MCN
Molecular and Cellular Neuroscience 20, 198 –210 (2002) doi:10.1006/mcne.2002.1131
Regulation of tau RNA Maturation by Thyroid Hormone Is Mediated by the Neural RNA-Binding Protein Musashi-1 Ana Cuadrado,* Luis F. Garcı´a-Ferna´ndez,* Takao Imai, † Hideyuki Okano, † and Alberto Mun˜oz* ,1 *Instituto de Investigaciones Biome´dicas “Alberto Sols,” Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto´noma de Madrid, E-28029 Madrid, Spain; and †Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
The tau gene encodes a microtubule-associated protein expressed by neuronal and glial cells. Abnormal deposits of Tau protein are characteristic of several neurodegenerative disorders. Additionally, mutations affecting tau pre-mRNA alternative splicing of exon 10 are associated with frontotemporal dementia and Parkinsonism linked to chromosome 17. In rodents, this process is developmentally regulated by thyroid hormone (T3) causing the predominance of exon 10-containing transcripts. Here we demonstrate that musashi-1 (msi-1) gene is induced by T3 during rat brain development and in N2a cells. T3 increases msi-1 mRNA level in an actinomycin D-sensitive, cycloheximide-resistant fashion without affecting its halflife, which suggests a transcriptional effect. Both ectopic Msi-1 expression and T3 treatment increased the proportion of exon 10-containing tau transcripts. Furthermore, antisense msi-1 expression inhibited T3 action. Our results show that msi-1 mediates the posttranscriptional regulation of tau expression by T3.
INTRODUCTION The tau gene encodes a microtubule-associated protein which is important for stabilization and organization of axonal microtubules and therefore crucial for neuronal morphology and polarity, neurite outgrowth, and axonal transport (Lee et al., 1988; Goedert et al., 1989; Ca´ceres and Kosik, 1990). Six Tau isoforms are produced in the human brain by alternative splicing of the single tau gene (Spillantini and Goedert, 1998). Al-
1 To whom correspondence and reprint requests should be addressed. Fax: 34-91-585 4587. E-mail:
[email protected].
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ternative splicing of exon 10 causes the production of proteins with four (4Rtau) or three (3Rtau) repeats which function as microtubule binding domains. The expression of Tau isoforms is species-specific: only 4Rtau polypeptides are expressed in the adult rodent brain, while in adult human brain the six isoforms coexist in a ratio of 4Rtau to 3Rtau isoforms of about 1, which suggests a tight regulation of the alternative splicing (Hong et al., 1998). Abnormal accumulation of Tau protein in filamentous cytoplasmic inclusions is a hallmark of an expanding family of neurodegenerative diseases collectively known tauopathies, which include Alzheimer’s disease (AD), Down’s syndrome, Pick’s disease (PiD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis/Parkinsonism-dementia complex of Guam, progressive subcortical gliosis (PSG), sporadic frontotemporal dementia (FTD), and familial FTD and Parkinsonism linked to chromosome 17 (FTDP-17) (Spillantini and Goedert, 1998; Lee and Trojanowski, 1999). While Tau deposits almost exclusively affect neurons in AD, they are also present in glial cells in FTDP-17, CBD, PSG, PiD, and PSP (Spillantini et al., 1997; Goedert et al., 1999; Yasuda et al., 2000; Garcı´a and Cleveland, 2001). Recently, missense and intron mutations in the tau gene have been shown to cause inherited FTDP-17 (Hutton et al., 1998; Varani et al., 1999; Grover et al., 1999; Hasegawa et al., 1999; Yasuda et al., 2000). These mutations increase the accumulation of tau transcripts containing exon 10 and thus the ratio of 4R to 3R isoforms. In rodents, alternative splicing of tau pre-mRNA is developmentally regulated. The levels of RNAs containing 1044-7431/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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exon 10 increase during development, replacing the embryonic variants lacking this exon (Goedert et al., 1989). It is believed that the preferential production of tau RNAs containing exon 10 is associated with the stabilization of neuronal network in adulthood, whereas juvenile isoforms may be more appropriate for the plasticity of neurites during development (Goedert et al., 1989; Lee and Trojanowsky, 1999). Human and rat glial cells also express tau, albeit at a lower level (LoPresti et al., 1995; Muller et al., 1997; Komori, 1999). Thyroid hormone (triiodothyronine, or T3, and the prohormone thyroxine, or T4) plays an essential role in brain development. Lack of T3 during fetal and neonatal periods leads to mental deficiency in humans and multiple brain abnormalities in experimental animals (Dussault and Ruel, 1987; Porterfield and Hendrich, 1993). The classic mechanism of action of T3 is the regulation of gene transcription through the binding to specific nuclear receptors (TR␣1, TR1, TR2, and TR3 isoforms) which interact with specific nucleotide sequences (thyroid response elements, T3REs) present in target genes (Bernal and Guadan˜ o-Ferraz, 1998; Forrest and Vennstro¨ m, 2000). In addition, T3 exerts posttranscriptional regulatory effects on pre-mRNA splicing; mRNA stabilization, processing, and translation; or on posttranslational mechanisms; among them, the transition from juvenile to mature tau RNA isoforms in the developing rat brain (Aniello et al., 1991; Puymirat et al., 1995). However, the basis for these effects remain mostly unknown. Posttranscriptional mechanisms are a source of diversity and a point of regulation of gene expression in the central nervous system (CNS) (Rio, 1993). Several neural RNA-binding proteins are known, including Nova-1, PTB, KSRP, and the Hu/Elav and Musashi (Msi) subfamilies (Grabowski, 1998). msi-1 is expressed in neural stem cells, neuronal and astroglial progenitor cells, and also in differentiated astrocytes (Sakakibara and Okano, 1997; Kaneko et al., 2000). The Msi-1 protein has two ribonucleoprotein (RNP)-type RNA binding domains (RBDs) and exhibits binding preferences for G/A(U)nAGU sequences (Nagata et al., 1999). Conceivably, Msi-1 may participate in the posttranscriptional regulation of its target genes. In fact, Msi-1 acts as a translation repressor of the Tramtrack69 zinc finger in Drosophila (Okabe et al., 2001). Here we show that msi-1 expression is induced by T3 in the developing rat brain and in neuroblastoma N2a cells. Both T3 treatment and ectopic Msi-1 expression increase the proportion of tau transcripts containing exon 10 and thus the ratio of 4Rtau to 3Rtau isoforms in N2a cells. Moreover, antisense msi-1 RNA expression in
N2a cells inhibits this effect of T3. Our results indicate that msi-1 mediates the control of tau pre-mRNA maturation by T3 in rodents.
RESULTS msi-1 Expression Is Regulated by Thyroid Hormone in the Rat Brain The idea that T3 may regulate a subset of its target genes indirectly through the control of intermediate genes involved in posttranscriptional mechanisms, together with the identification of the mammalian homolog of the Drosophila splicing regulator Suppressor-ofwhite-apricot as a thyroid target gene in the rat brain (Cuadrado et al., 1999) and the pattern of expression of msi-1 (Sakakibara and Okano, 1997; Kaneko et al., 2000), led us to study the effects of this hormone on msi-1 in the developing rat brain. At embryonal day 19 no differences were found in msi-1 RNA levels by in situ hybridization between control and hypothyroid animals (not shown). In contrast, at postnatal days 1–15 (P1–P15), when msi-1 expression is high in glial cells (Sakakibara and Okano, 1997; Kaneko et al., 2000), thyroid deficiency caused a down-regulation of msi-1 RNA levels (Figs. 1A and 1B). At P5, this effect was clearly visible in the immature proliferating neuronal and glial precursors of the SVZ, and in the piriform and cerebral cortices, caudate putamen, thalamus, and hippocampus (Fig. 1A). At P15, msi-1 expression decreased in the cerebellum (Fig. 1B). These data indicate that msi-1 expression in both neurons and glial cells is down-regulated by hypothyroidism. The dependence of msi-1 RNA content on thyroid status was further analyzed by hormonal treatment in vivo. At P15 a short (8-h) treatment of hypothyroid rats with T3 restored msi-1 RNA levels in the cerebellum (Fig. 1B, right panel). To examine whether changes in thyroid status altered Msi-1 protein expression we performed immunohistochemistry studies. At P5, the intensity of staining was slightly higher in the cerebral cortex of animals which were made hyperthyroid by repeated T4 injections (Fig. 1C) and in the hippocampus (not shown). Most labeled cells in both areas showed astrocytic morphology (open arrowheads). Although a few neurons were also stained, most pyramidal neurons in the hippocampal CA1-3 regions and cortex layer V were unlabeled (solid arrowheads). In agreement with RNA data, hypothyroid rats showed fewer stained cells, with weaker staining (Fig. 1C). Similar results were found at P15 (not
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FIG. 1. msi-1 expression is regulated by thyroid hormone in the developing rat brain. Analysis by in situ hybridization and immunohistochemistry of msi-1 levels in coronal sections of brains of control, hypothyroid (Hypo), or T3-treated hypothyroid (Hypo ⫹ T3) animals. (A) At P5, a reduction in msi-1 RNA levels was found in the subventricular zone (SVZ), caudate putamen (CPu), and piriform cortex (Pir) of
Musashi-1 Mediates tau RNA Maturation by T3
