Neuroserpin is post-transcriptionally regulated by thyroid hormone

Molecular Brain Research 123 (2004) 56 – 65 www.elsevier.com/locate/molbrainres

Research report

Neuroserpin is post-transcriptionally regulated by thyroid hormone Cristina Navarro-Yubero a, Ana Cuadrado a, Peter Sonderegger b, Alberto Mun˜oz a,* a

Instituto de Investigaciones Biome´dicas ‘‘Alberto Sols’’, Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto´noma de Madrid, E-28029 Madrid, Spain b Department of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland Accepted 31 December 2003

Abstract Neuroserpin is a serine protease inhibitor expressed in the developing and the adult nervous system. Studies with genetically modified mice indicate a role of neuroserpin in the regulation of anxiety. Mutations in the neuroserpin gene cause protein polymerization and formation of inclusion bodies leading to progressive myoclonic epilepsy and neurodegeneration. Here we demonstrate that neuroserpin expression is regulated by thyroid hormone (T3). Neuroserpin RNA levels are down-regulated in cortical layers II/III and VIa, the hippocampus, the retrosplenial cortex and the medial habenular nucleus, but not in cortical layer V or other areas of the hypothyroid rat brain. Concordantly, neuroserpin RNA and protein expression was induced by T3 in rat PC12 cells containing appropriate thyroid hormone receptor levels. In runon assays T3 did not affect the transcription rate of the neuroserpin gene, indicating that regulation was post-transcriptional. Moreover, T3 increased in vitro binding of cytoplasmic proteins to neuroserpin 3V-UTR RNA and caused biphasic regulation of the stability of this transcript in PC12 cells. Ectopic neuroserpin expression induced neurite extension in PC12 cells and enhanced neuritogenesis triggered by nerve growth factor. In summary, these results indicate that neuroserpin expression is post-transcriptionally regulated by T3 at the level of RNA stability. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Hormones and development Keywords: Neuroserpin; Thyroid hormone; Brain; Gene regulation; PC12 cells; RNA stability

1. Introduction Neuroserpin is a serine protease inhibitor of the serpin family that was initially identified as an axonally secreted protein from chicken dorsal root ganglion and motoneuron cultures [40,50,54]. In mice neuroserpin is expressed by neurons of the central and peripheral nervous systems (CNS, PNS) during development and in adult life [28]. In vitro, neuroserpin is a potent inhibitor of tissue plasminogen activator (tPA), urokinase-type PA (uPA) and plasmin [24,40,41]. Whether these serine proteases represent the cognate in vivo targets of neuroserpin remains to be determined [5].

* Corresponding author. Tel.: +34-91-585-4452; fax: +34-91-5854401. E-mail address: [email protected] (A. Mun˜oz). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2003.12.018

The biological role of neuroserpin has not been fully elucidated. Its spatio-temporal expression pattern indicates a role in both neural development and adult neural function. In the adult predominant expression in the neocortex, hippocampal formation, olfactory bulb and amygdala, has prompted speculation about its role in synaptic plasticity [28]. Recent behavioral studies with neuroserpin-overexpressing and neuroserpin-deficient mice revealed impaired explorative behavior and neophobia [35]. Some neuroserpin (SERPINI1) gene mutations cause changes in protein conformation and polymerisation leading to the formation of inclusion bodies (Collins bodies) in CNS neurons [15]. The resulting pre-senile autosomal dominant dementia was named FENIB (Familial Encephalopathy with Neuroserpin Inclusion Bodies). It belongs to the serpinopathies, which in turn are part of a larger group known as conformational neurodegenerative diseases, caused by the aggregation of specific proteins, including Alzheimer, Parkinson and prion diseases and frontotemporal dementias

