Morphological and functional alterations in the substantia nigra pars compacta of the Mecp2-null mouse

Neurobiology of Disease 41 (2011) 385–397

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Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i

Morphological and functional alterations in the substantia nigra pars compacta of the Mecp2-null mouse Nicolas Panayotis ⁎, Michel Pratte, Ana Borges-Correia, Adeline Ghata, Laurent Villard, Jean-Christophe Roux INSERM, UMR_S 910, Faculté de Médecine de La Timone, 27 Boulevard Jean Moulin, 13385 Marseille, France Aix-Marseille Université, Faculté de Médecine de La Timone, 27 Boulevard Jean Moulin, 13385 Marseille, France

a r t i c l e

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Article history: Received 26 August 2010 Revised 4 October 2010 Accepted 7 October 2010 Available online 14 October 2010 Keywords: Rett syndrome A9 Caudate–putamen Mecp2 Tyrosine hydroxylase Dopamine

a b s t r a c t Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the MECP2 gene, in which older patients often develop parkinsonian features. Although Mecp2 has been shown to modulate the catecholaminergic metabolism of the RTT mouse model, little is known about the central dopaminergic neurons. Here we found that the progression of the motor dysfunction in the Mecp2-deficient mouse becomes more severe between 4 and 9 weeks of age. We then studied the phenotype of the dopaminergic neurons of the substantia nigra pars compacta (SNpc). We found a major reduction in the number of tyrosine hydroxylase (Th)-expressing neurons, as well as a reduction in their soma size, by 5 weeks of age. We showed that this deficit is not due to apoptosis and that the remaining neurons express a mature dopaminergic phenotype. A reduction in the Th-staining intensity was also found in the caudate–putamen (CPu), the main dopaminergic target for SNpc. We found that the amount of activated-Th (pSer40-Th) is slightly reduced at 5 weeks of age in the Mecp2-deficient mouse, but that this amount is affected more importantly by 9 weeks of age. Neurochemical measurements revealed a significant reduction of dopamine content at 5 and 9 weeks of age in the CPu whereas SNpc contents were preserved. Finally, we found that chronic L-Dopa treatment improved the motor deficits previously identified. Altogether, our findings demonstrate that Mecp2deficiency induces nigrostriatal deficits, and they offer a new perspective to better understand the origin of motor dysfunction in RTT. © 2010 Elsevier Inc. All rights reserved.

Introduction Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene (Amir et al., 1999). Female patients are affected with an incidence of 1/ 15,000 live births (Laurvick et al., 2006, Chahrour and Zoghbi, 2007) and develop normally from birth to 6–18 months of age before the onset of deficits in autonomic functions, cognition, motor functions (stereotypic hand movements and impaired locomotion) and autistic features Abbreviations: RTT, Rett syndrome; Mecp2, methyl-CpG binding protein 2; SNpc, substantia nigra pars compacta; Th, tyrosine hydroxylase; pSer40-Th, serine40phosphorylated tyrosine hydroxylase; mDA, midbrain dopaminergic; Pitx3, pairedlike homeobox gene 3; Vmat2, vesicular monoamine transporter 2; Aadc, aromatic amino acid decarboxylase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Nurr1, nuclear receptor subfamily 4, group A, member 2; En1, engrailed gene 1; CaMKII, calmodulin kinase II; PKA, protein kinase A; Bdnf, brain-derived neurotrophic factor; ANOVA, analysis of variance; S.E.M., standard error of the mean; P, postnatal day; HPLC-EC, High Pressure Liquid Chromatography/Electrochemical Detection; DA, dopamine; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; L-Dopa, 3,4dihydroxyphenylalanine. ⁎ Corresponding author. INSERM U910, Faculté de Médecine la Timone, 27 Bd Jean Moulin, 13385 Marseille, France. Fax: +33 491 804 319. E-mail address: [email protected] (N. Panayotis). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2010.10.006

(Hagberg et al., 2002). Based on early clinical evaluations and postmortem studies, contradictory findings were reported concerning a possible alteration of bioaminergic systems in RTT (Roux and Villard, 2010). Nevertheless, using different techniques, several deficits have been described in brainstem catecholaminergic nuclei (A1/C1 and A2/ C2) (Viemari et al., 2005), in the peripheral sympathoadrenergic and chemoafferent systems (Wang et al., 2006; Roux et al., 2008; Ladas et al., 2009) and the pontine nucleus locus coeruleus (Taneja et al., 2009; Roux et al., 2010). All of these alterations were associated with a significant impairment of autonomic and cognitive functions in mice (Viemari et al., 2005; Chahrour and Zoghbi, 2007; Roux et al., 2008; Bissonnette and Knopp, 2008). A recent report described a deficit in the midbrain catecholaminergic metabolism and altered transcription profiles in Mecp2-deficient mice (Samaco et al., 2009). In that study, a conditional loss of Mecp2 in brain areas that synthesize biogenic amines was shown to induce motor impairments. However, to date, it has not been determined whether the Mecp2-null mouse, which displays motor defects reminiscent of the cardinal features of RTT pathology, exhibits a gradual morphofunctional alteration of midbrain dopaminergic (mDA) brain nuclei. The mDA area substantia nigra pars compacta (SNpc — A9) regulates the elaboration of motor strategies (Blandini et al., 2000). A dopaminergic deficit induces motor defects in primates and rodents, and Parkinson's disease in humans (Jenner, 2008). Previous studies have

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shown that older RTT patients show «Parkinsonian features» and a stiff gait (FitzGerald et al., 1990; Neul and Zoghbi, 2004). We used a Mecp2deficient mouse (Guy et al., 2001) to study the nigrostriatal pathway. This pathway originates from the SNpc and projects to the caudate– putamen (CPu), its main rostral target. We evaluated the impact of a Mecp2-deficiency on locomotion and motor coordination. Previous studies described motor impairments in RTT mouse models (Ricceri et al., 2008). We confirmed the fact that the Mecp2−/y mice (Bird strain) exhibits progressive postnatal deficits in motor behavior and studied this phenomenon further. We evaluated the integrity of the nigrostriatal pathway and described dopaminergic deficits occurring in the SNpc and CPu of Mecp2−/y mice during the development. We also showed that an L-Dopa treatment improved some of the motor deficits previously identified. Taken together, our findings demonstrate that Mecp2 deficiency induces nigrostriatal deficits and they offer a new perspective to better understand the origin of motor dysfunctions in the Mecp2deficient mouse.

each trial, the rotarod was accelerated from 4 to 40 rpm in 300 s. Latency to fall was recorded in number of seconds. At each age tested, the best of the three trials was recorded. If a mouse clinging on the rod completed a full passive rotation we push down the lever and record the latency. In this case, passive rotation was considered a failure in performance like falling (Brown et al., 2005). Open field activity Open field activity was measured in an arena made of clear Perspex (38 × 30 cm). The test session lasted 15 min, and data were recorded using the Ethovision 2.3.19 tracking system (Noldus Information Technology). Velocity (cm/s) and total distance moved (cm) were recorded. Velocity calculations on Ethovision were obtained using an input filter setting the minimal distance moved (0.6 cm) so that ambulations shorter than this value were never taken into account to calculate the velocity. Immunohistochemistry