shown). At this age, in the cerebellum Msi-1 protein was predominantly located in the Bergmann glial cells (open arrowheads) of the Purkinje cell layer (Fig. 1D, left panel). In hypothyroid rats immunostaining was much weaker and none of the Bergmann cells were stained (Fig. 1D). Treatment of hypothyroid rats with T3 induced Msi-1 expression in 8 h, and induction was much higher after 24 h treatment with T4 (Fig. 1D). To measure the changes in Msi-1 levels we used Western blotting. Extracts from the cerebral cortex, hippocampus, and cerebellum of hypothyroid rats contained approximately half the protein found in the corresponding regions of control animals from P0 to P15 (Fig. 1E). T4 treatment from P15 to P19 restored Msi-1 levels in the cerebellum. Together, these data indicate that msi-1 expression in the developing rat brain is under thyroid control. Mechanism of msi-1 Regulation by Thyroid Hormone in N2a Cells To examine how T3 regulates the msi-1 gene we used neuroblastoma N2a cells expressing high levels of hormone receptors. First, Northern blots showed that T3 increased msi-1 RNA levels in N2a ⫹ TR cells and also in N2a ⫹ TR␣ cells, but not in the parental N2a cells (Fig. 2A). Thus, both receptors have the same regulatory effect on msi-1, which was maximum (3- to 4-fold) 8 h after hormone addition (Fig. 2B). The stimulatory action of T3 in N2a ⫹ TR cells was specific, as other agents acting through related nuclear receptors such as dexamethasone, retinoic acid, or 1␣,25-dihydroxyvitamin D3 did not induce the msi-1 gene (Fig. 2C). Western blotting showed that the induction of msi-1 RNA by 24 h T3 treatment correlated with a similar (2.5-fold) increase in the amount of cellular Msi-1 (Fig. 3A). Maximum levels of Msi-1 were achieved using physiological T3 concentrations (5–25 nM; Fig. 3B). Immunoblotting of subcellular fractions revealed that Msi-1 was
201 predominantly cytosolic (8-fold higher than in the nucleus taking into account the protein content of each fraction) in untreated N2a ⫹ TR cells (Fig. 3C). T3 treatment increased Msi-1 concentration in both nuclear and cytosolic compartments (3.2- and 3.7-fold, respectively) without affecting its subcellular distribution (Fig. 3C). We used confocal laser microscopy to confirm the cellular localization of Msi-1. The nuclear-restricted U2B⬙, a component of the basal spliceosome complex which is highly concentrated in coiled bodies and nuclear speckles (Carmo-Fonseca et al., 1992), was studied as an internal control. In untreated N2a ⫹ TR cells Msi-1 staining was weak, diffuse, and predominantly cytoplasmatic (Fig. 3D, upper left panel). Forty-eight hours’ treatment with T3 increased Msi-1 levels in both the cytoplasm and nucleus (Fig. 3D, lower left panel), where it partially colocalized with U2B⬙ in irregular strongly stained areas which were close to but not coincident with coiled bodies (Fig. 3D, lower right panel, white arrow). As expected, U2B⬙ was strictly nuclear irrespective of T3 treatment (Fig. 3D, middle panels). The dual localization of Msi-1 in nuclear and cytosol was also found in cultures of primary astrocytes (Fig. 3E). In these cells, Msi-1 colocalizes with GFAP in the cytosol but not in the nucleus, which contains abundant Msi-1 and lacks GFAP (Fig. 3E). Transcription (actinomycin D, ActD) and translation (cycloheximide, CHX) inhibitors were used to analyze the mechanism of the T3-induced increase in msi-1 expression. The blockade of T3 action by ActD (Fig. 4A) and the lack of effect of CHX (Fig. 4B) suggested that the increase in msi-1 RNA can be the result of a direct effect on msi-1 transcription. This was also supported by the study of msi-1 mRNA stability in N2a ⫹ TR cells. To this end, N2a cells were first treated with T3 for 16 h and then incubated with ActD in the presence or absence of T3. No differences were found in the half-life of msi-1 mRNA (about 7 h) in cells treated or not with
hypothyroid animals. (B) At P15, msi-1 RNA expression was reduced in the hypothyroid cerebellum, but then partially restored by T3 treatment. Scale bar ⫽ 1.7 mm (A) and 1.5 mm (B). (C) Sections of cortex layers III–V showing intensely stained cells with astrocytic morphology (open arrowheads) in control and hyperthyroid animals and a reduction in staining in hypothyroid animals. In these animals, layer V pyramidal neurons are mostly unlabeled (solid arrowheads). (D) Low (upper panels) and high (lower panels) magnification of cerebellar sections from control, hypothyroid (Hypo), T3-treated (8 h) hypothyroid (Hypo ⫹ T3), and T4-treated (24 h) hypothyroid (Hypo ⫹ T4) animals. In control animals Bergmann glial cells were intensely stained (open arrowheads), but Purkinje cells were not (solid arrowheads). Abbreviations: EGL, external granular layer; ML, molecular layer; PcL, Purkinje cell layer; IGL, internal granular layer. Scale bars ⫽ 250 m (C), 1.5 mm (D, upper panels), and 375 m (D, lower panels). (E) Western blot analysis of Msi-1 expression in the hypothyroid rat brain. Protein extracts (30 g) from cerebral cortex (Cx), hippocampus (Hp), and cerebellum (Cb) of control (C), hypothyroid (H), and T4-treated (4 days) hypothyroid (H ⫹ T4) rats of the indicated ages were analyzed. Upper panel shows a representative result; each lane contains an homogenate of three samples. Lower panel shows a quantification of data from three experiments (nine animals per point). C, white columns; H, black columns; H ⫹ T4, dashed columns.