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[10,32]. FENIB patients show deficits in attention and concentration, and audiovisual impairment [15]. Four disease-causing mutations of neuroserpin have been found so far [16]. All are located in the shutter region, which is essential for the inhibitory mechanism of neuroserpin, and they impair its conformational stability. The mutations G392E, H338R and S52R and G392E severy affect the conformational stability of the molecule. Proteins encoded by genes bearing these mutations aggregate and cause inclusions in almost all neurons. The resulting neurodegeneration was associated with early progressive myoclonus epilepsy. The S49P mutation is less disruptive: inclusion bodies occur in fewer neurons and clinical symptoms appear later [6,16,55]. The mechanisms regulating neuroserpin expression are largely unknown. In mouse hippocampal neurons, elevated extracellular KCl induces neuroserpin gene transcription while zif/268 has a repressive effect [7]. We have reported that neuroserpin is regulated at the level of RNA stability by binding of HuD, an RNA-binding protein, to AU-rich sequences in its 3V-UTR [13]. Recently, we found that HuD is transcriptionally repressed by thyroid hormone (triiodothyronine, T3) in neuronal PC12 cells and in the rat brain [14]. These data led us to examine whether neuroserpin expression is under T3 control. We now show that thyroid hormone deficiency decreases neuroserpin RNA levels in several areas of the rat brain. In other areas, such as cortical layer V, where HuD protein is up-regulated, neuroserpin RNA did not decrease in the hypothyroid state. In PC12 cells we found that T3 induces neuroserpin RNA and protein expression without affecting the rate of gene transcription. From previous studies, T3 was expected to decrease neuroserpin RNA stability through the inhibition of HuD protein levels. However, we found that T3 causes an early and transient decrease in neuroserpin RNA 3V-UTR, followed by an increase in its stability. Our data suggest that T3 regulates neuroserpin expression post-transcriptionally through the control of its RNA half-life by a mechanism that involves HuD and probably other proteins.

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water of the dams from the 9th day after conception until the animals were killed. Hypothyroid animals showed the characteristic arrest of body weight increase (around 25% on P15) and low circulating levels of both, thyroxine and T3 measured by specific radioimmunoassay [37] analogous to those reported [47]. 2.2. In situ hybridization Under deep pentobarbital anaesthesia, three rats of each experimental group 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% pformaldehyde in 0.1 M sodium phosphate (pH 7.4) and cryoprotected in 4% p-formaldehyde+30% sucrose (w/v) in phosphate-buffered saline (PBS) at 4 jC. Subsequently, 25 Am thick coronal sections were cut on a cryostat. In situ hybridization on floating sections was performed as described [11]. Hyperfilm h-MAX films (Amersham Pharmacia Biotech Europe, Barcelona) were exposed for 15 – 21 days, developed in Kodak D19 and fixed. 2.3. Cell culture, treatments, plasmids and transfections

2. Materials and methods

Rat PC12 pheochromocytoma cells and their derivatives PC12+TRa1 and PC12+v-erbA cells [38] which express exogenous TRa1 or v-erbA genes, were grown in Dulbecco’s modified Eagles’ medium supplemented with 10% horse serum, 5% fetal calf serum and 1 mM glutamine (all from GIBCO-Invitrogen, Paisley, UK). T3, Dexamethasone (Dex), all-trans retinoic acid (RA), nerve growth factor (NGF) and G418 were purchased from Sigma. 1a,25-dihydroxyvitamin D3 (vitD3) was a gift from Productos Roche (Madrid). To generate cells stably expressing neuroserpin, PC12 cells were transfected using Lipofectamine (Invitrogen) with the pCDNA3.1 vector plasmid (Invitrogen) containing the open reading frame of murine neuroserpin. Stable cell lines expressing neuroserpin were obtained following selection with the aminoglycoside antibiotic G418 (800 Ag/ ml). For neuroserpin 3VUTR-RNA expression, a fragment corresponding to nucleotides 788 to 2944 of its cDNA was subcloned into the BamHI and XhoI sites of pCDNA3.1 and used to transfect PC12+TRa1 cells.

2.1. Rats

2.4. RNA extraction and northern analysis

Wistar rats were raised in our animal facilities. The maintenance and handling of the animals were as recommended by the European Communities Council Directive of November 24, 1986 (86/609/EEC). All efforts were made to minimize animal suffering, reduce the number of animals used and utilize alternatives to in vivo techniques. Congenital hypothyroidism was induced as described [2]: 2-mercapto-1-methylimidazole and potassium perchlorate (0.02% MMI, Sigma, St. Louis, MO, USA; 1% KClO4, Merck, Darmstadt, Germany) were administered in the drinking

To prepare total RNA from PC12 cells we used TriReagent according to the instructions of the manufacturer (Molecular Research Center, Cincinnati, OH). RNA was fractionated in formaldehyde agarose gels and blotted onto nylon membranes (Nytran; Schleicher and Schuell, Dassel, Germany) following standard methods [49]. As control for RNA loading, the filters were stained with 0.02% methylene blue in 0.3 M sodium acetate. Radioactive probes were prepared by the random priming procedure using Ready-togo kit (Amersham Pharmacia Biotech).