Materials and methods Animals Experiments were performed on the B6.129P2(c)-Mecp2tm1 + 1Bird mouse model for RTT (Guy et al., 2001). The mice were obtained from the Jackson Laboratories and maintained on a C57Bl/6 background. The experimental procedures were carried out in keeping with the European guidelines for the care and use of laboratory animals (Council Directive 86/609/EEC). Both pre-symptomatic and symptomatic mice were analyzed at different developmental stages. A total number of 77 Mecp2−/y and 72 WT mice were used in the study. The Mecp2−/y (null male) and WT (wild-type male) mice were studied at postnatal days 24 (P24; n = 11 WT; n = 8 Mecp2−/y), 35 (P35; n = 12 WT; n = 10 Mecp2−/y) and 55 (P55; n = 17 WT; n = 14 Mecp2−/y) for histology and western blot studies. For the behavioral tests, animals from postnatal days 24 (P24; n = 14 WT; n = 10 Mecp2−/y), 30 (P30; n = 14 WT; n = 12 Mecp2−/y) and 50 (P50; n = 14 WT; n = 8 Mecp2−/y) were used. For the neurochemical analysis, we used 6 Mecp2−/y and 10 WT mice from P35 and 9 Mecp2−/y and 8 WT mice from P55. For the pharmacological treatment, 10 Mecp2−/y-treated and 8 Mecp2−/y vehicle-treated (vehicle) mice were studied. Mecp2-deficient mice were compared to their respective wild-type littermates. Breeding and genotyping were performed as previously described (Viemari et al., 2005). Behavioral tests Mice subjected to a behavioral test battery were studied at P24, P30 and P50. Environmental parameters were carefully set to obtain the more reliable data. Room temperature was 19–23 °C, humidity was 45%–65%, and light/dark cycle was 12:12 h with lights on at 08:00 a.m. Each group of mice was housed in their environment until behavioral testing was completed. We tested our animals during the same time on the test days. Tests were always carried out in the following order: grip strength assessment, accelerating rotarod on day 1 and open field activity on day 2. Brief descriptions of each test are given later. Grip strength A Bioseb grip strength meter (Panlab) was used to measure the mice's grip strength. Two types of measurements were performed: forelimb measurement and forelimb and hind limb measurement. Five measures of each were taken and means were calculated from the three best trials. Accelerating rotarod The Panlab LE-8200 apparatus (380 × 260 × 240, Panlab) was used. Mice were subjected to 3 trials, with 5 min inter-trial intervals. During

Mice were deeply anaesthetized with pentobarbital (100 mg/kg) and transcardially perfused (NaCl for 1 min, followed by a buffered 4% paraformaldehyde for 10 min). Brains were removed and postfixed for 5 h and cryoprotected in 20% sucrose for another 24 h, then frozen at − 80 °C. Brains were cut into 20 μm coronal sections that encompassed the midbrain, using a cryostat (Microm Microtech, France). Sections were collected in separate sets for immunohistochemistry, so that each set contained every fifth serial section. One set of sections was immunostained for tyrosine hydroxylase (Th), a second set was used to carry out Th/Pitx3 double-immunostaining and a third set was stained for Th/Mecp2. The staining protocol has previously been described in detail (Roux et al., 2007; Dura et al., 2008). Briefly, sections were rehydrated, permeabilized (0.1% Triton X-100 PBS), blocked (7% normal goat serum) and incubated overnight at room temperature with the appropriate primary antibody. Sections were subsequently incubated with the secondary antibody, and then mounted in Shandon Immu-Mount™ antifade (Thermo Fisher Scientific). Tyrosine Hydroxylase (Th) was probed with a rabbit polyclonal antibody (1: 1000, Institut Jacques Boy, Reims, France) or a mouse monoclonal antibody (1: 1000, Millipore/Chemicon, MAB318), pSer40 Th with a rabbit polyclonal antibody (1:400, Cell Signaling, #2791S). Pitx3 was probed with a rabbit polyclonal antibody (1: 200, Zymed Laboratories, 38-2850). Mecp2 was probed with a rabbit polyclonal antibody (1: 500, Upstate Biotechnologies) or a mouse monoclonal antibody (1: 1000, Sigma-Aldrich). Vmat2 and Aadc were probed with rabbit polyclonal antibodies (respectively, 1:500, Millipore/Chemicon, AB1767 and 1:500, Abcam, AB3905). Goat anti-rabbit or goat anti-mouse Alexa 488 and goat anti-rabbit or anti-mouse Alexa 546 (1: 200, Molecular Probes) were used as secondary antibodies. DAPI (4.6-diamino-2-phenolindol dihydrochloride) counterstaining was performed on all sections, in order to visualize the nucleus. SNpc catecholaminergic cell counting Th-immunolabeled slides were digitized and recorded using an epifluorescence microscope (DM 5000B, Leica) equipped with a digital camera (DFC 300 FX, Leica). The SNpc region was defined by drawing a line from the most lateral tip of the SN to the most ventral point of the midline of the brain section, as previously described (Sgado et al., 2006) using the mouse brain stereological atlas (Paxinos and Franklin, 2001). SNpc cells were scored if they were ventral to this line. Medial VTA cells were not scored. Cell counts were performed on 10× magnification pictures using the nuclear-biased sampling procedure (Weibel, 1979). A neuron was considered to be Th-positive only if it met 3 criteria: 1) the cytoplasm was Th-immunoreactive, 2) part of the nucleus (DAPI positive) was located inside the counting frame but did not touch the

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delineated using the ImageJ software defined the soma boundaries. The program measured the area and expressed it in square micrometers.

avoidance lines and 3) a portion of the nucleolus was in focus between the top and bottom boundaries of the counting frame, or outside the guard zone. We used coronal sections of 20 μm thickness to avoid any potential double counting. This protocol allowed us to count the Thpositive neurons on the first set of sections, with a computer-assisted analysis program (ImageJ, NIH). Briefly, images were converted to 8-bit, then thresholded, and a region of interest (ROI) counting area was drawn on the image. From each animal, 3 regularly spaced sections from the central and largest portion of the nucleus were selected for quantitative analysis. Cell numbers are expressed as the total number of Th-positive neurons counted in the middle SNpc (mid-SNpc). For each animal, the total neuron number was counted in the right and left SNpc, and the mean of these two values calculated. The sections rostral and caudal to this landmark were included for microscopy and showed that the trend toward decrease of Th-expressing neuron number extended throughout the rostro-caudal extent of the nucleus and that the level selected for quantitative analysis was representative for the entire pars compacta. Mecp2-deficient mice and wild-type mice were analyzed blind for genotype with the same protocol.

A terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed on frozen brain sections. For this purpose, we used an in situ cell death detection kit (Roche Applied Science) to label apoptotic cell nuclei with Texas Modified Rhodamine. Sections were then processed with an epifluorescence microscope (DM 5000B, Leica). According to manufacturer's protocol, negative controls were included by incubating frozen sections with the labeling solution but not the enzyme solution. Positive samples were obtained by digesting the section with deoxyribonuclease grade I (Qiagen) for 30 min at 25 °C to induce DNA strand breaks prior to labeling. A TUNEL-Th double labeling was performed in order to identify dying catecholaminergic cells. Anti-cleaved caspase3 immunohistochemistry was performed using a rabbit antibody (#9661, Cell Signaling).

SNpc Th/Pitx3 double-quantification

Western blotting

Pitx3 and Th-staining was quantified using a second set of sections. Neurons were considered Pitx3-positive if they exhibited a clear nuclear staining colocalizing with the DAPI staining. Th-positive neurons were scored using the same criteria as previously defined. Quantification of both Pitx3-positive and Th-positive neurons was performed using an ImageJ colocalization tool (Institut Jacques Monod, Imaging Service, Paris). The mean number of Pitx3/Th double positive neurons, Th-positive/Pitx3-negative and Th-negative/Pitx3positive neurons per slice was determined and compared for each genotype on a total of 100 neurons in the SNpc structure. For each animal, we calculated the mean number of neurons in the right and left SNpcs and calculated the mean of these two values. As previously mentioned, Mecp2-deficient mice and wild-type mice were compared blind for genotype with the same protocol.

