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Published as doi: 10.1096/fj.07-9814.
 (The FASEB Journal. 2008;22:1083-1093.)
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(The FASEB Journal. 2008;22:1083-1093.)
© 2008 FASEB

Mitogen- and stress-activated protein kinase-1 deficiency is involved in expanded-huntingtin-induced transcriptional dysregulation and striatal death

Emmanuel Roze*,{dagger}, Sandrine Betuing*, Carole Deyts*, Estelle Marcon*, Karen Brami-Cherrier{ddagger}, Christiane Pagès*, Sandrine Humbert§, Karine Mérienne|| and Jocelyne Caboche*,1

* Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7102, Paris, France;

{dagger} Service de Neurologie, Hôpital Saint-Antoine, Assitance Publique-Hôpitaux de Paris, Paris, France;

{ddagger} Université Pierre et Marie Curie-Paris 6, INSERM, UMRS 839, Paris, France;

§ Institut Curie, CNRS, UMR 146,Orsay, France; and

|| Institut de Génétique et de Biologie Moléculaire et Cellulaire, Département de pathologie moléculaire; INSERM, U596; CNRS, UMR 7104, Illkirch, France

1Correspondence: Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7102, 9 quai St. Bernard, 75005, Paris, France. E-mail: jocelyne.caboche{at}snv.jussieu.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Huntington’s disease (HD) is a neurodegenerative disorder due to an abnormal polyglutamine expansion in the N-terminal region of huntingtin protein (Exp-Htt). This expansion causes protein aggregation and neuronal dysfunction and death. Transcriptional dysregulation due to Exp-Htt participates in neuronal death in HD. Here, using the R6/2 transgenic mouse model of HD, we identified a new molecular alteration that could account for gene dysregulation in these mice. Despite a nuclear activation of the mitogen-activated protein kinase/extracellular regulated kinase (ERK) along with Elk-1 and cAMP responsive element binding, two transcription factors involved in c-Fos transcription, we failed to detect any histone H3 phosphorylation, which is expected after nuclear ERK activation. Accordingly, we found in the striatum of these mice a deficiency of mitogen- and stress-activated kinase-1 (MSK-1), a kinase downstream ERK, critically involved in H3 phosphorylation and c-Fos induction. We extended this observation to Exp-Htt-expressing striatal neurons and postmortem brains of HD patients. In vitro, knocking out MSK-1 expression potentiated Exp-Htt-induced striatal death. Its overexpression induced H3 phosphorylation and c-Fos expression and totally protected against striatal neurodegeneration induced by Exp-Htt. We propose that MSK-1 deficiency is involved in transcriptional dysregulation and striatal degeneration. Restoration of its expression and activity may be a new therapeutic target in HD.— Roze, E., Betuing, S., Deyts, C., Marcon, E., Brami-Cherrier, K., Pagès, C., Humbert, S., Mérienne, K., Caboche, J. Mitogen- and stress-activated protein kinase-1 deficiency is involved in expanded-huntingtin-induced transcriptional dysregulation and striatal death.


Key Words: Huntington’s disease • polyglutamine • histone modifications • nucleosomal response • c-Fos


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HUNTINGTON’S DISEASE (HD) IS AN autosomal dominant neurodegenerative disorder characterized by chorea, psychiatric disturbances, and cognitive impairment (1) . It results from an abnormal CAG repeat expansion in exon 1 of the HD gene, which is translated into an abnormally long polyglutamine tract at the N terminus of huntingtin protein (Htt; ref. 2 ). Together with the formation of characteristic aggregates, one important feature of HD is the severe atrophy of specific brain areas, medium spiny neurons of the caudate-putamen (striatum) being the most susceptible to degeneration (3 4 5) . The exact cause of this selective neuronal death in HD is unknown. Among the molecular mechanisms by which mutant Htt induces cell death, transcriptional dysregulation may be critical (6) . Gene expression profiling studies (7 , 8) have shown decreased levels of mRNAs encoding immediate early genes in the striatum of R6/2 mice, a transgenic mouse model expressing the first exon of the human huntingtin gene containing an expanded CAG repeat (9) .

Classically, the current intense investigation of the molecular mechanisms that underlie transcriptional dysfunction in HD has been focused on the mechanisms that regulate transcription factors activity. However, new concepts have recently emerged, pointing out the role of chromatin remodeling in the modulation of gene expression in neuronal cells (10 11 12 13 14 15 16) As a corollary, alteration in chromatin remodeling can lead to neuronal dysfunctions and may be involved in the pathophysiology of neurodegenerative diseases, including in HD. Chromatin remodeling is an epigenetic mechanism leading to DNA decompaction. It requires posttranslational modifications of highly basic proteins called histones (H3, H4, H2A, and H2B; refs. 17 , 18 ). Position-specific modifications of the histone N-terminal tails at gene promoters define a "histone code" and control, at least in part, the transcriptional level of these genes (17 , 19) . In mouse models of HD, treatments that globally increase histone acetylation and/or decrease methylation levels result in improved phenotype (20 21 22 23 24 25) . Phosphorylation of histone H3 is also involved in chromatin remodeling. In particular, this phosphorylation event is critical to induce the nucleosomal response at the c-fos promoter and hence c-Fos expression in neuronal cells (10 , 11) . The kinase responsible for H3 phosphorylation is mitogen- and stress-activated protein kinase 1 (MSK-1), a kinase activated downstream of the mitogen-activated protein (MAP) kinase/extracellular regulated kinase (ERK) signaling pathway (26) . Although H3 phosphorylation has been shown to play a key role in neuronal plasticity (11 12 13 14 15 16) , no study has yet addressed its role in transcriptional dysregulation in HD.