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2.5. Western blotting Thirty micrograms of protein cell extract was routinely utilized for Western blot analysis of neuroserpin (10 Ag for the other proteins). Lysates and subcellular fractionation were prepared as described [21]. Protein lysates were electrophoresed in 10% polyacrylamide gels and transferred to nylon membranes (Immobilon P, Millipore, Bedford, MA, USA). The filters were blocked in 5% skimmed milk in TBS+0.1% Tween-20 or 3% BSA in TBS+0.5% Tween20 (for neuroserpin and HuR) and incubated overnight at 4 jC with the primary antibody in the same buffer (in the case of TIA1 and TTP antibodies, with TBS alone). Blots were washed from three to six times for 10 min in TBS+0.1% (or 0.5%) Tween-20, and incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Blots were developed by a peroxidase reaction using the ECL detection system (Amersham Pharmacia Biotech). The primary antibodies used were the following: neuroserpin (goat polyclonal G47 raised against recombinant murine neuroserpin and affinity-purified on murine neuroserpin coupled to CNBractivated Sepharose, 1 Ag/ml), HuR (mouse monoclonal antibody from Santa Cruz Biotechnology, Santa Cruz, CA, USA, 0.2 Ag/ml), AUF1 (rabbit polyclonal antibody from

Phoenix Pharmaceuticals, 0.8 Ag/ml), TTP (goat polyclonal antibody from Santa Cruz Biotechnology, 0.2 Ag/ml), TIA1 (goat polyclonal antibody from Santa Cruz Biotechnology, 0.4 Ag/ml) and TIAR (mouse monoclonal antibody from BD Transduction Laboratories, San Diego, USA, 0.25 Ag/ml). 2.6. Nuclear run-on transcription assays We followed the procedure described by Lo´pez-Carballo et al. [33]. Briefly, cells were lysed at 4 jC in 20 mM Tris – HCl, pH 8.0, 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.15 mM spermine, 0.5 mM h-mercaptoethanol, and 0.1% Nonidet P40. After washing in cold buffer B (50 mM HEPES-NaOH, pH 8.0, 5 mM MgCl2, 0.5 mM dithiothreitol, 1 Ag/ml bovine serum albumin (BSA), 25% glycerol) nuclei were pelleted, resuspended in the same buffer B and stored at 70 jC in aliquots of 5106. Nytran N45 membranes (Schleicher and Schuell) containing 1 Ag of purified cDNA fragments of neuroserpin, h-actin or GAPDH, or 5 Ag of linearized empty pBS SK+vector were prepared with the aid of a slot blot apparatus (Manifold II, Schleicher and Schuell). Transcription reactions were performed with 50 Al of transcription mix (50 mM HEPES-NaOH pH 8.0, 2

Fig. 1. In situ hybridization analysis of neuroserpin RNA expression in the brain of control and hypothyroid rats at postnatal day 15. (A) Coronal sections showing lower neuroserpin RNA levels in the cerebral cortical layer II/III and VIa, hippocampus (Hi), medial habenular nucleus (MHb), and retrosplenial (RSCx) cortex of hypothyroid animals, but not in cerebral cortex layer V (V). (B) Higher magnification showing the lack of down-regulation in cortex layer V. Scale bar: 2 mm applies to (A); 0.45 mm applies to (B). Abbreviations were as in Paxinos and Watson [43].

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mM MgCl2, 2 mM MnCl2, 1 Ag/ml BSA, 300 mM NH4Cl); 5 Al of 10 AM CTP, ATP, GTP mix; 100 ACi of (a-32P)-UTP (Amersham; 800 Ci/mmol); 40 units of RNasin (Promega, Madison, WI, USA); and 50 Al of nuclei (5106) in buffer B. After incubation for 20 min at 30jC, nuclear DNA was digested with 10 units of RQ1 DNase (Promega). Purification of labeled RNA and hybridization of membranes were as described [33].