Selected brain areas (midbrain DA area = SNpc and ventral tegmental area; caudate–putamen = striatal region) were microdissected with a micropunch needle, using a protocol adapted from Roux et al. (2003). Tissues taken from both hemispheres were extracted by sonication and proteins isolated in a lysis buffer containing 20 mM Tris–HCl pH = 7.5, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100 and complete protease inhibitor tablet (Roche). Protein concentrations were determined using the BCA (bicinchoninic acid) method. After a denaturating step at 96 °C for 5 min, proteins (20 μg) were separated on an 8% SDS-polyacrylamide gel and transferred onto a PVDF membrane (Amersham Pharmacia Biotech) by liquid electroblotting (Mini Trans-Blot Cell, Bio-Rad) for 1 h at 100 V. Non-specific binding was prevented by pre-incubating the membrane with 5% nonfat dry milk in TBS Tween 0.1% for 1 h at room temperature. Primary antibodies for Th (Instituts Jacques Boy, 1:5000), pSer40-Th (Cell Signaling, 1:5000) and Gapdh (Millipore, 1:50,000) were diluted in the same solution and incubated overnight at 4 °C. After extensive washing of the membrane with 5% nonfat dry milk TBS Tween 0.1%, appropriate peroxidase-conjugate antibodies were incubated for 2 h at room temperature. Bound antibody was detected with an enhanced chemiluminescence reagent kit (Supersignal West Femto or Pico, Pierce). Digital images were obtained using an imager (BioSpectrum AC Imaging System, Biochemi HR Camera) and signal quantified on 16-bit images using the ImageJ software from NIH.

Staining intensity immunoquantification Immunoquantification of the staining level was performed using the protocol previously described in detail (Roux et al., 2008). In order to compare the intensity of staining in Mecp2-deficient mice and their respective controls, great care was taken at all stages. Fixation, freezing, cutting, staining and scanning of the images were performed under the same conditions, using the same solutions and timing, and by alternating tissues coming from the two genotypes. The linearity of the camera response was verified, and the fluorescence intensity was carefully selected in order to avoid reaching saturation. Densitometric analysis of the staining level was performed on 8-bit images using ImageJ software (http://rsb.info.nih.gov). The integrated density was calculated as the sum of the values of the pixels in a cytoplasmic region of interest. Because Th and phospho-Th-staining can vary slightly in a given SNpc, we analyzed pictures at a higher magnification and for each section we measured the cytoplasmic Th-staining level of at least 50 Thimmunopositive cells in the SNpc. For each section, we subtracted the mean of the background staining (Th-immunonegative) from the specific staining (Th-immunopositive). We then calculated the average of the successive rostro-caudal sections for each animal. Immunoquantification was performed by successively alternating the wild-type and the Mecp2-deficient paired mice. SNpc morphometric analysis We analyzed the soma area of SNpc Th-positive neurons (n =50 cells) using magnified pictures (×20). For each neuron, a region of interest

Detection of apoptosis

Biochemical analysis P35 (n = 6 Mecp2−/y, n = 10 WT) and P55 (n = 9 Mecp2−/y, n = 8 WT) mice were killed by cervical dislocation, and their brains were dissected out within the first 2 min post-mortem. The dorsolateral caudate–putamen and midbrain were microdissected using a punching needle (0.5 mm Ø) and kept at − 80 °C until biochemical analysis. Briefly, brain area dissection was performed on cryostat brain sections with the help of a 5× magnifying lens, following their stereotaxic coordinates (Paxinos and Franklin, 2001). Chemicals Dopamine, DOPAC, HVA, 1-octanesulfonic acid (OSA), triethylamine and ethylene-diamine-tetra-acetic acid (EDTA) disodium salt were purchased from Sigma, sodium dihydrogen phosphate and citric acid from Merck and methanol from Prolabo. Ultrapure water was obtained with a Milli-Q system (Millipore, Bedford, MA, USA). Standard solutions of each monoamine or metabolite were stored at −20 °C at 1 mmol/l.

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Tissue extraction Tissue extraction was made at 0 °C with 60 μl of perchloric acid containing 1.34 mmol/l EDTA and 0.05% w/v sodium bisulphite, spiked with 2 nmol/l DHBA used as internal standard. Sonication was applied twice 10 s. After homogenization, extracts were centrifuged at 14,000 rpm at +4 °C for 20 min. 35 μl of supernatant was analyzed the same day using high performance liquid chromatography (HPLC). The remaining supernatant was collected and frozen at −20 °C. It was used for overloaded peaks and diluted 4- or 10-fold for a second HPLC analysis (mainly for DA determination in striatum). HPLC The HPLC system was composed of a Hitachi L-7000 series equipped with a degasser, a L-7100 pump, an L-7200 autosampler thermostated at 10 °C and a Decade Intro electrochemical detector fitted with a 3 mm glass carbon working electrode, an Ag/AgCl reference electrode and a 25 μm spacer (Antec, Leyden, The Netherlands). Separations were performed using a 250 mm×4.6 mm i.d. C18 5 μm Beckman Ultrasphere column equipped with two Phenomenex C18 filters in a security guard system. The mobile phase was pumped at a microflow rate of 0.8 ml/min and composed of 0.7 M sodium phosphate, 0.1 mmol/l EDTA, 1.1 mmol/l OSA, 3.1 mmol/l triethylamine, and 14% methanol, pH adjusted to 3.12 with 1 mmol/l citric acid, and it was filtered with 0.45 μm cellulose acetate membranes before use. The eluted fractions were detected at an oxidation potential of 700 mV vs reference electrode. The column and the detection cell were housed within the Faraday cage of the electrochemical detector that was set to 25.5 °C. The day of the analysis, 35 μl of the samples was placed in the autosampler and kept at +10 °C before injection. The injection volume was 30 μl. The retention times were, 13.5 min, 16.5 min 32 min for DOPAC, DA and HVA respectively. Pharmacological treatments Mecp2−/y mice received a chronic oral administration of L-Dopa (Sigma-Aldrich) at a concentration of 15 mg/kg/day diluted in the drinking water or the water only (vehicle group). The dose was selected on the basis of its ability to induce a major effect on DA metabolism. Investigators blind of the treatment performed behavioral experiments and data analysis. To assess the behavior in motorrelated tasks, Mecp2−/y mice were treated with L-Dopa (n = 10) or vehicle (n = 8) from P30 to P60. Behavioral data represents Mecp2−/y L-Dopa-treated vs Mecp2−/y vehicle-treated motor performances at P30 (n = 10 treated, n = 8 vehicle), P50 (n = 7 to 6 treated, n = 7 vehicle) and at P60 (n = 6 to 3, n = 7 vehicle). Statistical analysis We evaluated whether our data distribution fitted with a Gaussian representation using a K–S Kolmogorov–Smirnov Normality test. If valid, we further applied a 2-way analysis of variance (ANOVA) with genotype or age as factors, followed by Fisher's protected least square difference test. If not valid, we used an adapted non-parametric Mann– Whitney test or a Kruskal–Wallis test to compare genotypes. The results are reported as mean +/− standard error of the mean (S.E.M.). A p-value b 0.05 was considered to be statistically significant. Results Motor behavior in the Mecp2-deficient mouse is progressively altered The grip strength test measures muscle strength and neuromuscular integration in relation to the grasping reflex in the forelimbs and hind limbs. Grip strength significantly increased in WT from P24 to P30 for forelimbs (WT P24, 61+/− 3 g; WT P30, 74.8+/− 4.2 g; p b 0.001) and forelimb and hind limb (WT P24, 92.8+/− 3.8 g; WT P30, 129.4 +/ − 5.5 g; p b 0.001). WT strength increased with age, between P30 and