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the same effect in N2a ⫹ TR cells and if so, given that Msi-1 is a RNA-binding protein and shows a partial nuclear localization, whether this effect occurs through msi-1 up-regulation, N2a cells were transfected with an expression vector for the mouse msi-1 gene encoding a tagged Msi-1 protein (Flag-Msi-1). In N2a cells we detected two major endogenous Msi-1 polypeptides of 38 and 40 kDa (Fig. 5A). In transfected cells we also found the exogenous (⬃42-kDa) Flag-Msi-1 protein (Fig. 5A). Splicing of tau pre-mRNA exon 10 was analyzed by quantitative RT–PCR using primers which allow the amplification of the region from exon 9 to exon 12 (Hutton et al., 1998; Grover et al., 1999; Hasegawa et al., 1999; Yasuda et al., 2000). N2a cells transfected with a control plasmid expressed an equivalent amount of exon 10-containing (exon 10⫹, amplified fragment of 367 bp) and exon 10-lacking (exon 10⫺, amplified fragment of 274 bp) tau transcripts (Fig. 5B). Following msi-1 transfection there was a two- to three-fold increase in the ratio of exon 10⫹/exon 10⫺ RNAs (Fig. 5B, lower panel). Consistent with its effect in the rat brain, T3 gradually increased the ratio of exon 10⫹/exon 10⫺ tau transcripts two- to threefold after 4 days treatment in N2a ⫹ TR cells (Fig. 5C). In contrast, T3 had no effect in control cells (Fig. 5D). The effect of T3 was consistently found in a wide range (20 – 45) of PCR cycles (not shown).
FIG. 2. T3 induces msi-1 RNA expression in N2a cells. Twenty micrograms of total RNA were loaded per lane. (A) Effect of T3 on msi-1 RNA levels in parental N2a, N2a ⫹ TR␣, and N2a ⫹ TR cells. Cells were treated with 150 nM T3 for 2 days as indicated or not at all. (B) Time course of msi-1 RNA induction by T3. Lowest panel shows the data from three independent experiments after normalization to -actin RNA levels. Open circles: untreated N2a ⫹ TR cells; solid circles: T3-treated N2a ⫹ TR cells; open squares: untreated N2a ⫹ TR␣ cells; solid squares: T3-treated N2a ⫹ TR␣ cells. (C) Effect of 150 nM T3, 1 M vitD3, Dex, or RA on msi-1 RNA expression in N2a ⫹ TR cells.
T3, indicating that the hormone has no effect on its stability (Fig. 4C). Thyroid Hormone and Ectopic Msi-1 Expression Increase the Proportion of Exon 10-Containing tau Transcripts in N2a ⴙ TR Cells T3 regulates tau pre-mRNA splicing in the rodent brain (Aniello et al., 1991). To examine whether T3 has
msi-1 Mediates the Effect of T3 on the Maturation of tau mRNA To elucidate whether msi-1 mediates the action of T3, we examined the effect of antisense msi-1 expression on the modulation by T3 of tau mRNA isoforms accumulation. Tranfection efficiency was over 85% as assessed by transfecting an GFP expression vector (not shown). Expression of antisense msi-1 RNA was confirmed in Northern blots (Fig. 6A, left). Immunoblotting assays showed that the T3-induced increase in Msi-1 protein was inhibited, but not totally blocked in N2a ⫹ TR cells transfected with antisense msi-1 cDNA (Fig. 6A, right). Likewise, antisense msi-1 did not reduce basal Msi-1 in parental N2a cells (Fig. 6B). A putative explanation for this is the consistent finding that antisense msi-1 expression caused 25–30% cell death in both cultures, which indicates that a threshold of msi-1 expression is required for cell survival (Fig. 6B). Remarkably, in N2a ⫹ TR cells RT–PCR analysis revealed that transfection of antisense msi-1 cDNA inhibited the increase by T3 of exon 10⫹ tau transcripts (Fig. 6C). This result
Musashi-1 Mediates tau RNA Maturation by T3
strongly suggests that msi-1 mediates the regulatory effect of T3 on the maturation of tau mRNA (Fig. 6D).