In the present study, we found a defective phosphorylation of histone H3 in the striatum of R6/2 mice, despite a strong nuclear activation of the ERK pathway. We then asked whether MSK-1 deficiency could account for this defective H3 phosphorylation. To test this, we studied MSK-1 activation and expression in R6/2 mice and postmortem brains of HD patients. Finally, we evaluated, in vitro, the consequences of MSK-1 overexpression on H3 phosphorylation, c-Fos expression, and Exp-Htt-induced striatal death.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
R6/2 mice [B6CBA-TgN (HDexon1) 62], which express exon 1 of the human mutant HD gene containing 115–150 CAGs under the control of the human IT15 gene promoter, were obtained from the Jackson Laboratory (Bar Harbor, ME, USA), by crossing ovarian transplant hemizygous females with males of their background strain B6CBAF1/J. Genotyping of transgenic mice was performed on tail DNA by polymerase chain reaction (PCR; ref. 9 ). The mice were housed in groups with a 12-h light/dark cycle and food and water ad libitum. Animal care was conducted in accordance with standard ethical guidelines (U.S. National Institutes of Health publication no. 85–23, revised 1985, and European Committee Guidelines on the Care and Use of Laboratory Animals), and the experiments were approved by the local ethics committee.

Human tissue
Human brain samples 1–12 were obtained from Dr. E. Hirsch, INSERM U289 brain bank at the Salpêtrière Hospital (Paris, France), according to standard legislation. Samples 1–3 were cerebral cortex from controls. Samples 4–6 were cerebral cortex from symptomatic proved HD patients with family history. Samples 7–9 were caudate nucleus from controls. Samples 10–12 were caudate nucleus from proved symptomatic HD patients with family history.

Immunohistochemistry
Tissue from wild-type controls and R6/2 mice were processed in parallel for immunohistochemistry. The immunohistochemical procedure was adapted from previously described protocols (27) . Mice were rapidly anesthetized by intraperitoneal injection of pentobarbital 250 mg/kg (Sanofi, Paris, France) before intracardiac perfusion of 4% PFA in 0.1 M Na2HPO4/NaH2PO4 buffer, pH 7.5, delivered with a peristaltic pump at 25 ml/min for 5 min. Brains were then postfixed overnight in the same solution and stored at 4°C. Sections (30 µm) were cut with a vibratome (Leica Microsystems, Rueil-Malmaison, France) and kept in a solution containing 30% ethylene glycol, 30% glycerol, and 0.1 M phosphate buffer at –20°C until processing for immunohistochemistry. To detect phosphorylated proteins, 0.1 mM NaF was included in all buffers and incubation solutions.

P-ERK and P-Elk-1 immunohistochemistry
On day 1, free-floating sections were rinsed in Tris-buffered saline (TBS; 0.25 M Tris and 0.5 M NaCl, pH 7.5), incubated for 5 min in TBS containing 3% H2O2 and 10% methanol, and then rinsed three times for 10 min each in TBS. After 15 min of incubation in 0.2% Triton X-100 in TBS, the sections were rinsed three times in TBS. They were then incubated with the primary antibody overnight (polyclonal phospho-Thr 202/Tyr204-ERK; Cell Signaling, Beverly, MA, USA 1:400; monoclonal phospho-Ser-383-Elk-1 1:250; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. On day 2, the sections were incubated for 2 h at room temperature with the secondary biotinylated antibody (Vector Laboratories, Peterborough, UK), using a dilution twice that of the first antibody in TBS. After washes, the sections were incubated for 90 min in avidin-biotin-peroxidase complex (ABC) solution (final dilution, 1:50; Vector Laboratories). The sections were then washed in TBS and twice in TB (0.25 M Tris, pH 7.5) for 10 min each, placed in a solution of TB containing 0.1% 3,3'-diaminobenzidine (DAB; 50 mg/100 ml), and developed by adding H2O2 (0.02%). After being processed, the tissue sections were mounted on gelatin-coated slides and dehydrated through alcohol to xylene for light microscopy.

P-MSK, MSK, P-CREB, and P-H3 immunohistochemistry
On day 1, free-floating sections were rinsed in TBS (0.25 M Tris and 0.5 M NaCl, pH 7.5) and incubated for 15 min with 0.2% Triton X-100 in TBS. After three rinses, floating sections were saturated for 1 h with 3% BSA, 0.2% Triton in TBS. After three rinses, floating sections were saturated for 1 h at room temperature (for p-CREB, P-MSK, and MSK immunochemistry only) with 3% BSA. Then, the sections were rinsed three times in TBS and incubated with the primary antibody (phospho-Thr 581 MSK 1:750, Cell Signaling; MSK, 1:1500, Sigma-Aldrich, St. Louis, MO, USA; P-CREB 1:750, Upstate Biotechnology, New York, NY, USA; phospho-Ser-10-histone H3 1:500, Upstate Biotechnology; overnight at 4°C in TBS. On day 2, after three rinses in TBS, sections were incubated for 2 h at room temperature with a secondary Cy3-conjugated antibody 1:500 (Amersham Biosciences, Piscataway, NJ, USA), except for P-CREB immunohistochemistry, for which sections were incubated for 2 h with the secondary biotinylated antibody (anti-rabbit 1/200, Vector Laboratories) and then, after three rinses, with Cy3-conjugated streptavidin (1:300, Sigma-Aldrich). After three rinses in TBS, tissue sections were mounted under cover slips using Vectashield (Vector Laboratories) for fluorescence microscopy. For P-CREB immunohistochemistry, PBS with 0.2% Triton X-100 and 1% BSA was used instead of TBS for the entire processing.

