www.elsevier.com/locate/ynbdi Neurobiology of Disease 20 (2005) 93 – 103
Unraveling substantia nigra sequential gene expression in a progressive MPTP-lesioned macaque model of Parkinson’s disease F. Bassilana,a N. Mace,a Q. Li,b J.M. Stutzmann,a C.E. Gross,c L. Pradier,a J. Benavides,a J. Me´nager,a and E. Bezardc,T a
Sanofi-Aventis, Vitry sur Seine, France Lab Animal Research Center, China Agricultural University, Beijing, China c Basal Gang, CNRS UMR 5543, Universite´ Victor Segalen-Bordeaux 2, 146 rue Le´o Saignat, 33076 Bordeaux Cedex, France b
Received 4 November 2004; revised 24 January 2005; accepted 10 February 2005 Available online 24 March 2005
Taking advantage of a progressive nonhuman primate model mimicking Parkinson’s disease (PD) evolution, we monitored transcriptional fluctuations in the substantia nigra using Affymetrix microarrays in control (normal), saline-treated (normal), 6 days-treated (asymptomatic with 20% cell loss), 12 days-treated (asymptomatic with 40% cell loss) and 25 days-treated animals (fully parkinsonian with 85% cell loss). Two statistical methods were used to ascertain the regulation and real-time quantitative PCR was used to confirm their regulation. Surprisingly, the number of deregulated transcripts is limited at all time points and five clusters exhibiting different profiles were defined using a hierarchical clustering algorithm. Such profiles are likely to represent activation/deactivation of mechanisms of different nature. We briefly speculate about (i) the existence of yet unknown compensatory mechanisms is unraveled, (ii) the putative triggering of a developmental program in the mature brain in reaction to progressing degeneration and finally, (iii) the activation of mechanisms leading eventually to death in final stage. These data should help development of new therapeutic approaches either aimed at enhancing existing compensatory mechanisms or at protecting dopamine neurons. D 2005 Elsevier Inc. All rights reserved. Keywords: Microarray; Affymetrix; Nonhuman primate; RT-PCR; Neurodegeneration
Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder of which the principal pathological characteristic is the loss of dopamine (DA) neurons of the substantia nigra pars compacta (SNc) (Hassler, 1938). Parkinsonian signs appear when dopaminergic neuronal death exceeds a critical threshold: 70–80% of striatal nerve terminals and 50–60% of SNc pericarya T Corresponding author. Fax: +33 556901421. E-mail address:
[email protected] (E. Bezard). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2005.02.005
(Bernheimer et al., 1973). The nature of the etiology of the process underlying clinical deterioration remains unknown, but PD is characterized by its progressiveness (Hoehn and Yahr, 1967). Although cell death processes and compensatory mechanisms, responsible for the gradual appearance and worsening of clinical signs, are better understood through the identification of genetic mutations responsible for familial cases of PD (Dawson and Dawson, 2003b) and the use of neurotoxin-based animal models (Berg et al., 2001; Blum et al., 2001; Dawson and Dawson, 2003a; Giasson and Lee, 2001), our understanding of PD progression remains close to zero. Indeed, most experimental approaches have ignored the progressive nature of PD and have not taken into account the possibility that cell death mechanisms may vary accordingly to the extent of degeneration. Until recently, it has proved difficult to ascertain both the nature of such a sequence of events and its relationship with any subsequent plastic response of the SN. In the absence of animal models that replicate the progressive degenerative processes and the difficulty of longitudinal studies in man, it has been impossible to investigate such a question of paramount importance. Recently, an animal model of PD, which is able to address these issues, has been developed (Bezard et al., 2003). In this model, repeated administration of low doses of N-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the nonhuman primate (Macaca fascicularis) initiates a process of neurodegeneration reminiscent of that seen in PD (Bezard et al., 2001c, 2003). The novelty of the model accrues from the fact that, during the first 13–15 days of the protocol, despite significant dopaminergic loss, symptoms, are not apparent. During this period, several mechanisms compensate for the increasing loss of DA, suppressing the appearance of symptoms (Bezard et al., 1999, 2001a,b,c). The model is thus extremely attractive for defining neural mechanisms, and potential biomarkers, that relate to the progression of PD. High-density oligonucleotide arrays offer the opportunity to survey the transcriptional profiles of large numbers of genes and
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Material and methods
prefrontal cortex and substantia nigra (SN) were visually defined with aid of two stereotactic atlases (Martin and Bowden, 1996; Szabo and Cowan, 1984). Once dissected, structures were immediately placed in 10 volumes of RNAlater stabilization reagent. After incubation overnight in the reagent at 2 to 88C, the tissue was removed from the RNAlater stabilization reagent and transferred to 808C for storage. The present study reports the result obtained with the dorsal SN, i.e. mostly corresponding to the pars compacta of the SN (SNc) since SN was dissected in order to separate the dorsal from the ventral part. Thus, the material used for the present microarray experiments was enriched into DA neurons with less GABA neurons.