30 min at 25 jC and digested with 7.5 U/reaction RNase T1 (Calbiochem, La Jolla, CA) for 15 min at 37 jC. Complexes were resolved by electrophoresis, through native gels (7% acrylamide in 0.25 M Tris-borate-EDTA buffer) without loading buffer (160 V, 2 h, 4 jC).

2.7. Preparation of labeled RNA transcripts

3.1. Neuroserpin expression is altered in the hypothyroid rat brain

DNA templates for neuroserpin 3V-UTR transcript (1,565 nucleotides) were synthesized by PCR using the following oligonucleotides corresponding to nucleotides 1343-2908: T71343 (5V-GTAATACGACTCACTATAGGGCGAGTACAAAGAAAGCAGG-3V) and 2908a (5V-TATTCTTCCTTACAGGC-3V). RNA transcripts were synthesized using T7 RNA polymerase (Promega) and purified as described [34]. 2.8. RNA electrophoretic mobility shift assays Reaction mixtures (10 Al) containing 1 Ag of tRNA, 5 fmol of RNA (195,000 cpm) and 10 Ag of protein were incubated in reaction buffer (15 mM HEPES pH 7.9, 10 mM KCl, 10% glycerol, 0.2 mM dithiothreitol, 5 mM MgCl2) for

3. Results

We analyzed the effect of hypothyroidism on neuroserpin expression during rat brain development. In situ hybridization analysis revealed that neuroserpin RNA expression mostly coincides with that previously described in the mouse brain [28]. At postnatal day 15, when the number and occupancy of T3 receptors (TR) are maximal [19], hypothyroidism reduced neuroserpin RNA levels in several brain areas including cerebral cortex (layers II/III and VIa), hippocampus, medial habenular nucleus and retrosplenial cortex (Fig. 1). In other areas such as cortex layer V, however, no changes were found. Remarkably, in cortex layer V HuD is the only member of the HuD/Elav gene family to be expressed [39] and it is up-regulated at both RNA and protein levels in the hypothyroid rat brain [14].

Fig. 2. Northern blot analysis showing the regulation by T3 of neuroserpin RNA expression. Twenty Ag of total RNA was loaded per lane. Sizes of the two neuroserpin mRNAs and the fold increase in neuroserpin RNA levels are indicated. The 18S ribosomal RNA stained with methylene blue was used as control. (A) Effect of T3 levels in parental PC12, PC12+TRa1, and PC12+v-erbA cells. Cells were treated or not with 150 nM T3 for 24 h as indicated. (B) Dose – curve of neuroserpin mRNA induction by T3 in PC12+TRa1 cells. Cells were treated for 24 h with the indicated T3 concentrations. (C) Effect of 24 h treatment with 150 nM T3 or 1 AM dexamethasone (Dex), 1a,25-dihydroxyvitamin D3 (VitD) or all-trans retinoic acid (RA) on neuroserpin RNA expression.

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Fig. 3. Time-course of induction of neuroserpin RNA and protein by T3. (A) Northern blot analysis of neuroserpin RNA induction by T3. PC12+TRa1 cells were treated or not with T3 (150 nM) for the indicated periods. Twenty Ag of total RNA was loaded per lane. Sizes of the neuroserpin mRNAs are indicated. The bottom shows the 18S ribosomal RNA stained with methylene blue. (B) Quantification of 3.0 kb neuroserpin mRNA level. Mean values and standard deviations obtained in three experiments are shown. (C) Western blot analysis of the induction of neuroserpin protein by T3. Thirty micrograms of cytoplasmic extract was loaded per lane. Size of neuroserpin protein is indicated. The bottom shows the signal of an unrelated band as control. (D) Quantification of neuroserpin protein levels. Mean values and standard deviations obtained in three experiments are shown.