Mounted tissue sections were observed under a Leica DM LB microscope. Immunoreactivity was quantified with IMAGE PRO PLUS 4.5.0.19 image analysis software (Media Cybernetics, Silver Spring, MD, USA). A basal threshold was established and then applied to immunoreactivity in HD and control brain sections. Neurons with immunoreactivity above this threshold were counted as immunoreactive cells. For P-CREB immunolabeling, global immunofluorescent staining was also measured using Image J software. Data are expressed as mean ± SE. Statistical analysis was based on Student’s t test implemented with Excel software. Differences were considered significant if P < 0.05.

Western blot analysis
Mouse striata were dissected and homogenized in lysis buffer containing 50 mM Tris-HCl pH 8.0, 10% glycerol, 5 mM EDTA, 150 mM KCl, 1 mM Pefabloc, a cocktail of protease inhibitors, and 1% Triton X-100. They were incubated for 15 min on ice and centrifuged at 13,000 rpm for 20 min at 4°C. Human brain samples were homogenized in Nonidet P-40 lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EGTA, 1% Nonidet P-40, 10 mM β-glycerophosphate, 5 mM NaF, 1 mM NaPPi, 2 mM DTT, 1 mM sodium vanadate, and 100 µM PMSF) and cleared by centrifugation at 6000 g (15 min; 4°C). Supernatants were collected and analyzed by SDS-PAGE. Primary antibodies rabbit polyclonal anti-MSK-1 1:1000 (Cell Signaling), rabbit polyclonal anti-ERK1/2 K-23 1:1000 (Santa Cruz Biotechnology), and mouse monoclonal anti-β-tubulin 1:5000 (Sigma-Aldrich) were used and revealed with appropriate anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Jackson Laboratories) and the ECL chemiluminescent reaction (Pierce).

Real-time reverse transcription-PCR
Reverse transcription (RT) was performed on 1 µg of total striatal RNA by using SuperScriptII (Invitrogen, Carlsbad, CA, USA) and random hexamers according to the manufacturer’s instructions. PCR amplification of cDNA was run on a Light-Cycler instrument. PCR primers used for the detection of MSK-1, the housekeeping gene 36B4, and c-fos were MSK-1-forward: 5'-TGCTGAAGGTCCTAGGAACT-3' and MSK-1-reverse: 5'-GCATACAGCTTTCCAGCATC-3'; 36B4-forward: 5'-GAGGTCACTGTGCCAGCTCA and 36B4-reverse: 5'-GAAGGTGTACTCAGTCTCCA-3'; and cFos-forward: 5'-CAACGCCGACTACGAGGCGTCAT-3' cFos-reverse: 5'-GTGGAGATGGCTGTCACC G-3'.

Neuronal cell culture and treatments: in vitro analysis
Striata were dissected out from 14-day-old Swiss mouse embryos (Janvier, France) and c57 black6 MSK-1 knockout or MSK-1 wild-type mouse embryos, as described previously (28) . Briefly, after dissection and dissociation of striatal neurons, cell pellets were suspended in Neurobasal media (B27 supplement, Life Technologies, Gaithersburg, MD, USA; 500 nM L-glutamine; 60 µg/ml pennicilin G; and 25 µM β-mercapto-ethanol) and then plated in 24-well or 6-well (Nunc multiwell) plates or 8-well Lab-Tek II-Chamber Slides (Nunc, Roskilde, Denmark). Transient transfection of striatal cells were performed with Lipofectamine 2000 (Invitrogen) using Htt or Exp-Htt constructs (provided by the Huntington’s Disease Foundation Resource Bank, University of California, Los Angeles, CA, USA) as described previously (29) . These constructs correspond to pcDNA3 containing CMV promoter controlling the expression of the entire exon 1 of the human huntingtin gene (IT15), with 25 (25Q-Htt) or 103 (103Q-Htt) continuous CAA or CAG repeats. A sequence encoding enhanced green fluorescent protein (EGFP) was inserted in frame at the C terminus of each construct. The GFP-MSK-1 construct (36) was a generous gift from Rachel Toth (MRC Phosphorylation Unit, Dundee, UK). Three and a half hours after transfection, the medium was removed and replaced by the complete neurobasal medium. After 16 or 24 h, cells were treated with glutamate at 100 µM and then fixed in 24-well plates or 8-well Lab-Tek II-Chamber Slides with PBS containing 4% paraformaldehyde for 40 min at room temperature and then incubated with methanol/acetone (50/50) for 10 min at 4°C. Then, after three washes with PBS, plates were treated with blocking buffer (normal goat serum 10% in PBS). Rabbit antibodies raised against MSK-1 1:500 (Sigma-Aldrich) phospho Ser-10-histone H3 1:1000 (Upstate Biotechnologies), c-Fos 1:7000 (Santa Cruz), or mouse antibodies raised against MAP2 1:1000 (Sigma-Aldrich) were incubated overnight at 4°C in PBS. Plates were rinsed in PBS and incubated with appropriate secondary antibodies for 2 h at room temperature. For each experiment, cells were analyzed under a fluorescence microscope (Leica) directly in the wells. Neuronal retraction was evaluated by manually tracing the neurites using the software ImageJ (version 1.34s; National Institutes of Health, Bethesda, MD, USA). The percentage of surviving transfected neurons was analyzed 16 and 24 h after transfection. Transfected striatal neurons containing condensed or fragmented nuclei (DNA labeling with Hoechst) were scored as dying cells. The percentage of MSK-1, Phospho-H3, or c-Fos immunoreactive transfected neurons was analyzed 16 and 24 h after transfection. The percentage of transfected neurons containing Exp-Htt aggregates was also analyzed 16 and 24 h after transfection with the GFP tag. For each condition, a minimum of 100 transfected neurons per experiment were counted (4 independent experiments). Data are expressed as mean ± SE. Statistical analysis was based on Student’s t test implemented with Excel software. Differences were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
c-fos transcription is down-regulated in the striatum of R6/2 mice despite a strong activation of ERK along with Elk-1 and CREB
We first confirmed previous results (7 , 8) , showing that basal mRNA levels of c-fos are down-regulated in striatal extracts of R6/2 mice at 12 wk (Fig. 1 A). Since the MAP kinase/ERK signaling pathway has been shown to play a key role in this transcriptional regulation in the striatum (27 , 30 , 31) , we analyzed ERK activation in R6/2 mice. We used immunocytochemical detection of an anti-active antibody that specifically recognizes its dually phosphorylated form (32 ; Fig. 1B ). Very few immunoreactive cells were present in wild-type mice and at early stages in R6/2 mice (Fig. 1B, D ). At 12 wk, P-ERK-immunoreactive cells significantly increased in the dorsal part of the striatum of R6/2 mice, specifically (Fig. 1B, D ). At this stage, combined P-ERK immunofluorescence and nuclear staining with Hoechst dye showed a nuclear labeling of P-ERK (Fig. 1C ). Furthermore, total ERK protein levels remained unchanged in R6/2 mice when compared to their wild-type littermates (data not shown). Elk-1 is a transcription factor directly activated by ERK in response to glutamate in striatal sections and on electrical cortico-striatal stimulation in vivo (31 , 33) . On its phosphorylation by activated ERK, Elk-1 becomes a transactivator of genes carrying a serum response element (SRE) in their promoter, including c-fos (34) . The activation of Elk-1 was detected in sections adjacent to those used for ERK analysis at 9 and 12 wk by immunohistochemical detection with a phosphor-Ser-383-Elk-1 (P-Elk-1)-specific antibody (31 , 33) . As observed with P-ERK, the number of P-Elk-1-positive cells significantly increased at 12 wk in the dorsal striatum of R6/2 mice (Fig. 2 A). CREB is also critically involved in c-fos transcription by transactivating the cAMP/calcium-responsive element (CRE) in its promoter. CREB phosphorylation was measured at 12 wk (the time of peak ERK and Elk-1 activation) in the striatum of R6/2 mice by immunofluorescence detection with a phospho-Ser-133 CREB (P-CREB)-specific antibody (31) (Fig. 2B ). P-CREB immunoreactivity was moderate in striatal and cortical neurons of wild-type mice and significantly increased in both the dorsal and ventral parts of the striatum of R6/2 mice at 12 wk (Fig. 2C, D ).