Animals
Behavioral assessment
Experiments were conducted on twenty-one female cynomolgus monkeys (M. fascicularis, CAS, Beijing, PR China; mean age = 3.0 F 0.2 years; mean weight = 2.8 F 0.2 kg). Animals were housed in individual primate cages under controlled conditions of humidity (50 F 5%), temperature (24 F 18C) and light (12h light/12h dark cycles, time lights on 8:00 am), food and water were available ad libitum and animal care was supervised by veterinarians skilled in the healthcare and maintenance of nonhuman primates. Experiments were carried out in accordance with European Communities Council Directive of 24 November 1986 (86/609/EEC) for care of laboratory animals. All efforts were made to minimize animal suffering and to use the minimum number of animals necessary to perform statistically valid analysis. To maximize data obtained from these animals, brain tissues acquired in the present experiment will be used for further experiments on the mechanism of the progressive nature of PD.
In order to follow the progression of the syndrome, animal behavior was assessed daily (2:00 p.m.) on a parkinsonian monkey rating scale using videotape recordings of monkeys in their cages and clinical neurological evaluation as previously described (Bezard et al., 2001c). For each group, however, the pertinent data are the assessments done the day of sacrifice. During each session, two examiners evaluated the animals’ levels of motor performance, coaxing them to perform various tasks by offering appetizing fruits. A third examiner, watching a video recording, made an independent and blind assessment. The minimum disability score was 0 and the maximum score was 25 (Bezard et al., 2001c).
allow complex biology especially as it relates to disease processes, making possible the unraveling of the basis of PD progression (Marvanova et al., 2003). In this model-driven study, in contrast to hypothesis-driven experiments, we have taken advantage of our progressive nonhuman primate paradigm to monitor transcriptional fluctuations in the SN using human Affymetrix microarray technology, previously shown to display statistically satisfactory human and macaca cross-hybridization properties (Marvanova et al., 2003).
Experimental protocol Three untreated monkeys were killed at the end of the study and were termed controls. Three animals were treated daily for 15 days with saline, that is, they received the same number of injection as that used to induce parkinsonism, and were termed saline group. The remaining 15 monkeys were treated daily (9:00 am) with MPTP hydrochloride (0.2 mg/kg, i.v., Sigma, St. Louis, MO) dissolved in saline according to a previously described protocol (Bezard et al., 1997, 2001c). This protocol describes a reproducible MPTP cumulative dosing regime that leads to the first appearance of parkinsonian clinical signs after 15 F 1 injections (i.e. a cumulative dose of 3.0 F 0.2 mg/kg) (Bezard et al., 1997, 2001c). Five presymptomatic monkeys were killed at day 6 (i.e. after 6 injections; D6 group), five presymptomatic monkeys at day 12 (i.e. after 12 injections; D12 group) and the remaining five fully parkinsonian monkeys at day 25 (i.e. after 15 injections and 10 days of symptom progression and stabilization; D25 group). All animals were killed by sodium pentobarbital overdose (150 mg/kg, i.v.) and the brains were removed quickly after death. Each hemisphere was immediately sliced into 1 mm-thick sections on a refrigerated table and dipped into RNAlater RNA Stabilization Reagent (Qiagen). These sections were then microdissected (by E.B.) using microsurgical tools under binocular magnifying glasses from the most rostral to the most caudal sections. Boundaries of structures, caudate nucleus, putamen, subthalamic nucleus, globus pallidus pars externalis and pars internalis, motor thalamic nuclei, cerebellar cortex, motor cortex, supplementary motor area,
RNA preparation Dorsal SN samples were homogenized in QIAzol Lysis Reagent (Qiagen, Hilden, Germany) (1 ml/100 mg tissue). Total RNA was isolated using RNeasy Mini Kit (Qiagen) and its quality and concentration were directly analyzed on an RNA LabChip Agilent using the 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany). Microarray procedure The probe synthesis (Affymetrix, Inc.) was performed according to the standard protocol outlined in the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, USA). Ten micrograms of Total RNA was converted to cDNA by using SuperScript Choice System (Invitrogen Life Technologies, Carisbad, USA), with T7-(dT)24 oligomer (Proligo Paris, France). Second-strand synthesis was performed using T4 DNA polymerase, and cDNA was isolated by phenol/chloroform extraction with Phase Lock Gel (Eppendorf, Hamburg, Germany) and concentrated by ethanol precipitation. Three independent in vitro transcriptions (IVT) using the same cDNA were performed to produce biotin-labeled cRNA using a BioArray HighYield RNA Transcript Labeling Kit (Affymetrix). Biotinylated cRNAs were pooled, cleaned up by an RNeasy Mini Kit column (Qiagen) and then fragmented according to the Affymetrix protocol, giving biotinylated cRNA fragments of 50–200 bp in length. Fifteen micrograms of randomly fragmented and biotin-labeled cRNA was hybridized to a human U133A microarray (Affymetrix) for 16 h at 458C with constant rotation at 60 rpm. The array was then automatically washed and stained with streptavidin–phycoerithrin conjugate (Molecular Probes, Eurogene, OR, USA) on an Affymetrix fluidics station. Finally, the probe array was scanned