P50 for forelimb strength (WT P30, 74.8 +/− 4.2 g; WT P50, 109 +/ − 4.9; p b 0.001) and forelimb and hind limb muscle strength (WT P30, 129.4 +/− 5.5 g; WT P50, 206.4 +/− 11.9 g; p b 0.001). The overall distribution of grip strength differed significantly between WT and Mecp2−/y, with Mecp2−/y mice showing weaker grip strength than WT, both in forelimb measurement (pb 0.05) and forelimb and hind limb measurement (pb 0.001). However, we found a significant difference between WT and Mecp2−/y only at P50, for forelimb muscle strength (WT P50, 109 +/− 4.9 g; Mecp2−/y P50, 78.3 +/− 5.9 g; p b 0.001) as well as forelimb and hind limb strength (WT P50, 206.4 +/− 11.9 g; Mecp2−/y P50, 146.1 +/− 12.1 g; p b 0.001) (Fig. 1A). The purpose of the rotarod test is to assess the rodent's sensorimotor coordination. Neither WT nor Mecp2−/y mice improved their performance on the accelerated rotating rod from P24 to P50 (Fig. 1B). Regardless of age, however, the latency to fall was significantly shorter in Mecp2−/y than in WT mice (pb 0.01). Mecp2−/y performed worse than WT at P30 (WT, 171.4 +/− 17.8 s; Mecp2−/y, 117.5 +/− 16.6 s; p b 0.05) and P50 (WT, 186.2 +/− 19 s; Mecp2−/y, 131.4 +/− 14.2 s; p b 0.05). We observed that P24 WT animals ameliorate progressively their performances on the rotarod during the 3 successive trials of the test day (WT P24, p b 0.001). At the same age, Mecp2−/y animals presenting no obvious phenotype exhibit the same motor learning (Mecp2−/y, p b 0.05). Thereafter, we noticed that P35 and P55 WT and Mecp2−/y do not ameliorate between the 3 consecutive trials (WT P35, p N 0.05–Mecp2−/y P35, p N 0.05; WT P55, p N 0.05–Mecp2−/y P55, p N 0.05). Thus, as soon as P35, animals complete the task previously acquired with a constant level of performance. These last results suggest that the poorer Mecp2−/y motor performance at P35 and P55 are not due to an impairment of their motor learning. The open field test is designed to measure more complex behavioral responses such as locomotor activity, hyperactivity, and exploratory behaviors. The mean velocity, irrespective of age, was greater in WT than in Mecp2−/y mice (WT, 7.4 +/− 0.2 cm/s; Mecp2−/y, 6.3 +/ − 0.4 cm/s; Mann–Whitney test, p b 0.05). However, no differences were found when we compared age-matched mice. There was no difference in the total distance moved when WT and Mecp2−/y mice were compared (Fig. 1C). The results of behavioral tests designed to investigate motor and coordination performances suggest that Mecp2−/y mice suffer a postnatal deterioration in their muscle strength and in the coordination of movements, and that they are slower than WT mice. This is consistent with there being a progressive alteration of motor function in Mecp2−/y mice. The Th-positive neuron number is reduced from P35 in the mid-SNpc of Mecp2−/y mice Rett syndrome patients and Mecp2-deficient mice display a progressive, postnatal neurological phenotype (Chahrour and Zoghbi, 2007). Mecp2−/y mice are globally normal until P30 and then begin to suffer cognitive and motor dysfunctions (Guy et al., 2001). We hypothesized that the appearance of the symptomatic motor phenotype could be linked to an SNpc alteration. To test this hypothesis, we examined the Thexpressing neurons in the SNpc. Immunofluorescent Th labeling showed lower staining in the Mecp2−/y SNpc (Fig. 2). We therefore quantified the number of Th-immunoreactive neurons in this brain structure. As expected, we did not observe a significant difference in the number of Th-positive neurons at different ages in wild-type mice, neither between P24 and P35 (WT P24, 362.6+/− 14.6 vs WT P35, 354.4+/−12.9, p=0.69), nor between P35 and P55 (WT P35, 354.4+/−12.9 vs WT P55, 354.1+/−15.9; p=0.99). At P24, comparison of WT to Mecp2−/y animals showed that the number of SNpc Th-positive neurons is not significantly different between the two genotypes (number of neurons: WT, 362.6+/ −14.6 vs Mecp2−/y, 356.9+/−24.1; n =7 mice for WT, n =5 mice for Mecp2−/y; p=0.6). At P35, a significant reduction in Th-positive neurons was apparent in the Mecp2−/y SNpc (number of neurons: WT, 354.4+/

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Fig. 1. Mecp2-deficiency causes progressive postnatal defects in locomotor and coordinated behaviors. Histograms showing the behavioral performances in wild-type (WT, black line) and Mecp2-deficient (Mecp2−/y, gray line) animals at postnatal days P24 (n= 14 for WT; n = 10 for Mecp2−/y), P30 (n= 14 for WT; n = 12 for Mecp2−/y) and P50 (n= 14 for WT; n = 8 for Mecp2−/y). A. Grip strength test. Test results are expressed in grams. There is a statistically significant difference between the two genotypes for forelimb and forelimb+ hind limb at P50. B. Rotarod. Results are expressed as latency to fall in seconds. At P24, WT and Mecp2−/y behave similarly. In WT, the latency to fall increases progressively with age. Mecp2−/y mice fall off the rotating rod sooner than wild-type controls at P30 and P50. C. Open field test. The mean mouse velocity (expressed in cm/s) is reduced in Mecp2−/y compared to WT, independent of age. The total distance the mice covered in the apparatus during the 15-minute session is not statistically different between the two genotypes at any age, although a tendency to poorer results is visible with the Mecp2−/y mice. A high variability among the Mecp2−/y group is observed for this test. (*p b 0.05, **p b 0.01).

−12.9 vs Mecp2−/y, 277.5+/−16.2; n=9 mice for WT, n=7 mice for Mecp2−/y; pb 0.01). At P55 we also found a reduction in the Thpositive neurons in the Mecp2−/y SNpc (number of neurons: WT, 354.1+/ −15.9 vs Mecp2−/y, 297.3+/−11.4; n=10 mice for WT, n=8 mice for Mecp2−/y; pb 0.05), but no decrease was apparent in Mecp2−/y mice between P35 and P55 (Mecp2−/y P35, 277.5+/−16.2 vs Mecp2−/y P55, 297.3+/−11.4) (Fig. 3A). Thus, Mecp2−/y mice display a progressive postnatal decrease in the Th-positive neuron number in SNpc. This reduction is first apparent at P35 and remains at P55. However, we observed the number of Th neurons to be stable between these two stages. Decreased intensity of tyrosine hydroxylase staining in Mecp2-deficient SNpc and caudate–putamen Given that the number of Th neurons in the SNpc of Mecp2−/y animals decreases, we examined the Th integrated density in the SNpc and the caudate–putamen, its main target. We found that at the fully symptomatic stage (P55), the Th integrated density in the SNpc decreased significantly in the Mecp2−/y mice (WT, 54,402 +/ − 9523 pixels vs Mecp2−/y, 33,996 +/− 3691 pixels; Mann–Whitney test, p b 0.05; n = 3 mice for WT, n = 3 mice for Mecp2−/y). For the caudate–putamen analysis, measurements were taken throughout the entire structure. We compared Mecp2−/y male and WT controls. Th integrated density in the caudate–putamen was significantly lower in Mecp2-deficient mice (WT, 10.156 +/− 1.183 megapixels vs