DISCUSSION We show here that the neural RNA-binding protein Msi-1 mediates the regulatory effect of T3 on tau RNA maturation. We found that msi-1 RNA and protein expression in the rat brain and in N2a cells is regulated by T3 and that a rise in the level of Msi-1 by either T3 treatment or ectopic msi-1 expression increases the proportion of mature exon 10⫹ tau transcripts in neuronal N2a cells. Furthermore, the inhibition of T3 action on tau RNA maturation by antisense msi-1 expression demonstrates that Msi-1 is involved in this process. The recent finding that intronic and missense exonic mutations affecting tau pre-mRNA alternative splicing of exon 10 are responsible for FTDP-17 has emphasized the importance of alternative splicing in tauopathies (Hutton et al., 1998; Varani et al., 1999; Grover et al., 1999; Hasegawa et al., 1999; Yasuda et al., 2000). Some FTDP-17 mutations increase the ratio of 4Rtau to 3Rtau in neurons and glial cells 1.5- to 2-fold and result in a partial loss and a toxic gain of function (Hong et al., 1998; Goedert et al., 1999). An increase in the ratio of 4Rtau/3Rtau polypeptides is sufficient for the development of neuronal and glial filamentous tauopathy. Interestingly, the change in the ratio of Tau isoforms caused by mutations in FTDP-17 patients corresponds to the T3-regulated shift in the expression from juvenile to mature Tau isoforms occurring naturally during rodent brain development (Aniello et al., 1991). Since Tau pathology is important in human neurodegenerative disorders characterized by dementia, as exemplified by the identification of tau as the FTDP-17 gene, T3 and msi-1 may be involved in the generation or progression of these disorders. Thus, it would be interesting to examine Msi-1 expression in FTDP-17 patients, particularly those who do not harbor mutations affecting tau mRNA exon 10 splicing. The predominant modulation of msi-1 expression by T3 in glial cells after birth suggests a relation between thyroid dysfunction and tauopathies affecting these cells such as sporadic FTD, CBD, PSP, PSG, or PiD (Garcı´a and Cleveland, 2001). Interestingly, families with missense or splice site mutations that affect tau exon 10 have intraneuronal and glial Tau inclusions consisting predominantly of 4Rtau isoforms; in contrast, families with missense mutations outside exon 10, which affect all Tau isoforms, have only neuronal Tau inclusions. Together with these find-
203 ings, our data suggest that the effect of Msi-1 on tau pre-mRNA splicing may be more critical in glial cells than in neurons, where this process may be under a complex regulation. The fact that the same change in the ratio of tau mRNA isoforms is induced by treatment of N2a cells with T3 or by overexpression of an exogenous msi-1 gene and the finding that expression of an antisense msi-1 RNA inhibits this effect demonstrate that the effect of T3 is mediated by the induction of msi-1. The finding that T3 increases Msi-1 content similarly in nucleus and cytoplasm is consistent with the detection of Msi-1 staining in both cellular compartments in the developing rat brain and primary astrocytes and is compatible with a role of Msi-1 in the regulation of tau pre-mRNA splicing. Interestingly, the human tau gene pre-mRNA contains a sequence (GUGAGU) at the begining of intron 10 highly homologous to the Msi-1 consensus binding sequence (G/A(U)nAGU) found in vitro (Kaneko et al., 2000). Remarkably, several mutations found in FTDP-17 patients that increase the inclusion of exon 10 affect this sequence. They may act by altering the proposed stem–loop structure that putatively constitutes an intron splicing silencer (D’Souza and Schellenberg, 2000). It is so tempting to speculate that increased levels of Msi-1 might have an analogous inhibitory effect on exon 10 splicing. Furthermore, Msi-1 is located in the vicinity of coiled bodies which are predicted to be stores of splicing factors (Lewis and Tollervey, 2000). However, attempts to analyze the putative binding of Msi-1 to this sequence in tau premRNA have been up to now precluded by the inability to obtain soluble full-length protein. This has precluded RNA-protein binding studies. Moreover, though our data suggest a direct or indirect role of Msi-1 in tau pre-mRNA splicing, we cannot rule out that Msi-1 is affecting other processes such as the stabilization of specific tau mRNA isoforms, as described for HuD (Aranda-Abreu et al., 1999). In the cytoplasm, Msi-1 represses translation in Drosophila (Okabe et al., 2001) and of mouse numb mRNA by binding to the 3⬘-UTR region (Imai et al., 2001). Examples of other RNA-binding proteins with multiple regulatory roles are the Drosophila Sex-lethal and Saccaromyces cerevisiae ribosomal L32 proteins, which regulate splicing in the nucleus and translation of target mRNAs in the cytoplasm (Gebauer et al., 1997). T3 is unlikely to control its numerous target genes by direct regulation of their transcription. Rather, it is easier to envisage that the regulatory effects of T3 during development are exerted through a cascade, in which a
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FIG. 3. Induction of Msi-1 protein by T3 in N2a ⫹ TR cells. Twenty micrograms of protein extracts were loaded per lane. (A) Time course of Msi-1 induction by T3. Cells were treated with 150 nM T3 for the indicated times or not at all. Two experiments were performed. Lower panel shows the quantification of mean values. (B) Dose–response curve of Msi-1 induction by T3. Cells were treated with the indicated T3 concentrations for 24 h or not at all. Two experiments were performed. Lower panel shows the quantification of mean values. (C) Western blot analysis of the amount of Msi-1 present in cytosol or nucleus of N2a ⫹ TR cells upon T3 (150 nM) treatment for 48 h. Antibodies against -actin and histone H1 0 were used to ensure purity of subcellular fractions. (D) Confocal microscopy analysis of Msi-1 expression and induction by T3. The expression of Msi-1 and of the nuclear U2B⬙ proteins was analyzed in cells treated (lower panels) or not (upper panels) with 150 nM T3 for 48 h. Areas of colocalization appear yellowish. Arrows point to the structures where Msi-1 accumulates within the nucleus. Arrowheads indicate the coiled bodies. Asterisks in right panels indicate the perinuclear, putative Golgi area which is basically devoid of Msi-1 staining. Scale bar ⫽ 10 m. (E) Confocal microscopy analysis of Msi-1 and GFAP expression in rat primary astrocytes. Areas of colocalization appear in yellow. Arrows show Msi-1 expression in the nucleus (delineated by dotted lanes in upper photos). Scale bar ⫽ 10 m.
