supplementary Material

Supplementary Material is available at HMG online.

Acknowledgements

We thank Dr. Thomas Cooper at Baylor of Medicine, Houston, USA, for sharing reagents and the EpA960 mouse line. We also thank Drs. Tang Tang and Yi-Shuian Huang for manuscript comments; Ms. Sue-Ping Lee at the Institute of Molecular Biology, Academia Sinica and IBMS image core facility, for helping with image acquisition and quantification. G.-S. Wang was supported by grants from the Ministry of Science and Technology, Taiwan and the Institute of Biomedical Sciences, Academia Sinica, Taiwan, and T.-Y. Kuo was supported by a Postdoctoral Fellowship from Academia Sinica.

Conflict of Interest statement. None declared.

Funding

Ministry of Science and Technology, Taiwan (NSC101-2321-B-001-017), Institute of Biomedical Sciences, Academia Sinica, Taiwan