Mecp2−/y, 6.747 +/− 0.545 megapixels; n = 3 mice for WT, n = 3 mice for Mecp2−/y; p b 0.05) (Figs. 3B–C). These observations indicate that a dopaminergic deficit occurs in the SNpc and the caudate– putamen of the Mecp2−/y mouse, which are, respectively, the source and the target of the nigrostriatal pathway. Mecp2-deficient dopaminergic SNpc neurons have a smaller soma area Neuropathology studies on RTT brains (Bauman et al., 1995) and recent histological results in Mecp2-deficient mice (Taneja et al., 2009) reported, respectively, that neurons in the SN of patients and the Locus coeruleus of Mecp2-null are abnormal. We compared the size of the soma area of SNpc neurons in WT and Mecp2−/y male mice at P24, P35 and P55, using fluorescent imaging on Th-stained brain sections. In order to take into account the inter-individual variability between the two groups (WT vs Mecp2−/y), we averaged the soma areas for each animal and expressed the value as the mean of the WT vs Mecp2−/y group (Fig. 3D). As expected, we did not find statistically significant differences between P24 and P35, nor between P35 and P55, for WT (Kruskal–Wallis test, p = 0.54). At P24, the mean soma area in WT and Mecp2−/y were respectively 283.4 +/− 46.6 μm2 and 261.9 +/− 19.9 μm2. By comparing WT and Mecp2−/y, we found that the distribution in the two groups did not differ significantly (Mann– Whitney test, p N 0.99; n = 4 mice for WT, n = 3 mice for Mecp2−/y). At P35, we observed a significant decrease in the soma area of Th-positive SNpc neurons in Mecp2−/y compared to age-matched controls (WT,

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279.8 +/− 3.5 μm2 vs Mecp2−/y, 224.8 +/− 3.8 μm2; Mann–Whitney test, p b 0.05; n = 3 mice for WT, n = 3 mice for Mecp2−/y). At P55, we also observed a significant decrease in the soma area of Mecp2−/y Thpositive SNpc neurons (WT, 280.2 +/− 8 μm2 vs Mecp2−/y, 211 +/ − 17.5 μm2; Mann–Whitney test, p b 0.05; n = 3 mice for WT, n = 3 mice for Mecp2−/y). These results show that the size of the soma area is reduced at pathology onset, but that there is no further decrease between P35 and P55.

Th protein content is decreased in the Mecp2-deficient midbrain area A reduction in the number of Th-positive neurons would be expected to modify the catecholamine content. We used a micropunch technique to extract proteins from the SNpc, the ventral tegmental area (VTA) and the striatal region (caudate–putamen). We quantified the Th protein levels using western blot analysis. Our results show that Mecp2−/y mice (n = 3) have 30% less tyrosine hydroxylase protein in the midbrain dopaminergic area compared to wild-type animals (n = 4) (p b 0.05). However, in the caudate– putamen, no statistically significant difference was found (27% less than WT, n = 4 mice for WT and n = 3 for Mecp2−/y, p = 0.08) (Supplementary Fig. S1). Although results were similar, a significant variability in Th protein content in the caudate–putamen was observed. Our results indicate that, in Mecp2−/y mice at P55, the Th protein content is reduced in the midbrain dopaminergic area, but not in its target, the caudate–putamen area.

The decrease in Th-positive neuron number is not due to cell death By cell counting, we show that there is a postnatal decrease in the number of Th-positive neurons in the SNpc. This prompted us to investigate whether apoptosis is the cause, by means of a cell death detection assay using the terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL), and by anti-caspase3 immunohistofluorescent techniques coupled with Th-staining. This allowed us to examine cell death in catecholaminergic neurons in the SNpc (Supplementary Fig. S2). We compared 3 WT vs 3 Mecp2−/y mice at both P35 and P55. We detected no apoptotic cells in the SNpc of the Mecp2−/y mice.

The mature dopaminergic fate is not affected in Th-expressing neurons in Mecp2-deficient SNpc

Fig. 2. Decrease in Th protein levels in the Mecp2-deficient SNpc. Representative crosssectional photomicrographs of P55 WT (A–C) and Mecp2−/y (E–G). SNpc co-immunolabeled for Th (green), Mecp2 (red) and counterstained with DAPI (blue). D–H. Co-labeling with Th and Mecp2 in WT (D) and Mecp2−/y (H) SNpc (scale bar A–D and E–H = 200 μm). I–K. Representative photomicrographs of WT (I) and Mecp2−/y (K) mice co-labeled with Th and Mecp2 at a high magnification. J–L. Inset zoom of I and K. Arrows indicate neurons colabeled by Th and Mecp2 with characteristic Mecp2 nuclear staining and surrounding somatic Th expression. As expected, Mecp2 staining is absent in the SNpc of Mecp2−/y mice (E to L). The Th-staining level is significantly reduced in the Mecp2-deficient mice. (Scale bar I, K = 100 μm and 62 μm for J and L).

To investigate a possible modification in the phenotype of the mature dopaminergic (mDA) neuron population in SNpc, we carried out a double labeling of Th and Pitx3 in the SNpc. The paired-like homeobox protein Pitx3 is an important transcription factor as regards the development and function of mDA neurons (Smidt et al., 2004). Basically, Pitx3 is widely expressed at E11.5 in mice, but is only found in mDA neurons after birth (Smidt et al., 1997). In our study, the number of neurons that expressed both Th and Pitx3 did not differ between WT and Mecp2−/y at the late symptomatic stage (P55) (WT, 73.2 +/− 10.8 vs Mecp2−/y, 61.6 +/− 7.4; n = 10 mice for WT, n = 8 mice for Mecp2−/y; p = 0.38). The number of neurons that expressed Pitx3 but did not express Th increased in Mecp2−/y animals at P55, although it did not reach statistical significance (WT, 8.5 +/− 2.4 vs Mecp2−/y, 14.3 +/ − 1.6; n = 10 mice for WT, n = 8 mice for Mecp2−/y; p = 0.064). The proportion of Th-positive and Pitx3-negative neurons was not statistically different when the two genotypes were compared (WT, 18.1 +/ − 2.8 vs Mecp2−/y, 19.7 +/− 3.2; n = 10 mice for WT, n = 8 mice for Mecp2−/y; p = 0.69) (Figs. 4A–B). These results suggest that Pitx3 is still fulfilling its normal role in the maintenance of Th-positive neurons in the Mecp2−/y SNpc.

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Fig. 3. Immunohistological and densitometric analysis of nigrostriatal dopaminergic neurons. A. Number of Th-positive neurons in the middle SNpc of Mecp2−/y (white bars) compared to their respective WT controls (black bars) at P24 (n = 7 for WT; n = 5 for Mecp2−/y), P35 (n = 9 for WT, n = 7 for Mecp2−/y) and P55 (n = 10 for WT, n = 8 for Mecp2−/y). The number of Th-positive neurons in the SNpc is not affected at P24 but is reduced at P35 and P55. There is no further reduction between P35 and P55 in the Mecp2−/y SNpc. B. Representative photomicrographs of coronal sections through the WT and Mecp2−/y brain at the level of the caudate–putamen immunostained for Th (scale bar = 500 μm). C. Immunoquantification of the integrated density of Th-staining (in pixels) in the SNpc and in the caudate–putamen (in megapixels) of P55 WT (black bar, n = 3) and Mecp2−/y (white bar, n = 3). D. Left. Illustration of P24, P35 and P55 SNpc Th-positive somas (scale bar = 50 μm). Right. Quantification of soma area (in μm2) in P24 (n = 4 for WT, n = 3 for Mecp2−/y), P35 (n = 3 for WT, n = 3 for Mecp2−/y) and P55 (n = 3 for WT, n = 3 for Mecp2−/y). (*p b 0.05, **p b 0.01, n.s.: non-significant).