Figure 1
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  		Figure 1. Down-regulation of c-fos correlates with an hyperactivation of ERK in the striatum R6/2 mice. A) Real-time RT-PCR analysis of total RNA from 12-wk-old R6/2 and wild-type (Wt) mouse striata, using primers specific for c-fos. Primers amplifying 36B4 were used as internal controls. R6/2 mRNA levels are quantified as a percentage of Wt mRNA, after normalization to the internal control level. Each bar represents the mean value ± SE (R6/2: n=4; Wt: n=4). B) Immunohistochemical detection of phospho-Thr 202/Tyr204-ERK (P-ERK)-positive cells from Wt and R6/2 mice. Shown are the dorsal striatum (DSt) and parietal cortex (PCx) of R6/2 mice at 9 and 12 wk of age (R6/2–9W and R6/2–12W) and their Wt littermates. For Wt mice, brain sections at the same age as R6/2 mice were compared. C) Immunofluorescent detection of P-ERK in the DSt of R6/2 mice at 12 wk (R6/2–12W) using an anti-rabbit Cy3-coupled secondary antibody (red, middle). Right: fusion of P-ERK immunoreactivity and nuclear labeling (Hoechst staining shown at left). Note the nuclear localization of P-ERK in most cells (white arrows). D) P-ERK-positive neurons were counted in the dorsal and ventral (VSt) parts of the striatum and the parietal cortex at 9 and 12 wk (9w and 12w). Statistical comparisons for a given region at a given age between Wt and R6/2 mice were based on Student’s unpaired t test; *P < 0.05 (n=4 to 6 independent mice for each strain at each stage). Note the increase in P-ERK-immunoreactive cells in the dorsal striatum of R6/2 mice at 12 wk.


Figure 2
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  		Figure 2. Elk-1 and CREB hyperphosphorylation in the striatum of R6/2 mice. A) P-Elk-1-positive cells were counted from Wt and R6/2 mice at 9 and 12 wk of age. B) Immunohistochemical detection of P-CREB-positive cells from Wt and R6/2 mice at 12 wk of age. Shown are the dorsal striatum and parietal cortex (PCx) of R6/2 mice at 12 wk of age (R6/2–12W) and their Wt littermates at the same age. C) P-CREB-positive cells were counted from Wt and R6/2 mice at 12 wk of age. D) Measurements of global immunofluorescent staining were performed using Image J software. Quantification and statistical comparisons were performed as indicated in Fig. 1D (A, C, D ); *P < 0.05; **P < 0.01; ***P < 0.005 (6 mice of each strain).

Lack of histone H3 phosphorylation in the striatum of R6/2 mice is correlated with down-regulation of MSK-1
Having shown that a strong activation of the signaling pathway involved in c-fos regulation was associated with c-fos down-regulation in the striatum of R6/2 mice at 12 wk, we hypothesized that a deficiency in histone H3 phosphorylation could account for this down-regulation. To analyze this, we used a phospho-Ser-10-H3-specific antibody (P-H3), which showed increased levels of immunoreactivity in response to glutamate in vitro (35) . Thus, in primary cultures of striatal neurons, both immunocytochemical (Fig. 3 A) and biochemical detection of P-H3 levels (Fig. 3B ) showed a significant increase on glutamate stimulation. Using this antibody, we failed to detect any phosphorylation of H3 in striatal sections of R6/2 mice (Fig. 3C , middle). As a positive control, we found an increase in P-H3 levels in the striatum of cocaine-treated wild-type mice (15 ; Fig. 3C , right).