3.2. T3 increases neuroserpin expression in PC12 cells The mechanism of regulation of neuroserpin expression by T3 was studied in rat PC12 cells. Northern blots revealed that T3 induces a near three-fold increase in the expression of both neuroserpin mRNAs (3.0 and 1.7 kb) in cells containing appropriate TR levels (PC12+TRa1) but not in wild-type PC12 cells that express low levels of TR or in PC12+v-erbA cells containing the v-erbA oncogene which encodes a mutant version of TRa1 receptor unable to bind T3 [38] (Fig. 2A). The induction of neuroserpin transcripts

was detectable at physiological (nanomolar) hormone levels (Fig. 2B). The effect of T3 was specific since other hormones such as dexamethasone, 1a,25-dihydroxyvitamin D3 or all-trans retinoic acid, which also act through nuclear receptors, failed to induce neuroserpin expression (Fig. 2C). The induction of neuroserpin RNA was detected 4 h after T3 treatment and increased progressively to 6-fold at 72 h (Fig. 3A and B). This effect was paralleled by a similar and slightly delayed increase in neuroserpin protein expression, which was around 12-fold at 72 h after T3 addition (Fig. 3C and D).

Fig. 4. Run-on analysis of the effect of T3 on neuroserpin gene transcription. Nuclei from PC12+TRa1 cells treated or not with T3 (150 nM) for 4 or 8 h (not shown) were subjected to the in vitro transcription reaction as described in Materials and methods. As negative controls we used h-actin and GAPDH genes, and also the empty pBS SK+ vector. HuD gene was used as positive control. Right panel shows the quantification of the transcription rates of neuroserpin and h-actin genes normalized to that of GAPDH.

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3.3. T3 regulates neuroserpin gene post-transcriptionally Run-on assays showed that the transcription rate of neuroserpin RNA expression was lower than that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and h-actin genes used as controls, and unaffected by 4 or 8 h treatment with T3 (Fig. 4 and not shown). This, indicates that induction of neuroserpin RNA by T3 was not due to an increase in the rate of gene transcription. As positive control of a gene transcriptionally regulated by T3 we used HuD [14]. Next, we examined whether the induction of neuroserpin expression by T3 was the result of the modulation of neuroserpin RNA stability. First, we found by RNA electrophoretic mobility shift assays that T3 treatment of PC12+TRa1 cells increased the binding of cytoplasmic proteins to neuroserpin RNA 3V-UTR (Fig. 5A). We studied this effect in intact PC12+TRa1 cells by transfection with a plasmid encoding this region (pCDNA3-neuroserpin 3VUTR). T3 treatment had a biphasic effect: while at early times (1 –2 h) it reduced neuroserpin 3V-UTR levels, later (4– 6 h) it increased its stability (Fig. 5B). Concordantly, the half-life of endogenous neuroserpin RNA in PC12+TRa1 cells increased from 20 h to more than 28 h upon T3 treatment but could not be determined precisely due to the cytotoxic effect of actinomycin D. Since HuD protein binds the 3V-UTR of neuroserpin RNA and increases neuroserpin RNA stability when over-

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expressed [13], and its expression is repressed by T3 in PC12+TRa1 cells [14], we examined whether the increase in neuroserpin RNA steady-state levels by T3 was caused by the regulation of other RNA-binding proteins. By Western blotting, we studied the cellular content of AU-binding factor (AUF1), tristetraprolin (TTP), T-cell internal antigen-1 (TIA-1) and TIA-1-related (TIAR) proteins. T3 caused only a small increase in the cellular content of these proteins except in the case of TIA-1, whose expression was transiently down-regulated (Fig. 5C). In addition, expression of other members of the Elav/Hu family was investigated: while HuR protein expression changed only minimally in comparison to HuD (Fig. 5C), that of HuB and HuC was undetectable in Northern blots in both untreated and T3-treated cells (not shown). 3.4. Ectopic neuroserpin expression induces neuritogenesis in PC12 cells Stable neuroserpin expression induces neurite-like processes in anterior pituitary AtT-20 cells [25] while decreasing the number of PC12 cells extending neurites and the total length of neurites [42]. We stably transfected our parental PC12 cells with an expression vector for neuroserpin that also encodes the neor gene. Both parental and neuroserpin-overexpressing (around two-fold) cells were then incubated in the presence or absence of nerve growth factor (NGF).