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aminergic markers normally present in fully mature mDA neurons (Ang, 2006; Smidt and Burbach, 2007). We studied the vesicular monoamine transporter 2 (Vmat2), a protein involved in the uptake of catecholamines in synaptic vesicles, and the second enzyme in the catecholaminergic synthesis pathway, aromatic amino acid decarboxylase (Aadc), involved in the conversion of L-Dopa to DA. Our results show that almost all the Th-positive neurons counted (n= 50 cells per animal) were positive for Vmat2 (Ratio Vmat2/Th, WT, 0.96 +/− 0.04 vs Mecp2−/y, 0.94 +/− 0.03; n = 3 mice for WT, n = 3 mice for Mecp2−/y) and Aadc-positive (Ratio Aadc/Th, WT, 0.9 +/− 0.04 vs Mecp2−/y, 0.9 +/ − 0.03; n = 3 mice for WT, n = 3 mice for Mecp2−/y). We found a few Th-negative and Vmat2-positive neurons in both WT and Mecp2−/y animals. Such cells have already been described in the SN in several studies (Weihe et al., 2006). We found no difference in the number of neurons expressing Th and Aadc or Th and Vmat2, when we compared WT and Mecp2−/y animals (Supplementary Figs. S3, S4 and S5). This indicates that these cells are probably able to synthesize dopamine and charge vesicles for subsequent synaptic release in Mecp2−/y animals. Active tyrosine hydroxylase levels decrease at P55 in Mecp2-deficient SNpc

Fig. 4. Assessment of the Pitx3 phenotype of Th-expressing neurons in the Mecp2−/y SNpc. A. Representative cross-sectional photomicrographs of the SNpc co-immunolabelled for Pitx3 (green), Th (red) and DAPI (blue) counterstaining. Th/DAPI overlay show that Th is cytoplasmic, as expected. Th/DAPI and Th/Pitx3 merged images confirm that the Pitx3-staining is nuclear. Arrows depict DAPI-positive cells that exhibit faint but positive Pitx3-staining, but lack the Th enzyme (scale bar = 50 μm). B. Quantification of the number of Th+/Pitx3+, Pitx3+/Th− and Th+/Pitx3− cells over a total population of 100 SNpc neurons in P55 WT (black bars, n = 10) and Mecp2−/y (white bars, n = 8). n.s.: non-significant.

Th-positive neurons present at P55 in Mecp2-deficient SNpc express mature markers We examined Th-expressing neurons in Mecp2-deficient SNpc at P55, in order to determine whether they expressed the catechol-

Although the number of Th-positive neurons decreases between P24 and P35 in Mecp2-deficient SNpc, it is not significantly different between P35 and P55. However, the dopamine content continues to decrease in the whole brain after disease onset (Ide et al., 2005). Th activity is regulated by various mechanisms (Kumer and Vrana, 1996), including phosphorylation of serine 40 (pSer40; Dunkley et al., 2004). We hypothesized that a decrease in the pSer40-Th content could participate to nigrostriatal deficits progression in the Mecp2-deficient mouse between P35 and P50. To investigate this possibility, we double-labeled SNpc neurons for Th and pSer40-Th (i.e. active tyrosine hydroxylase) (Fig. 5A). We observed that Th-positive neurons also express the active Th isoform in WT and Mecp2−/y (data not shown), thus indicating that all Th-expressing neurons express the active Th isoform. We next focused on the pSer40-Th-staining intensity per neuron (as an index of the protein content). We calculated the integrated density of Thstaining in 50 SNpc cells at P35, WT (n = 3) and Mecp2−/y (n= 3) and at the fully symptomatic stage (P55) WT (n= 3) and Mecp2−/y (n= 3) (Fig. 5B). At P35, phospho-Th-staining is lower for SNpc neurons in Mecp2−/y (14% less than WT level). Based on the integrated density of pSer40-Th-staining, neurons constitute two distinct populations that correlate to genotype. The mean integrated density was slightly lower when we compared the two populations at P35 (WT, 15,436 +/ − 440.78 pixels vs Mecp2−/y, 13,192 +/− 372.16 pixels, p b 0.001). At a later stage (P55), using phospho-Th-staining, we found a clear reduction in density in Mecp2−/y (56.5% less than WT at the same age) (WT, 11,674 +/− 225.91 pixels vs Mecp2−/y, 5077 +/− 145.21 pixels; p b 0.0001). This result indicates that the amount of pSer40 is reduced in the Mecp2-deficient mouse when the first symptoms appear, but that there is a further reduction at later stages of the disease, when the phenotype becomes more severe. We further confirmed by western blot on midbrain dopaminergic area microdissected samples (including the SNpc and the VTA) that the amount of pSer40-Th is reduced in P55 Mecp2−/y compared to their WT littermates (37% less than WT, n = 4 mice for WT and n = 3 mice for Mecp2−/y, p b 0.05) (Fig. 5C). Mecp2-deficient mice exhibit dopaminergic disturbances in brain regions regulating motor strategies We have shown that Mecp2−/y mice exhibit impaired motor behaviors and a significant loss of the Th-positive phenotype in the nigrostriatal neurons. We measured dopamine and its metabolites in SNpc and CPu microdissected areas. Measurements of catecholamine contents using HPLC-EC revealed no statistical difference in the levels of DA (WT P35, 0.146+/−0.02 μmol/l; Mecp2−/y P35, 0.102+/−0.03 μmol/l; Mann–

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Fig. 5. Measurement of pSer40-Th levels in Mecp2−/y SNpc. A. Representative immunohistofluorescent images of P35 and P55 WT and Mecp2−/y brain sections at the level of the SNpc for Th (red), phosphoTh (pSer40, green) and Th/pSer40 merged images. At P35, no obvious modification of the pSer40 staining intensity is present in the two genotypes. At P55, pSer40 staining intensity is markedly reduced, indicating a reduction in protein content (scale bar = 100 μm). B. Graphs representing the distribution of WT (black bars) and Mecp2−/y (white bars) cells (n= 50 cells) according to the level of pSer40 staining in the soma. Left: integrated density of WT vs Mecp2−/y cells at P35. Right: integrated density of WT vs Mecp2−/y cells at P55. Black and red lines, respectively, represent polynomial fitting curves for Mecp2−/y and WT data. No direct comparison between P35 and P55 data was made, since they were generated from two independent experiments. C. The midbrain dopaminergic area (containing the SNpc and the VTA) was microdissected from WT (n= 4) and Mecp2−/y (n= 3) mice at P55. Extracted proteins were analyzed for their relative phospho-Th (pSer40-Th) content by western blot. 20 μg of proteins was loaded on SDS-8% PAGE and analyzed. Membranes were probed with a rabbit polyclonal pSer40-Th antibody (Cell Signaling). The bottom blot in panel C is a loading control probed with an antibody to Gapdh. Lower graph illustrates the quantitative values of the respective blot images. Data are expressed in arbitrary units as a percentage of the control values. (*p b 0.05).

Whitney test, pN 0.05), dihydroxyphenylacetic acid (DOPAC) (WT P35, 0.09+/−0.01 μmol/l; Mecp2−/y P35, 0.07+/−0.02 μmol/l; Mann–Whitney test, pN 0.05) and homovanillic acid (HVA) (WT P35, 0.14 +/ − 0.02 μmol/l; Mecp2−/y P35, 0.08 +/− 0.03 μmol/l; Mann–Whitney test, pN 0.05) in the SNpc of P35 Mecp2−/y mice (n=6) compared with their WT littermates (n=9) (Fig. 6A). At P55, the Mecp2−/y SNpc

contained a lower DA concentration (WT P55, 0.25+/−0.04 μmol/l; Mecp2−/y P55, 0.18 +/− 0.02 μmol/l; Mann–Whitney test, p N 0.05), however the difference did not reach significance. DOPAC levels were unaffected in Mecp2−/y (WT P55, 0.11+/−0.01 μmol/l; Mecp2−/y P55, 0.08 +/− 0.01 μmol/l; Mann–Whitney test, p N 0.05). Conversely, HVA levels were significantly reduced (WT P55, 0.12+/−0.009 μmol/l;

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Fig. 6. Measure of dopamine content in the SNpc and the CPu of Mecp2−/y mice at P35 and P55. HPLC analyses of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentration in (A, B) the midbrain (SNpc, higher panels) and (C, D) the caudate–putamen (CPu, lower panels) of WT (black bars) and Mecp2−/y (white bars) mouse at postnatal ages P35 and P55. P35 SNpc, n = 9 WT and n = 6 Mecp2−/y; P55 SNpc, n = 8 WT and n = 9 Mecp2−/y; P35 CPu, n = 10 WT and n = 6 Mecp2−/y; P55 CPu, n = 8 WT and n = 9 Mecp2−/y. (*p b 0.05, **p b 0.01).