Figure 3
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  		Figure 3. Lack of histone H3 phosphorylation in the striatum of R6/2 mice. A) Immunocytochemical detection of phospho-Ser-10-H3 (P-H3) in primary cultures or striatal neurons treated (Glu 30') or not (Cont) with glutamate during 30 min. Right: Hoechst staining corresponding to P-H3 immunocytochemical detection at Glu 30'. Note the high percentage of P-H3 immunopositive cells. B) Western blot detection of P-H3 (top) along with total histone H3 (bottom) after chromatin extraction of striatal neurons treated or not with glutamate (Glu 30' and Cont, respectively). C) Immunohistochemical detection of phospho-Ser-10-H3 (P-H3) in the Dst of Wt and R6/2 mice at 12 wk of age (R6/2–12W) (data are representative of 6 independent mice for each condition). The same experimental protocol was used for detection of P-H3 in the striatum of Wt mice treated with cocaine for 30 min (Wt-Coc) as a positive control.

MSK-1 is an important intracellular signaling component downstream of ERK (36) . Its kinase activity is critically involved in histone Ser-10-H3 phosphorylation and chromatin remodeling in both nonneuronal and neuronal cells (15 , 26 , 37 38 39 40) . We therefore investigated MSK-1 activation in R6/2 mice compared to their wild-type littermates. The Thr-581 residue of MSK-1 is phosphorylated downstream of ERK in response to mitogens and neurotransmitters (15 , 41 , 42) and is crucial for MSK-1 kinase activity on various substrates, including histone H3 Ser-10 (43) . An affinity-purified antiserum that specifically recognizes this phospho-Thr-581 residue (P-MSK-1; refs. 15 , 35 ) was used to analyze MSK-1 activation. Immunohistochemical detection of P-MSK-1 showed a significant basal level in wild-type mice at 12 wk and notably in the dorsal and ventral striatum (Fig. 4 A, B). By contrast, very few P-MSK-1-immunoreactive cells were found in the striatum of R6/2 mice at this stage (Fig. 4A, B ). We then postulated that down-regulation of total MSK-1 expression could account for the lack of MSK-1 activation in R6/2 mice. Immunocytochemical detection of total MSK-1 in wild-type and R6/2 mice at 12 wk showed strong expression of total MSK-1 in the striatum of wild-type mice (Fig. 4C, D ). Total MSK-1 immunoreactivity was markedly lower in R6/2 mice (Fig. 4C, D ), and this down-regulation was confirmed by Western blotting of striatal extracts (Fig. 4E, F ). RT-PCR analysis showed significantly lower levels of MSK-1 mRNA in striatal extracts from R6/2 mice compared to their wild-type littermates (Fig. 4F ).


Figure 4
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  		Figure 4. R6/2 mice show deficient MSK-1 activation and expression at 12 wk of age. A) Immunohistochemical detection of P-MSK-1-positive cells was performed from sections of Wt and R6/2 mice at 12 wk of age. Shown are the DSt and PCx. B) Quantification of P-MSK-1-positive cells. C) Immunohistochemical detection of total MSK-1-positive cells. D) Quantification of MSK-1-positive cells. Statistical analysis was performed as described in Fig. 1D(B, D ). Note the strong decrease in both P-MSK-1- and MSK-1-immunoreactive cells in the striatum of R6/2 mice when compared to Wt littermates. E) Striatal extracts from Wt and R6/2 mice at 12 wk of age (3 mice of each strain) were processed for Western blotting with antibodies directed against MSK-1 and tubulin. The bands corresponding to each protein are shown in the same blot. Note the decreased MSK-1 protein signal in striatal extracts from R6/2 mice when compared to Wt, contrasting with the similar tubulin expression. F) Left: Relative MSK-1 protein levels were quantified on Western blots in A by measuring the ratio to tubulin. Right: Real-time RT-PCR analysis of total RNA extracted from 12-wk-old R6/2 (R6/2) and Wt mouse striata, using primers specific for MSK-1. Primers amplifying 36B4 were used as internal controls. mRNA levels in R6/2 striata are expressed as a percentage of levels in the Wt mouse striata, after normalization to the internal control level. Each bar represents the mean value ± SE (R6/2: n=4; Wt: n=4). F, G) Statistical analyses; Student’s unpaired t test; *P < 0.05 for striatal extracts from R6/2 vs. Wt mice.

MSK-1 is down-regulated in human caudate nucleus from HD
MSK-1 deficiency in the striatum of R6/2 mice could be relevant of a process involved in HD pathophysiology. It was thus important to analyze the level of MSK-1 expression in postmortem brain samples from HD patients (Fig. 5 A). Using anti-MSK-1 immunoblotting, we analyzed the cerebral cortex and caudate nucleus. Anti-tubulin antibody was used as a control for protein levels, and a MSK-1/tubulin ratio was measured for quantification (Fig. 5B ). MSK-1 was expressed in both cortex and caudate nucleus in control samples. Interestingly, in caudate nucleus, but not cerebral cortex samples, we found a significant decrease of MSK-1 expression in HD patients when compared to control. Although the postmortem samples represent late stages of the disease, no profound modifications in the levels of calbindin, a specific marker of medium spiny neurons, were found in these extracts (44) . This suggests that the decreased levels of MSK-1 in postmortem samples are not merely a reflection of cell death.