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at 3-Am resolution using the GeneChip System confocal scanner (Hewlett-Packard, Santa Clara, CA) with a 488-nm emission and detection at 570 nm, controlled by Affymetrix Microarray Suite 5.0 (Affymetrix). Real-time PCR 2 Ag of total RNA from each group was reverse transcribed with oligo(dT)16 (Genset) following the Applied Biosystems RT reaction procedure. The final reverse transcription reaction included template, 1 cDNA first-strand synthesis buffer, 5.5 mM of MgCl2, 0.5 mM of each dNTP, 0.4 U/Al of RNAse inhibitor, 2.5 AM of oligo(dT) and 1.25 U/AL Multiscribe reverse transcriptase was brought to 100 Al with water. Samples were then incubated for 10 min at 258C, followed by 30 min at 488C and then heated at 958C to denature the enzymes and stop the reaction. Gene-specific primers corresponding to the gene represented on the chips were designed using Vector NTI software (Informax, North Bethesda, USA). Primers amplified fragments of 100 to 200 bp. FastStart DNA Master SYBR Green I mix (Roche diagnostics, Mannheim, Germany) was mixed with 1 Al of cDNA and primers at 0.4 AM in a final volume of 20 Al and amplified. A negative control without cDNA template was run with every assay to assess the overall specificity. The PCR was run on a LightCycler (Roche diagnostics) as follows: 1 cycle of 958C for 8 min, followed by 40 cycles of 958C for 15 s, 658C for 10 s and 728C for 10 s. This was followed by melt curve analysis beginning at 658C and increasing by 0.18C/s to 958C. None of the primer pairs demonstrated more than one peak of fluorescence, indicating a single gene product without primer–dimer formation. Housekeeping genes were used to control RNA and check that they were detected at the same level in each group. The mean housekeeping gene concentration was determined once for each cDNA sample and used to normalize all other genes tested from the same cDNA sample. Analysis Raw data are available for download in a MIAME-compatible format at http://www.neurophy.u-bordeaux2.fr/sn_microarray. Data normalization and processing Each microarray was standardized according to the lowest sum of present values. The expression threshold is the 99th percentile of all expression values of the absent calls throughout all microarrays, which is 250. All absent calls expression values were set to zero for subsequent steps and data mining. To estimate false positive expression ratios, we have calculated for each qualifier, the ratio of the expression value to the value for one of its replicates. Since there were three replicates per condition, three ratios were calculated per treatment group. Again, the 99th percentile (1.6) was taken as the threshold. Selection criteria Parametric approach: All analyses were set with a V 5%. With the triplicates of the five conditions, we have performed a one way ANOVA test. Using the 15 values’ residues, normality was assessed using a Shapiro–Francia test. To assess homogeneity of variances, we used a Cochran test. Only qualifiers that passed the three tests were considered in the parametric analysis. When significant, a post hoc analysis using the Dunnett’s test was carried out using the absolute control condition as reference. Non-
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parametric approach: As a complementary approach to parametric analysis and since not all the aforementioned values were considered as having a normal distribution, we also used a Kruskal–Wallis (KW) test. Non-Gaussian regulation of transcription is classically not taken into account in microarray experiments but still exists requiring combination of statistical approaches to ensure complete survey of transcription regulation. User defined supplement conditions: To exclude false positives, values for which the absolute control condition and the saline condition were significantly different in the Dunnett’s test were excluded. For the KW test, the following condition was added: when a ratio A/B was calculated with A N B then the median of A should be 1.6 fold B’s maximal value. On top of that, any MPTP treated group should be at least 1.6 fold of one of the 2 controls (absolute or saline) and at least 1.4 fold of the other. Assessment of present qualifiers: Each condition was performed as a triplicate. We have considered as bpresentQ the qualifiers that were given a present call according to Affymetrix software in all three replicates. Databases used Annotation using Ensembl: In order to be able to compare our dataset to others, we have used the EBI’s Affymetrix Ensembl annotation (Kasprzyk et al., 2004). Pathways using Locuslink: We have linked the Unigene accession number given in the Affymetrix table to the ones contained in the Locuslink database (http:// www.ncbi.nlm.nih.gov/LocusLink/). This allowed retrieval of complementary information like the Gene Ontology used in an attempt to understand metabolic pathways.