Mecp2−/y P55, 0.09+/−0.005 μmol/l; pb 0.01) in the SNpc of Mecp2−/y mice (n=9) compared with their WT littermates (n=8) (Fig. 6B). To examine the possibility that morphological SNpc deficits may affect terminal dopaminergic release in rostral brain areas, we measured the amount of DA and its metabolite in the caudate–putamen. As it was previously shown, DA, DOPAC and HVA levels in the SNpc are approximately 10% of the corresponding CPu values (Robertson et al., 1991). We observed that from P35 (Fig. 6C), the levels of DA were significantly reduced in Mecp2−/y (n=6) compared to WT (n=10) (WT P35, 1.23+/−0.02 μmol/l; Mecp2−/y P35, 0.64+/−0.157 μmol/l; Mann– Whitney test, pb 0.01). The levels of DOPAC were not altered (WT P35, 0.27+/−0.022 μmol/l; Mecp2−/y P35, 0.2+/−0.06 μmol/l; Mann–Whitney test, pN 0.05) but the HVA contents were clearly diminished (WT P35, 0.29+/− 0.023 μmol/l; Mecp2−/y P35, 0.18 +/− 0.05 μmol/l; Mann– Whitney test, pb 0.01). At P55 (Fig. 6D), we found that the striatal DA levels are reduced in Mecp2−/y (n=9) compared to WT (n =8) (WT P55, 2.46 +/− 0.19 μmol/l; Mecp2−/y P55, 1.74+/−0.18 μmol/l; p b 0.05). DOPAC contents were not different between Mecp2−/y and WT (WT P55, 0.38 +/− 0.039 μmol/l; Mecp2−/y P55, 0.32 +/− 0.032 μmol/l; Mann–Whitney test, pN 0.05). HVA levels were reduced in the CPu (WT P55, 0.36 +/− 0.044 μmol/l; Mecp2−/y P55, 0.23 +/− 0.022 μmol/l; pb 0.05). These observations are in good agreement with previous studies showing that dopamine content is decrease in the whole Mecp2deficient brain (Ide et al., 2005; Samaco et al., 2009).

should improve the phenotype of the Mecp2-deficient animals. We treated Mecp2-deficient animals from P30 to P60 with L-Dopa or the vehicle (i.e. drinking water) (Fig. 7). We first evaluated the impact of this treatment on the locomotion using an open field arena. We did not detect significant differences in the velocity and total distance moved between the two groups at P30 (percentage of the vehicle group; velocity: 96.9 +/− 5.5%; distance moved: 85.5 +/− 8.3%; Mann–Whitney test, p N 0.05) and P50 (percentage of the vehicle group; velocity: 124.9 +/− 18.2%; distance moved: 146.26 +/ − 35.6%; Mann–Whitney test, p N 0.05). At P60, the Mecp2−/y animals receiving a chronic L-Dopa treatment exhibited better performances than the vehicle group in the open field test (velocity: 149.75 +/ − 12.7%; distance moved: 199.65 +/− 29.4%; Mann–Whitney test, p b 0.05 for both parameters). However, we observed no benefit of LDopa treatment in the rotarod test (latency to fall at P30: 130.6 +/ − 16.4%; at P50: 60.35 +/− 22.1%; at P60: 122.5 +/− 28%; Mann– Whitney test, p N 0.05), the grip strength tests for the forelimb strength (P30: 98.3 +/− 6.6%; P50: 97.3 +/− 6.4%; P60: 99.7 +/ − 14.1%; Mann–Whitney test, p N 0.05) and the grip strength test for the forelimb + hind limb strength (P30: 100.7 +/− 5.2%; P50: 90 +/ − 7.2%; P60: 89.3 +/− 10.3%; Mann–Whitney test, p N 0.05). These results indicate that a chronic administration of L-Dopa is able to improve the performance of Mecp2−/y-treated mice in paradigms evaluating the voluntary motor behavior.

Behavioral effects of oral L-Dopa administration

Discussion

If the alteration of the nigrostriatal pathway, leading to a reduction of DA concentration in the CPu terminals, is responsible for the motor impairments, we reasoned that the stimulation of the DA metabolism

Previous studies have shown that the lack of Mecp2 results in catecholaminergic deficits in neurons located in the central and peripheral nervous systems (Viemari et al., 2005; Wang et al., 2006; Roux et al., 2008,

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Fig. 7. Impact of the oral L-Dopa treatment on the motor performances of Mecp2−/y mice. Histograms showing the behavioral performances in the vehicle group (Mecp2−/y untreated, white) and L-Dopa treated (Mecp2−/y L-Dopa, gray) animals. A. Open field test. The mouse velocity of Mecp2−/y mice treated with L-Dopa is unaffected at P30 (n = 10 LDopa, n = 8 vehicle) and P50 (n = 6 L-Dopa, n = 7 vehicle), and it is significantly increased at P60 (n = 3 L-Dopa, n = 7 vehicle). The total distance traveled by the mice in the open field arena during the 15-minute session is not statistically different between the two groups before P60. B. Rotarod. At P30, Mecp2−/y L-Dopa (n = 10) and Mecp2−/y vehicle (n = 8) behave similarly. At P50, the performance of the group treated with L-Dopa is reduced, but not statistically significant (n = 7 L-Dopa, n = 7 vehicle). At P60 (n = 6 L-Dopa, n = 7 vehicle), there is no difference between the two groups. C. Grip strength test. The forelimb and forelimb + hind limb grip strength measurements are unaffected by the L-Dopa treatment at all ages (P30: n = 10 L-Dopa, n = 8 vehicle; P50: n = 7 L-Dopa, n = 7 vehicle; P60: n = 6 L-Dopa, n = 7 vehicle). (*p b 0.05).

2010; Taneja et al., 2009;). In these structures, catecholamines are involved in the autonomic and arousal regulation. Here, we report that SNpc, the main source of dopamine in the brain, is also altered, participating in turn to the appearance of in vivo motor deficits. Alteration of motor behavior in Mecp2−/y mice The progression of the RTT phenotype in human females parallels that of Mecp2-deficiency in the mouse. Mecp2−/y mice are apparently unaffected until 3–6 weeks of age, when they exhibit stiff and uncoordinated gait, hypoactivity, tremor and hind limb clasping reminiscent of movement disorders seen in the patients. The symptoms of the Mecp2-deficient mouse subsequently worsen and finally lead to a drastic weight loss and death around 10 weeks of age (Chahrour and Zoghbi, 2007). Previous studies have assessed the motor performance of two Mecp2-null mice (Guy et al., 2001; Chen et al., 2001). However, for the Mecp2−/y mouse produced by Adrian Bird's group (Guy et al., 2001), this aspect has not been investigated fully. This strain exhibits growth retardation, alteration of coordinated behaviors in the rotarod test and lower postural capabilities (Guy et al., 2001; Santos et al., 2007). In the open field test conducted by Santos and colleagues, a reduction in the total distance traveled was found, but surprisingly high values were reported for movement speed (300 cm/s, whereas standard values are

around 10 cm/s; Bothe et al., 2004). We confirm that coordinated behaviors and grip strength are altered in Mecp2−/y mice at pathology onset. The locomotor behavior in the open field test revealed no differences in the total distance traveled when Mecp2−/y and WT mice were compared, at all ages investigated. However, movement velocity was lower in Mecp2−/y than in WT mice, independent of age. Consequences of a reduced number of Th-positive neurons for nigrostriatal function in Mecp2−/y SNpc In humans, a reduction in the number of Th neurons in the SNpc and a concomitant dopamine depletion in the striatum results in Parkinson disease (Lees et al., 2009). Several animal models based on the toxic lesion of the SNpc by 6-hydroxydopamine, MPTP, or rotenone, have highlighted the fact that this structure is fundamental for normal motor behavior (Dauer and Przedborski, 2003). The elaboration of motor strategies is dependent on the SNpc and is fine-tuned by a complex cortico-basal ganglia-thalamo-cortical loop (Parent and Hazrati, 1995). Here, we hypothesized that a dopaminergic deficit in the SNpc of the Mecp2−/y mouse could explain part of its motor phenotype. Indeed, we found that at P35 (i.e. when motor impairments become apparent) and P55, the Th-positive population of neurons is reduced in the Mecp2deficient SNpc. Taken together with the finding that Th-staining