Figure 5
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  		Figure 5. MSK-1 is down-regulated in HD postmortem caudate nucleus extracts. A) Cortical and caudate nucleus extracts from control and HD patients were processed for Western blotting with antibodies directed against MSK-1 (top) and β-tubulin (β-tub) (bottom). A band corresponding to 90 kDa was found at the expected molecular weight for human MSK-1. B) Relative MSK-1 protein levels were quantified on Western blotting in A by measuring the ratio to β-tubulin. Statistical analysis was based on Student’s t test. *P < 0.05 when comparing MSK-1 protein signal in HD caudate nucleus extracts with control caudate nucleus extracts.

Overexpression of MSK-1 restores P-H3 and c-fos down-regulation induced by Exp-Htt
To further investigate the role of MSK-1 in c-fos regulation in HD, we used an in vitro model system, previously set up in our laboratory (29) . Primary cultures of striatal neurons were transiently transfected with GFP-tagged constructs, expressing the exon 1 of the human huntingtin containing either normal (25Q-Htt) or expanded (103Q-Htt) huntingtin, as described previously (29 , 45 ; Fig. 6 A). MSK-1 expression showed similar levels in nontransfected and 25Q-Htt-transfected neurons at 16 and 24 h posttransfection (data not shown). When compared to 25Q-Htt overexpression, 103Q-Htt-transfected neurons showed significant decrease in MSK-1 expression (Fig. 6A ). Thus, at 24 h posttransfection, only 20% of 103Q-Htt-transfected neurons (vs. 30% for 25Q-Htt-transfected neurons) showed significant levels of MSK-1 expression (Fig. 6B ).


Figure 6
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  		Figure 6. Exp-Htt induces MSK-1 deficiency in vitro along with c-Fos and P-H3 down-regulation: restoration by MSK-1 overexpression. A) Immunocytochemical detection of MSK-1 protein (red) was performed on primary cultures of striatal neurons transfected with either normal Htt (25Q-Htt) or expanded Htt (103Q-Htt; detected by GFP). The nuclei were labeled using Hoechst staining (blue). B) Quantification of MSK-1-immunoreactive neurons among 25Q-Htt- and 103Q-Htt-expressing neurons 16 and 24 h after transfection. C) Top: c-Fos immunolabeling (red) in glutamate-treated striatal neurons. Note the lack of c-Fos staining in 103Q-Htt-expressing neurons (GFP staining). Bottom: 103Q-Htt was coexpressed with MSK-1 (GFP staining). Note the rescue of glutamate-induced c-Fos expression. D) Quantification of c-Fos positive 25Q-Htt- or 103Q-Htt-transfected neurons. Glutamate was applied during 120 min (glu 120'). When indicated, MSK-1 was coexpressed with either 25Q (white bars) or 103Q-Htt (black bars). Note the rescue of c-Fos expression induced by MSK-1 overexpression in the presence or not of glutamate. E) Quantification of P-H3 positive 25Q-Htt- or 103Q-Htt-transfected neurons. Glutamate was applied during 30 min (glu 30'). When indicated MSK-1 was coexpressed with either 25Q (white bars) or 103Q-Htt (black bars). Note the rescue of P-H3 expression induced by MSK-1 overexpression in the presence or not of glutamate. D, E) Data were analyzed from 3 independent experiments (100 transfected neurons per experiment) and expressed as mean ± SE. Statistical analyses were based on Student’s t test: ***P < 0.001, 25Q-Htt-expressing neurons vs. 103Q-Htt-expressing neurons; ###P < 0.001, neurons cotransfected with 103Q-Htt and MSK-1 vs. neurons transfected with 103Q-Htt alone.

We then wished to analyze whether our in vitro model system of HD could reproduce the down-regulation of c-Fos induction observed in vivo in the striatum of R6/2 mice (see Fig. 1A ). c-Fos expression was analyzed on glutamate treatment, 24 h after transfection of 25Q- or 1O3Q-Htt (Fig. 6C ), a time point when MSK-1 was significantly reduced by 103Q-Htt. Two hours of glutamate treatment induced c-Fos expression in ~45% of 25Q-Htt-transfected neurons (Fig. 6D ), which represents the same percentage as nontransfected neurons (data not shown; see ref. 35 ). Overexpression of 103Q-Htt significantly reduced glutamate-induced c-Fos expression (Fig. 6C ), and the percentage of transfected neurons that expressed significant levels of c-Fos was twice as low (Fig. 6D ). We then overexpressed MSK-1 in these cells. We observed a total rescue of glutamate-induced c-Fos expression in 103Q-Htt-transfected neurons (Fig. 6C, D ), with no variation of expression in 25Q-Htt-transfected neurons (Fig. 6D ). The overexpression of MSK-1 induced c-Fos expression in nontransfected (data not shown) as well as transfected neurons in the absence of glutamate treatment (Fig. 6D ).

We then analyzed levels of P-H3 immunoreactivity in neurons 24 h after transfection with either 25Q-Htt or 103Q-Htt (Fig. 6E ). In the absence of treatment, we could not detect any P-H3 immunoreactive neurons. On 30 min of glutamate treatment, we found a strong increase of P-H3 immunoreactivity in 25Q-Htt-transfected neurons that reached similar values to nontransfected neurons (data not shown; see ref. 35 ). The percentage of P-H3 positive neurons was twice as low in 103Q-Htt-expressing neurons (Fig. 6E ). We then over-expressed MSK-1, along with 25Q-Htt or 103Q-Htt, and found a total rescue of glutamate-induced P-H3 immunoreactivity in 103Q-Htt-transfected neurons. This induction was similar in transfected neurons in the absence of glutamate treatment (Fig. 6E ). Thus, overexpression of MSK-1 is able to induce phosphorylation of H3 in the presence or not of glutamate treatment.