set of transcriptionally regulated genes regulate secondary sets of genes. The finding that many T3 target genes do not contain T3REs supports this view (Bernal and Guadan˜ o-Ferraz, 1998). Few studies have reported regulatory effects of this hormone at posttranscriptional levels such as mRNA stabilization (Staton and Leedman, 1998; Forrest and Vennstro¨ m, 2000), or alternative splicing, as occurs in tau (Aniello et al., 1991). msi-1 is highly expressed from early embryonic development to
the third postnatal week (Sakakibara et al., 1996; Sakakibara and Okano, 1997). The period of thyroid responsiveness of msi-1 coincides with the peak of expression and occupancy of TR, when the brain is more sensitive to variations in thyroid hormone levels (Ferreiro et al., 1990). Given that we have no data at the msi-1 promoter level, our results suggest that T3 regulates either directly or indirectly msi-1 gene transcription. In summary, our data demonstrate a link between T3,
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FIG. 4. Thyroid hormone increases msi-1 gene transcription. (A) Northern blot showing the blockade by ActD of the increase in msi-1 RNA level by T3 in N2a ⫹ TR cells. Cells were pretreated with ActD (2 g/ml) 30 min before addition of T3 (150 nM). Single treatments with either T3 or ActD were done as controls. Lower panel shows the quantification of msi-1 RNA levels after normalization to -actin RNA content. The same results were obtained in two independent experiments. (B) Northern blot showing that CHX does not inhibit the increase in msi-1 RNA level by T3 in N2a ⫹ TR cells. Cells were pretreated with CHX (8 g/ml) 30 min before addition of T3 (150 nM). Single treatments with either T3 or CHX were done as controls. The small increase in RNA level by CHX may represent stabilization of ribosomal-bound transcripts. Lower panel shows msi-1 RNA after normalization to -actin RNA obtained in three independent experiments. (C) T3 does not affect msi-1 RNA turnover. N2a ⫹ TR cells were preincubated with 150 nM T3 for 16 h to increase msi-1 RNA. Then (time 0), cells were transferred to medium containing ActD (2 g/ml) either alone (open squares) or in addition to (solid squares) 150 nM T3, and RNA was extracted at the indicated times (4 to 10 h). The basal level of msi-1 RNA in untreated cells is also shown (open circle on the y axis). The quantification of msi-1 RNA after normalization to actin RNA is shown.
Msi-1, and the maturation of tau pre-mRNA and suggest that that alterations in msi-1 expression may be implicated in the generation of Tau inclusions in glial cells. In addition, they indicate that part of the wide regulatory actions of T3 may be mediated by genes involved in posttranscriptional mechanisms such as msi-1.
EXPERIMENTAL METHODS Rats Wistar rats raised in our animal facilities were used. The maintenance and handling of the animals were as recommended by the European Communities Council
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FIG. 5. Ectopic msi-1 expression and T3 treatment increase the proportion of mature tau RNA isoforms in N2a cells. (A) Western blotting analysis of the expression of exogenous Flag-Msi-1 protein in transfected N2a cells. Sizes of the endogenous and exogenous Msi-1 proteins are indicated. (B) RT–PCR analysis demonstrating an increase in the ratio of exon 10⫹ (367 bp) to exon 10⫺ (274 bp) tau mRNAs in Msi-1 overexpressing cells. -actin mRNA was used as an additional internal control for the PCR reaction and equal DNA loading. Quantitative RT–PCR analysis was performed as described under Experimental Methods. Lower panel shows the quantification of the results corresponding to three independent experiments. (C) N2a ⫹ TR cells expressing high levels of hormone receptors were treated with 150 nM T3 for the indicated days or not at all. The exon 10⫹/exon 10⫺ ratio of tau transcripts was analyzed by RT–PCR. -actin mRNA was used as an internal control. Lower panel shows the quantification of the results corresponding to five independent experiments: ns, statistically nonsignificant; **P ⬍ 0.05; ***P ⬍ 0.005. (D) Parental N2a cells expressing low levels of TRs were subjected to the same study as control. Lower panel shows the quantification of the results corresponding to three independent experiments.