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intensity in the caudate–putamen is also reduced, our results are the first report of a deficit in the nigrostriatal dopaminergic pathway in this mouse model. It should be noted, however, that the extent of Th-positive neuron depletion is not comparable to the severe depletion observed in animal models for PD or transgenic mice, lacking genes essential for the development and maintenance of midbrain DA neurons, such as Pitx3, Nurr1 or En1 (Smidt and Burbach, 2007; Sonnier et al., 2007). SNpc neurons have a smaller soma area in Mecp2−/y mice compared to WT Post-mortem investigations on RTT brains and Mecp2−/y studies revealed that SN neurons exhibit morphological abnormalities (Jellinger, 2003; Belichenko et al., 2009). We found that the soma area of SNpc neurons begins to be reduced from P35 onwards in Mecp2−/y animals. The same observation in the locus coeruleus (also affected in the Mecp2-deficient brain) has been linked to electrophysiological changes (Taneja et al., 2009; Roux et al., 2010). It would be interesting to investigate the electrophysiological properties of Mecp2-deficient SNpc neurons in the future. The phenotype of Th-expressing neurons in the Mecp2-deficient SNpc is mature We did not detect cell death in the brain areas in which we observed a reduction of the number of Th-expressing neurons. This suggests that, although the Th neuron number is reduced, the neurons themselves are not definitively dead. It is possible that these cells are still present but have lost their normal Th-positive phenotype. This decrease in Th expression occurs between P24 and P35 and remains at P55. Even if we cannot delineate the cause of such a variation, we can argue that the loss and gain of catecholaminergic phenotype during the postnatal maturation and functional changes have already been described and commented by several laboratories (Bezin et al., 1994; Ginovart et al., 1996; Marcel et al., 1998; Traver et al., 2006; Bjorklund and Dunnett, 2007). Thus, it was observed in aging and pathological conditions that a decline in SNpc neurons could be due to a downregulation of Th in surviving DA neurons. The authors proposed the Th phenotype switch is a consequence of DA neurons transcription factors misexpression (see Bjorklund and Dunnett, 2007 for review). Pitx3 regulates Th expression and the neurotransmitter phenotype of DA cells (Jacobs et al., 2009). However, we did not observed statistically significant variation in the number of neurons co-expressing Pitx3 and Th in the Mecp2-deficient SNpc. The Th-expressing neurons that remain at P55 in Mecp2−/y mice express Vmat2 and Aadc, suggesting that the phenotype of these cells is mature. A deficit in active tyrosine hydroxylase appears to aggravate the pathology at later stages The reduction in the number of Th-expressing neurons cannot, by itself, explain the aggravation of the motor deficits in the Mecp2−/y mice. It is not known if the remaining Th-expressing neurons in the Mecp2−/y SNpc can compensate for this reduction. Th activity can be modulated by several mechanisms (Kumer and Vrana, 1996). Th phosphorylation is responsible for maintaining catecholamine levels in tissues. This mechanism relies on the action of different kinases (CaMKII, PKA), which ultimately phosphorylate serine 40, increase Th activity and preclude catecholamines-dependent feedback inhibition (Dunkley et al., 2004). We demonstrate here that the amount of pSer40-Th is reduced at P35, but that a much larger reduction is observed at P55. These results suggest that the decrease in the number of Th-positive neurons constitutes the first step in dopaminergic dysfunction, a second step possibly being a decrease in the amount of active (phosphorylated) tyrosine hydroxylase. Taken together, these data indicate that Th neurons in the Mecp2−/y SNpc

are unlikely to be able to sustain normal activity. This is consistent with previous descriptions of a deficit in catecholamine content in the Mecp2-deficient mouse (Samaco et al., 2009; Roux and Villard, 2010). DA neurons in the mouse midbrain SN-VTA complex represent approximately 87% of the total number of DA neurons in the brain (Zaborszky and Vadasz, 2001). Accordingly, DA contents are progressively reduced in Mecp2−/y brain (Ide et al., 2005). Nigrostriatal dopaminergic disturbances support the manifestation of motor deficits in the Mecp2−/y mouse Our results show that the DA metabolism is preserved in the SNpc, when the motor behavior and SNpc integrity begins to deteriorate (P35), but is already reduced in its main target. The decrease of DA contents in the nigrostriatal terminals is in accordance with a reduction of the number of SNpc neurons that express active Th and a weaker Th-positive fiber density in the CPu. A significant reduction of HVA contents is observed in the SNpc between P35 and P55. Such a decrease could be explain by, 1) lower DA release from the SNpc, leading to a reduction of catabolites formation or 2) a reduction of the activity of degradation enzymes. This last phenomenon could in turn result in the maintenance of DA in the synaptic cleft. This DA is likely to reflect a somatodendritic release and participate to the SNpc cell firing feedback regulation (Cheramy et al., 1981). It is tempting to speculate that this event could in turn negatively regulate tyrosine hydroxylase phosphorylation. We propose that the decrease of DA contents detected in the Mecp2−/y brain by others (Ide et al., 2005; Samaco et al., 2009) could be due to an alteration of nigrostriatal neurons, given that there is a significant DA reduction in the CPu, a structure receiving the terminal endings of SNpc neurons. Interestingly, we were able to attenuate some of the motor deficits of the Mecp2−/y mice using L-Dopa, the better known of the molecules used to improve motor symptoms in Parkinson's disease. These improvements indicate that the DA nigrostriatal deficits play a role in the appearance of the motor deficits in the Mecp2-deficient animals. However, we cannot rule out the fact that other structures and neurotransmitter systems could also be involved given that motor strategies are not only elaborated and regulated by the basal ganglia, but also require brainstem and spinal networks (Kozlov et al., 2009). Taken together, our results support the notion that a lack of Mecp2 in the mouse leads to a global deficit in catecholamines and they demonstrate that DA neurons in the SNpc are affected in the Mecp2−/y mouse. However, the mechanism linking Mecp2-deficiency and the catecholaminergic metabolism has yet to be elucidated. A Mecp2binding site has been found in the Th promoter (Yasui et al., 2007), but functional studies are needed to further characterize this putative interaction. Based on our data, it seems that the absence of Mecp2 alters the SNpc dopaminergic system and contributes to the motor phenotype of the Mecp2-deficient animals. Alternatively, the defects observed in the SNpc could be due to the reduction of Bdnf levels previously described in the Mecp2-deficient animals (Wang et al., 2006). Indeed, Bdnf is a key factor for the survival and maintenance of mDA neurons (Oo et al., 2009). Although further studies are needed to explore these hypotheses, we believe that our data improve the knowledge of the pathophysiology of Mecp2-deficiency. We hope that they will also allow the development of alternative therapeutic strategies targeting the dopaminergic metabolism. Supplementary materials related to this article can be found online at doi:10.1016/j.nbd.2010.10.006. Acknowledgments This study was supported by INSERM, the Provence-Alpes-Côte d'Azur region, AFSR (Association Française du Syndrome de Rett), the E-rare EuroRett network, the Fondation Jérôme Lejeune and the DISCHROM project (FP7). We thank Dr. Sandrine Parrot and Dr. Luc

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