Fig. 5. Effect of T3 on the stability of neuroserpin RNA 3V-UTR. (A) RNA electrophoretic mobility shift assay showing that T3 treatment enhances the binding of cytoplasmic proteins to neuroserpin RNA 3V-UTR. Ten Ag of nuclear or cytoplasmic extracts of PC12+TRa1 cells treated for the indicated times with 150 nM T3 or left untreated were incubated in vitro with a transcript corresponding to the complete 3V-UTR of neuroserpin RNA as described in Materials and methods. The shifted bands are indicated. (B) Northern blot analysis showing that T3 controls the stability in intact cells of an exogenous transcript containing the 3V-UTR of neuroserpin RNA. PC12+TRa1 cells were transfected with an expression vector containing the entire 3V-UTR of neuroserpin RNA. The expression of the 2.1 kb exogenous transcript and 18S ribosomal RNA stained with methylene blue (bottom) are shown. (C) Western blot analysis showing the expression of several RNA-binding proteins in PC12+TRa1 cells treated or not with 150 nM T3 for the indicated times. AUF1, HuR, TTP, TIA1 and TIAR proteins were detected using specific antibodies as indicated in Materials and methods. The lower parts of the panels show the Coomassie blue staining. Numbers refer to fold-increase over values in untreated cells. Protein sizes are indicated.

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Fig. 6. Effect of increased neuroserpin expression on neurite outgrowth in PC12 cells. Cells were stably transfected with empty (control) or neuroserpin expression vectors. Phase-contrast micrographs of control (A) or neuroserpin-transfected (B) cells were taken 7 days after antibiotic selection in medium supplemented with 0.5% horse serum. Most neuroserpin over-expressing cells showed neurites (white arrows). At day 14 of antibiotic selection, control cells (C) showed progressive rounding and degeneration, while neuroserpin-transfected cells (D) displayed polygonal morphology. NGF (50 ng/ml) treatment during the second week induced neurites of variable length in control cells (E), and a wide neurite-inducing effect in neuroserpin-expressing cells (F). Scale bar: 340 Am.

A large number of PC12 cells ectopically expressing neuroserpin developed neurite-like processes after 1 week of antibiotic (G418) exposure (Fig. 6, compare A to B). During the second week, cell rounding and death was observed in mock-transfected cells while neuroserpin-expressing cells appeared healthy and showed a polygonal morphology and short spikes (Fig. 6, compare C and D). Addition of NGF to the culture medium during the second week rescued many parental cells from cell death (Fig. 6E) and induced strong morphological differentiation in neuroserpin-expressing cells, some of which developed long branched processes (Fig. 6F).

4. Discussion We had previously reported that HuD protein binds to neuroserpin RNA 3V-UTR and that its over-expression increases neuroserpin RNA lifetime [13], and also that T3 represses HuD expression in PC12 cells [14]. In this study we show that neuroserpin gene expression is regulated by T3 in the rat brain and in PC12 cells. At postnatal day 15, when the number and occupancy of TR are maximal [19], thyroid deficiency causes a reduction of neuroserpin RNA

in many brain areas but not in cortical layer V. One explanation for this finding is that hypothyroidism upregulates HuD protein in this layer, where it is the only member of the Elav/Hu family to be expressed [14]. In PC12+TRa1 cells neuroserpin RNA and protein are up-regulated by physiological concentrations of T3. Our data show that the regulation is post-transcriptional since no changes in the rate of transcription were found in run-on assays and T3 enhances the stability of neuroserpin RNA 3V-UTR in transfected cells. As T3 inhibits HuD expression in PC12 cells, this result indicates that the stability of neuroserpin RNA in these cells is controlled by additional proteins. Indeed, binding of proteins to the 3V-UTR of neuroserpin RNA increased after only 30 min of T3 treatment. The rapid destabilization of neuroserpin 3V-UTR found in transfected cells might be due to the reduction in HuD levels [14] (HuD has high affinity binding: Kd=8– 20 nM). This destabilization, however, does not affect neuroserpin RNA steady-state level as shown in Northern blots. Furthermore, this destabilization is transient, which suggests that it is compensated by binding of stabilizing proteins leading to the accumulation of neuroserpin RNA. These proteins remain uncharacterized. Small changes in the amount of several neural RNA-binding proteins such as