Directive of November 24th, 1986 (86/609/EEC). Embryonic and neonatal hypothyroidism were induced as described (Alvarez-Dolado et al., 2000): 2-Mercapto-1methylimidazole and potassium perchlorate (0.02% MMI, Sigma; 1% KClO 4, Merck) were administered in the drinking water of the dams from the 9th day after conception until the animals were sacrificed. Thyroid hormone treatment consisted of daily single intraperitoneal injections of 1.8 g thyroxine (T4)/100 g body wt starting 4 days before death. Rats were killed 24 h after the last T4 injection. T4 was used for the in vivo hormonal treatments because it crosses the blood– brain barrier more efficiently than T3 and is converted to T3 in the brain (Dickson et al., 1987). The results obtained in the animal studies referred to consistent findings based on the analysis of sections from at least three animals per experimental group. Hypothyroid animals showed the characteristic arrest of body weight increase (20 –25% on P15 and 50 – 60% on P25).
In Situ Hybridization Under deep pentobarbital anesthesia, rats were perfused through the heart with cold 4% p-formaldehyde in 0.1 M sodium phosphate (pH 7.4). The brains were quickly removed, postfixed in 4% p-formaldehyde in 0.1 M sodium phosphate (pH 7.4), and cryoprotected in 4% p-formaldehyde ⫹ 30% sucrose (w/v) in phosphatebuffered saline (PBS) at 4°C. Subsequently, 25-m-thick coronal sections were cut using a cryostat. In situ hybridization on floating sections was performed as described (Alvarez-Dolado et al., 2000). Hyperfilm -MAX films (Amersham Biosciences) were exposed for 15–21 days, developed with Kodak D19, and fixed. Immunohistochemistry Immunohistochemistry was as described (Sakakibara and Okano, 1997) using affinity purified rabbit polyclonal antimouse Msi-1 antibody and a secondary ratpreadsorbed goat antimouse antibody.
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FIG. 6. Expression of antisense msi-1 RNA inhibits the regulation by thyroid hormone of tau isoforms proportion. One million N2a ⫹ TR cells were transfected with 1 or 2 g of pCMV-␣msi-1 plasmid encoding msi-1 cDNA in antisense orientation or with the empty pCMV vector. (A, left) Northern blot (20 g total RNA per lane) showing the expression of endogenous and antisense msi-1 RNA 16 h after transfection. (A, right) Western blot showing the reduction of Msi-1 protein induction by T3 in cells transfected with antisense msi-1 DNA. Protein extracts were prepared 4 days after transfection. (B, top) Western blot showing that antisense msi-1 expression does not affect the basal level of Msi-1 protein in N2a cells. Protein extracts were prepared 4 days after transfection. (B, bottom) Micrograph showing the induction of cell death by antisense msi-1. (C) Ratio of tau mRNA exon 10⫹/exon 10⫺ isoforms expressed in parallel cultures of N2a ⫹ TR cells transfected with either empty pCMV vector or with antisense msi-1 (pCMV-␣msi-1) which were treated with 150 nM T3 daily or not at all. Quantitative RT–PCR was performed 4 days after transfection. Values corresponding to triplicates obtained in two independent experiments are shown: **P ⬍ 0.05; ***P ⬍ 0.005. (D) Scheme of the two-step regulation of the ratio of tau mRNA isoforms by T3 through the modulation of Msi-1 expression. T3 induces msi-1 gene probably at the transcription levels leading to increase Msi-1 level, which favors the synthesis of transcripts encoding 4R tau isoforms.
Western Blotting N2a cell protein extracts were prepared following the Dignam C method (Sambrook et al., 1989). Subcellular fractionation, was performed as described (Gonza´ lez et al., 2000). To prepare protein extracts from brain tissue, dissected regions were homogenized in 7 volumes of chilled high pH buffer [30 mM diethylamine, 1 mM EDTA (pH 11.5)], cleared by centrifugation at 900g for 15 min at 4°C, and finally centrifuged at 100,000g for 30 min at the same temperature. Aliquots of the supernatants were electrophoresed in 12% polyacrylamide gels and transferred to nylon PVDF membranes. The filters were washed, blocked with Blotto (5% skimmed milk in PBS, 0.1% Tween-20), and incubated overnight at 4°C with the mMsi-1 antibody (1:500 dilution). Blots were washed 3X 15 min in PBS, 0.1% Tween-20 and incubated with HRP-
conjugated antirabbit (for anti-Msi-1), HRP-conjugated antigoat (for anti--actin), or HRP-conjugated antimouse (for antihistone H10) for 1 h at room temperature. Blots were developed by a peroxidase reaction using the ECL detection system (Dupont). After exposure, Msi-1 bands were quantified by densitometric analysis of the autoradiographs. Histone H10 was detected using a monoclonal antibody donated by Prof. A. Alonso (Heidelberg). -Actin was detected with the sc-1615 (Santa Cruz Biotechology, Inc.).