Results Changes in motor behavior Changes in motor behavior were fully comparable to those previously reported with this administration protocol (Bezard et al., 2001c). Monkeys at day 6 (D6; n = 5) and day 12 (D12; n = 5) did not exhibit parkinsonian motor symptoms (parkinsonian score of 0 for all animals at both time points; P N 0.5 compared to both control and saline groups). Both the D6 and D12 groups were thus asymptomatic. Monkeys in the day 25 (D25; n = 5) group exhibited parkinsonian motor abnormalities as reflected by their clinical score (median 17 (range 14–18)). Monkeys were bradykinetic, adopting a flexed posture, with increased rigidity of the limbs and decreased vocalization. Their movements were less accurate, for example when reaching for fruit, and there were occasional episodes of freezing. Both postural tremor and some resting tremor were observed. Cross-hybridization ratio Out of the over 22,000 qualifiers comprised in the U133A Affymetrix microarray, 5251 (23%) were considered bpresentQ in the control group (see http://www.neurophy.u-bordeaux2.fr/ sn_microarray for raw data) The same experiment carried out with commercial whole human brain RNA yielded 9601 (43%) present qualifiers. The ratio M. fascicularis versus Homo sapiens was therefore about 55%, which is in the range of our previous report (Marvanova et al., 2003). After applying the very stringent threshold for the minimum expression value (which is 250), 2640 qualifiers remain.
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Methods The present study used 2 reference groups, i.e. control (nontreated; n = 3) and saline-treated (n = 3) animals. It is well known that simple manipulation of animals can affect gene expression, especially in models of chronic diseases. We thus used these two reference groups to exclude false positives caused by the experimental design. Thus, all qualifiers for which values were different between the control and saline-treated groups, as determined by post-hoc analysis following the ANOVA, were excluded from the final analysis. Overview of biological event’s Seventy-four regulated transcripts were selected using ANOVA followed by the 1.6 fold criterion, while 83 qualifiers were selected using the Kruskal–Wallis test (KW) followed by a median to maximum ratio. It is worth noting that only 41 were common to both approaches, which highlights complementarity of these approaches. Indeed, while ANOVA is more powerful, the KW is more flexible regarding normality or differences in group variances. For further analyses, these qualifiers were pooled. Some transcripts were represented by many qualifiers on Affymetrix microarray. This redundancy can be a positive factor because it provides additional replicates. It can also induce disequilibria sometimes, when comparing numbers of up and down-regulated transcripts. We have used the transcripts’ annotation to reduce redundancy by grouping qualifiers that correspond to the same genes. This step reduces the total number of regulated transcripts to 101 (Tables 1–6). Non-linear evolution over time The analysis does not display an increasing quantity of deregulated transcripts with the progression of the syndrome. Instead, we observe a two-step transcriptional regulation. There is an early response to degeneration after 6 days (i.e. after 20% cell loss) with 14 down-regulated and 25 up-regulated transcripts. Then, the situation seems to reach a steady state as reflected by the smaller amount of qualifiers regulated at 12 days (i.e. after 40% cell loss) with 7 down-regulated and 17 up-regulated transcripts. At 25 days, when at least 85% of neuronal degeneration has occurred (Bezard et al., 2001c), we observe another large transcriptional deregulation with 31 down-regulated and 24 up-regulated transcripts. This biphasic evolution that is dissociated from the progression of neuronal death in this model (Bezard et al., 2001c) could reflect a complex sequence of intricate events including responses to ongoing neurodegeneration, enhancement of compensatory mechanisms (Bezard et al., 2003), compensation attempts by the remaining neurons and neocolonization of SN by glial cells. Different time courses The time course of transcriptional regulation is more revealing. The 116 qualifiers were normalized using a z score calculation. The hierarchical clustering algorithm used was Ward’s method, using the half square Euclidian distance. It is worth noticing that we have used different methods as well as different distance calculations and that we obtained very similar results. This representation displays five main clusters (Fig. 1) that can be represented, in the same order, i.e. from top to bottom of Fig. 1, by 5 profiles (Fig. 2).