Role of MSK-1 in expanded-Htt-induced striatal death in vitro
We reasoned that, by altering expression of c-Fos, MSK-1 deficiency could account for a greater susceptibility to striatal death induced by expanded-Htt. Thus, striatal neurons from MSK-1 knockout mice, together with neurons from their wild-type littermates, were transiently transfected with either 25Q-Htt or 103Q-Htt cDNAs. Cell viability was analyzed using Hoechst staining. In striatal neurons from wild-type mice, 103Q-Htt induced a significant neuronal death 16 and 24 h after transfection (Fig. 7 A) as previously demonstrated in our model system (29 , 45) . The death induced by expanded-Htt was more pronounced in striatal neurons from MSK-1 knockout mice at 16 h posttransfection (Fig. 7A ). At 24 h posttransfection, no significant difference was found between wild-type and MSK-1 knockout mice. MSK-1 deficiency failed to affect 103Q-Htt-induced aggregate formation, which remained elevated at any stage (Fig. 7B ).


Figure 7
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  		Figure 7. MSK-1 deficiency aggravates striatal death induced by Exp-Htt. A) Primary culture of MSK-1+/+ or MSK-1–/– striatal neurons were transfected with either 25Q-Htt (white bars) or 103Q-Htt (black bars). The percentage of surviving neurons was determined 16 and 24 h after transfection by morphological criteria based on DNA labeling with Hoechst. B) Percentage of 103Q-Htt-transfected neurons with aggregates was determined 16 and 24 h after transfection. Data were analyzed from 3 independent experiments (100 transfected neurons per experiment) and expressed as mean ± SE. Statistical analysis was based on Student’s t test. **P < 0.01, 25Q-Htt-expressing neurons vs. 103Q-Htt-expressing neurons; ##P < 0.01, 103Q-Htt-expressing neurons from MSK-1+/+ neurons vs. 103Q-Htt-expressing neurons from MSK-1–/– neurons.

We then investigated whether MSK-1 overexpression might be neuroprotective in our in vitro model system. A GFP-tagged version of MSK-1 was transfected in striatal neurons along with 25Q-Htt or 103Q-Htt constructs (Fig. 8 A). Overexpression of MSK-1 totally protected striatal neurons against 103Q-Htt-induced death 16 h after transfection (Fig. 8A, B ). Thus, surviving neurons represented 92 vs. 62% when comparing neurons expressing 103Q-Htt alone and neurons cotransfected with MSK-1. At 24 h, when only 50% of 103Q-Htt-expressing neurons were surviving, MSK-1 overexpression still produced a strong protection with 83% survival (Fig. 8B ). At 24 h, cotransfection of MSK-1 and 103Q-Htt led to a marked decrease of 103Q-Htt-induced neuritic retraction (Fig. 8C ) and aggregate formation (Fig. 8D ).


Figure 8
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  		Figure 8. MSK-1 overexpression rescues expanded-Htt-induced striatal death in vitro. A) Primary cultures of striatal neurons were transfected with MSK-1 or cotransfected with either 25Q-Htt or 103Q-Htt together with MSK-1. Transfected neurons were revealed with GFP staining. Immunocytochemical detection of MAP2 (red) and Hoechst (blue) labeling were performed to analyze the neuronal integrity. Examples of neurons transfected with 103Q-Htt alone (top) or together with MSK-1 (bottom). Note that MSK-1 overexpression rescues from 103Q-Htt-induced DNA damage, neuritic retraction, and aggregate formation. B) The percentage of transfected surviving neurons was determined 16 and 24 h after transfection by morphological criteria based on DNA labeling with Hoechst. C) The percentage of mean neuritic length in 25Q-Htt and 103Q-Htt-transfected neurons was measured at 24 h. D) The percentage of 103Q-Htt-transfected neurons with aggregates was determined at 24 h. White bars = 25Q-Htt- or 103Q-Htt-transfected neurons; black bars = coexpression of MSK-1 and either 25Q-Htt or 103Q-Htt. Data were analyzed from 3 independent experiments (100 transfected neurons per experiment) and expressed as mean ± SE. Statistical analysis was based on Student’s t test. **P < 0.01, ***P < 0.001, 25Q-Htt-expressing neurons vs. 103Q-Htt-expressing neurons; #P < 0.05, ##P < 0.01, 103Q-Htt-expressing neurons vs. 103Q-Htt- + MSK-expressing neurons.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We identified a new molecular event involved in transcriptional dysregulation and striatal death observed in HD. We found a deficit of MSK-1 expression in the striatum of R6/2 mice and postmortem HD patients. In R6/2 mice, this deficit may account for the absence of phosphorylation of histone H3 and c-fos transcription despite a strong nuclear activation of ERK, along with Elk-1 and CREB phosphorylation. We further demonstrate that genetic suppression of MSK-1 increased Exp-Htt-induced striatal death. Conversely, MSK-1 overexpression protected striatal neurons from Exp-Htt-induced death.

Activation of ERK in the striatum of R6/2 mice was previously reported (46) . We confirmed this finding and further showed that this activation was nuclear and did not result from increased expression of ERKs. The role of ERK activation in the neurodegenerative processes remains controversial. It may be associated with either an increased sensitivity to neurodegeneration or a survival response of the cells. In HD, inhibition of the ERK pathway has been found to increase Exp-Htt-induced neuronal death in vitro (47) . Within the nucleus, ERK plays a pleiotropic role in gene transcription, acting through at least two transcription factors, Elk-1 and CREB. ERK-induced activation of Elk-1 and CREB correlates with c-fos induction in striatal neurons (27 , 30 , 31) . These two transcription factors act synergistically on the SRE and CRE sites of the c-fos promoter to induce its full transcriptional activation (48) . Despite the hyperphosphorylation of Elk-1 and CREB (49) observed in the striatum of R6/2 mice, we detected, as previously reported (7 , 8) , a significant decrease of c-Fos expression. Thus, the activation of these transcription factors, although necessary, does not appear to be sufficient to mediate transcription. We thus hypothesized that altered chromatin remodeling could impair the transactivating role of these transcription factors at the c-fos promoter.