Cell Culture, Treatments, Plasmids, and Transfections Mouse N2a neuroblastoma cells and their derivatives N2a ⫹ TR␣ (Pastor et al., 1994) and N2a ⫹ TR (Lebel
208 et al., 1994), which express exogenous TR␣1 or TR1 thyroid hormone receptor genes (donated by Dr. J. Bernal from our Institute and Dr. J. Puymirat from Quebec, Canada, respectively), were grown in Dulbecco’s modified Eagle’s medium (DMEM)-Hepes (pH 7.2) supplemented with 10% fetal calf serum and 2 mM glutamine (all from Gibco-BRL). Thyroid hormone, dexamethasone (Dex), and retinoic acid (RA) were purchased from Sigma. 1␣,25-Dihydroxyvitamin D3 (vitD3) was a gift from Productos Roche (Madrid). The expression of exogenous Msi-1 protein was induced by transfection of N2a cells with pCDNA3-Flag-msi-1, which contains the ORF of mouse msi-1 inserted at the XbaI site of pCDNA3-Flag vector (Invitrogen). For antisense expression the msi-1 cDNA was cloned in 3⬘–5⬘ orientation at the BamHI site of pCMV vector (donated by Dr. C. Lo´ pez-Otı´n, Oviedo) (pCMV-␣msi-1). N2a cells were transfected by the calcium phosphate technique. After a 16-h incubation, the cells were washed twice in PBS and incubated in fresh medium containing 10% T3-depleted serum with or without T3 (150 nM). For transfection of N2a ⫹ TR cells we used Lipofectamine Plus reagent (Gibco BRL). Primary cultures of embryonic day 18 rats were prepared as described (Hisahara et al., 1997) and cultured for 5 days before use. RNA Extraction and Northern Analysis To prepare total RNA from N2a cells we used standard methods (Sambrook et al., 1989). RNAs were fractionated in formaldehyde agarose gels and blotted onto nylon membranes. As controls for the amount of RNA, filters were rehybridized with a -actin cDNA probe. Radioactive probes were prepared by the random priming procedure using Ready-to-go kit (Amersham Biosciences). Confocal Microscopy It was performed with a BioRad MRC-1024 laser scanning microscope equipped with an Axiovert 100 invert microscope (Zeiss) at excitation wavelengths of 488 nm (for FITC) and 543 nm (for TxR). For N2a cells we used the described m-Msi-1 and a mouse monoclonal anti-U2B⬙ antibody (Habets et al., 1989) and for astrocytes a monoclonal antiglial fibrillary acidic protein (GFAP) antibody (GA5, Sigma). Secondary antibodies were a goat antirabbit IgG-TxR, a rabbit antimouse IgG-FITC (Jackson Immunoresearch), and an antirabbit IgG-Cy3 (Chemicon). Samples were examined with Plan-Apochromat 63X (N2a) or 40X (astrocytes) oil-immersion objectives.
Cuadrado et al.
RT–PCR Analysis of tau Exon 10 Alternative Splicing We followed a quantitative method extensively used in the literature (Grover et al., 1999; Hasegawa et al., 1999; Yasuda et al., 2000). Four micrograms of total RNA isolated from N2a cells were retrotranscribed using M-MLV reverse transcriptase (Promega). PCR was performed using primers designed for annealing to exonic sequences from exon 9 (forward, 5⬘ CTGAAGCACCAGCCAGGAGG 3⬘) and exon 12 (reverse, 5⬘ TGGTCTGTCTTGGCTTTGGC 3⬘). Amplification was carried out by 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, with a final 72°C extension phase for 7 min. PCR products were visualized in 2% agarose TBE gels. The size of exon 10⫹ PCR products was 367 bp, while that of exon 10⫺ products was 274 bp. As an internal control, we performed RT–PCR of -actin RNA using primers designed for annealing to sequences from 881 to 1237 positions of the mouse -actin mRNA.
ACKNOWLEDGMENTS This work was supported by Grants SAF98-0060 and SAF2001-2291 from Plan Nacional de Investigacio´ n y Desarrollo of Spain. We thank Drs. M.T. Berciano and M. Lafarga for their help with the confocal microscopy analysis, Drs. J. Puymirat and J. Bernal for providing us with N2a ⫹ TR and N2a ⫹ TR␣ cells respectively, Professor A. Alonso for the antihistone H1° antibody, and Drs. J. Avila and S. Sakakibara for critical reading of the article.
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