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AUF1, TTP, TIA1 or TIAR were observed following T3 treatment. Likewise, the cell content of the ubiquitously expressed HuR protein was only slightly affected by T3, and HuB and HuC were not detected in PC12 cells. We speculate that the relative amount of different RNA-binding proteins interacting with neuroserpin RNA may decide its lifetime, and that the regulatory effects of T3 on neuroserpin expression may depend in vivo on the different areas of the rat brain and in cultured cells on its interaction with other factors controlling the transcription of the gene and on the set of proteins modulating neuroserpin RNA stability that are expressed. Our previous and present data indicate that HuD is one of these proteins. Moreover, both neuroserpin and HuD genes are deregulated in chronic schizophrenia [23]. In the hypothyroid, stable expression of neuroserpin RNA in the cerebral cortical layer V may be caused by the up-regulation of HuD [14], whereas the inhibition observed in other brain regions may result from distinct unbalanced combinations of proteins interacting with neuroserpin RNA. T3 is critical for the development and function of the CNS, acting as a wide regulator of gene expression through binding to its nuclear receptors that function as ligand-dependent transcription factors [3,9,30,reviews]. In addition, several studies have reported post-transcriptional regulatory effects of T3 on the mRNA half-life of a number of genes including malic enzyme [4,53], 3-hydroxy-3-methylglutaryl CoA reductase [52], thyrotropin h subunit [27,31], RXRg [36], acetylcholinesterase [45] and uncoupling protein-1 [22]. We have described that T3 controls the expression of different RNA-binding proteins implicated in post-transcriptional processes in the CNS. SWAP (Suppressor-of-white-apricot), a splicing regulator, is under thyroid hormone control in the rat brain [11] and the RNA-binding protein Musashi-1 mediates the effect of the hormone on tau pre-mRNA splicing [12]. Further suggesting post-transcriptional gene regulatory actions, T3 modulates the expression of NAT-1, a translational repressor, in brain and liver [51]. mRNA turn-over is emerging as a crucial process in post-transcriptional gene regulation, as small differences in mRNA half-life can rapidly alter its abundance and, consequently, the amount of protein expressed [18,48]. The role and importance of neuroserpin regulation in its biological activity cannot yet be established. It is, however, known that synaptogenesis and neuronal plasticity and migration are under T3 control [8,17,reviews], and PAs have been implicated in the modulation and reorganization of synaptic connections in the developing and mature brain [26]. During embryogenesis the increase in tPA expression coincides with cellular migration and proliferation and CNS remodeling [20]. In the CNS, tPA activity is preferentially located at the growth cones [29], and its overexpression also induces PC12 cell migration [44]. Part of the regulation of these processes by T3 may be mediated through the control of proteases and proteases inhibitors, and at least one example has been reported: Reelin, an

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extracellular protein assigned an important role in neuronal migration and positioning, has been described to be under thyroid control [1] and to be a serine protease acting on several components of the extracellular matrix [46]. The finding that stable ectopic neuroserpin expression induces neurite extension and cell viability and promotes NGF action is consistent with the effects of HuD overexpression [13] and its effects in AtT-20 pituitary cells [25], but opposes another report that neuroserpin decreases neurite extension in response to NGF in PC12 cells [42]. Though in our experiments NGF was added after 1 week culture in low serum medium whereas it was present in serumcontaining medium from the beginning in the study by Parmar et al. [42], the reasons for this discrepancy are unclear. In summary, our data show that T3 regulates neuroserpin expression post-transcriptionally at the level of RNA turnover through a mechanism involving HuD and other as yet uncharacterized RNA-binding proteins.

Acknowledgements We thank Dr. M. Gorospe for providing us with the AUF1, TTP, TIA1 and TIAR antibodies, Dr. D. Barettino for his help with the run-on assays and Robin Rycroft for his help in the preparation of the English manuscript. C.N.-Y. was supported by a fellowship from Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo of Spain. This study was supported by Grant SAF2001-2291 from Ministerio de Ciencia y Tecnologı´a of Spain.

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