The crucial and exclusive role of histone H3 phosphorylation for the nucleosomal response at the c-fos promoter was previously demonstrated in neurons (10) . We recently showed that, within the striatum, this phosphorylation occurs through the activation of the ERK pathway and its downstream kinase MSK-1 (15 , 35) . We thus asked whether uncoupling between ERK signaling and chromatin remodeling at the c-fos promoter may be due to MSK-1 deficiency in HD. We found a decreased expression of MSK-1 in R6/2 mice and postmortem brain of HD patients in the striatum specifically. In the adult mouse brain, MSK-1 is expressed in ~60% of striatal neurons, strongly suggesting that these are projection neurons (50) , which make up the majority of striatal neurons. This specific expression pattern could be related to the vulnerability of these neurons in HD. We have previously demonstrated that genetic suppression of MSK-1 accounts for alteration of both histone H3 phosphorylation and c-Fos induction in response to glutamate in striatal neurons, in vitro (35) . Here we show that Exp-Htt produces similar alterations by decreasing MSK-1 expression, since overexpression of MSK-1 restored glutamate-induced H3 phosphorylation and c-fos induction in Exp-Htt-expressing neurons. Together, these findings support that MSK-1 deficiency causes an alteration of histone H3 phosphorylation and a dysregulation of c-fos transcription in HD.

Several recent articles emphasized the role of histone H3 phosphorylation in gene regulation related to neuronal plasticity. This phosphorylation event was found to be critical for plasticity associated with behavioral responses to cocaine in the striatum (15) , experience-dependant plasticity of the visual cortex (16) , and seizure-induced hippocampal plasticity (10 , 12 13 14) . These results suggested that histone H3 phosphorylation can modulate the transcription in neurons and underlie various cerebral functions. Conversely, we propose that alteration of histone H3 phosphorylation may result in cerebral dysfunctions, as observed in neurodegenerative diseases. In our in vitro model of HD, decreased c-Fos expression paralleled the alteration of histone H3 phosphorylation and was restored by MSK-1 overexpression. Since c-Fos plays a critical role for stress response and survival in neurons (51) , we hypothesized that MSK-1 deficiency could be involved in Exp-Htt-induced striatal death. In keeping with this hypothesis, Exp-Htt-induced striatal death was increased in striatal neurons of MSK-1 knockout mice, at least at early stages. At later stages, i.e., 24 h after the transfection, the lack of effect of MSK-1 deficiency may be explained by the down-regulation produced by Exp-Htt on endogenous MSK-1. Conversely, overexpression of MSK-1 protected striatal neurons from Exp-Htt-induced death. MSK-1 is likely to exert its neuroprotective effects not only through the restoration of c-Fos expression but also by activating histone H3 phosphorylation, and hence chromatin remodeling, required for the transcriptional regulation of other important genes for survival. Alternatively, MSK-1 is one of the CREB kinases at Ser-133 (15 , 52) and may thus induce activation of neuroprotective genes, whose transcription depends on CREB activation. However, because CREB phosphorylation remained high in the striatum of R6/2 mice, despite MSK-1 deficiency, one may hypothesize that other CREB kinases were responsible for this phosphorylation event in this HD model.

It has been previously shown that histone decacetylase (HDAC) inhibitors increase histone acetylation levels and improve the phenotype in various mouse models of HD (20 21 22) . The basal global levels of histone acetylation in these mice is controversial, as they were found to be either unchanged (20 , 25) or decreased (21 , 22) . A recent study (25) in R6/2 mice has shown that hypoacetylation of histone H3 occurred specifically at the promoter of genes known to be down-regulated in HD, despite a lack of global histone H3 hypoacetylation. Moreover, treatment with the HDAC inhibitor phenylbutyrate partly restored histone H3 acetylation at the promoter of these genes along with their transcription (25) . Alteration of histone methylation can be also important in the transcriptional dysregulation observed in HD (22 23 24) . We show here that altered phosphorylation of histone H3, resulting from MSK-1 deficiency, is involved in the down-regulation of c-Fos induced by Exp-Htt. We thus propose that histone modifications that are critical for the nucleosomal response can be specific to the promoter of the genes. Although acetylation of H3 and/or H4 can be critical for some HD dysregulated genes, H3 phosphorylation could be involved at the promoter of other genes. Alternatively, a combination of altered histone acetylation and phosphorylation at the promoter of some genes may account for impaired DNA decompaction and transcriptional dysregulation of these genes.

Our data strongly support that restoring MSK-1 expression or its related phosphorylation of histone H3 may represent an interesting therapeutic target for HD treatment. Drug association that allows the simultaneous targeting of various histone modifications may thus be promising.


   ACKNOWLEDGMENTS
 
This work was supported by Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie-Paris 6, and the Hereditary Disease Foundation (HDF) for J.C. We thank Dr. Simon Arthur for the generous gift of MSK-1 knockout mice and Rachel Toth for the MSK-1 construct. K.B.-C. was supported by Association pour la Recherche sur le Cancer. C.D. was a grant recipient from the High Q foundation, E.R. was the recipient of a grant "poste d’acceuil" from AP-HP and CNRS, and K.M. was supported by CNRS and Fondation pour la Recherche sur le Cerveau. We thank Frederic Saudou for critical reading of the manuscript.

Received for publication September 10, 2007. Accepted for publication October 25, 2007.


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B. Drobic, B. Perez-Cadahia, J. Yu, S. K.-P. Kung, and J. R. Davie
Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex
Nucleic Acids Res., February 3, 2010; (2010): gkq030v1 - gkq030.
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