Four profiles, i.e. 1, 3, 4 and 5, display a non-time-dependent shape. Indeed, up or down-regulated transcriptions occur only at a given time point without pre- or post-deregulation (Fig. 2). Profile 1 is characterized by an increased expression of its members at day 6, such as T-cell-factor-7-like-2 (Tcf7l2), suggestive of an attempt to protect the surviving neurons by overexpressing protective factors. Profile 3 shows massive up-regulation at day 25, suggesting a compensatory role for some of those transcripts such as cyclic AMPregulated phosphoprotein (M(r) = 21,000; ARPP-21), or a deleterious role for some others such as GPR37 commonly named Pael receptor (Pael-R). Profile 4 involves transcripts massively downregulated at 25 days, which may reflect the loss of 85% of DA neurons (Bezard et al., 2001c). It is striking that this profile remains basically unaffected in D6 and D12 groups in spite of degeneration that reaches 20% and 40% of DA neurons, respectively (Bezard et al., 2001c). In this cluster, we found for instance the dopamine transporter (DAT), the vesicular monoamine transporter 2 (VMAT2) and the nuclear receptor nurr-1 (NR4A2). On the other hand, other down-regulated transcripts such as Heat shock 70 kDa protein (Hsp70) may reflect an ongoing deleterious mechanism in surviving neurons. Profile 5 is constituted of transcripts massively downregulated at 6 and 12 days and still down-regulated, although less dramatically, at 25 days. In this cluster, we found, for instance, the slit homolog 2 protein precursor (SLIT2). Given the role in axon guidance and pruning of SLIT2, such an expression profile suggests a compensatory role by decreasing the synthesis of chemorepellents in the SN. Profile 2, however, exhibits different behavior (Fig. 2). There is a positive progression in transcriptional regulation up to day 12, followed by a decay at 25 days. This kind of biphasic response suggests the existence of some regulatory mechanism during the degeneration process up to symptom appearance. It is suggestive of an adaptive response by remaining neurons suggesting that tyrosine hydroxylase (TH) or Fatty Acid Binding Protein-3 (FABP3) overexpression have a compensatory role before symptom appearance. Real-time PCR confirmation Generating microarray data involves multiple steps both in vitro and in silico that may lead to a variable number of false positive results. Thus, if interpreting a fairly large number of regulated transcripts is reasonable, deriving hypotheses from single observations could be more risky. We have therefore performed real-time PCR experiments on transcripts representative of their cluster (Fig. 3) to validate the in silico observations. Taking into account both the little sequence information available for M. fascicularis and its genomic proximity to man, we have designed PCR primers from human transcripts when necessary. To increase the probability of success, these were designed in the coding regions. All transcripts so far mentioned and further discussed (see Fig. 4 for instance) display excellent correlation between microarray and real-time PCR expression changes.
Discussion The present work relates to the relationship between the transcription changes in the SN and the level of nigrostriatal degeneration in a progressive MPTP-lesioned macaque model of PD. Five clusters can be described as five temporal profiles that are
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Table 1 Up-regulated transcripts in D6 group AffyID
Article annotation
Gene/transcript
206373_at 209660_at 212761_at 216037_x_at 216035_x_at 212762_s_at 208672_s_at 205751_at 202483_s_at 203997_at 205336_at 219032_x_at 209159_s_at 209116_x_at 217232_x_at 211696_x_at 200745_s_at 217077_s_at 203865_s_at 205630_at 212953_x_at 205549_at 207853_s_at 221217_s_at 200708_at 217414_x_at 211699_x_at 209458_x_at 211745_x_at 214414_x_at 204018_x_at 200010_at 221691_x_at 222333_at 221805_at
ZINC FINGER PROTEIN ZIC 1 TRANSTHYRETIN PRECURSOR TRANSCRIPTION FACTOR 7-LIKE 2
ENSG00000152977 ENSG00000118271 ENSG00000148737
SPLICING FACTOR, ARGININE/SERINE-RICH 3 SH3-CONTAINING GRB2-LIKE PROTEIN 2 RAN-SPECIFIC GTPASE-ACTIVATING PROTEIN PROTEIN TYROSINE PHOSPHATASE, NON-RECEPTOR TYPE 3 PARVALBUMIN ALPHA OPSIN 3 NDRG4PROTEIN HEMOGLOBIN GAMMA-A AND GAMMA-G CHAINS
ENSG00000112081 ENSG00000107295 ENSG00000099901 ENSG00000070159 ENSG00000100362 ENSG00000054277 ENSG00000103034 ENSG00000161280
GUANINE NUCLEOTIDE-BINDING PROTEIN G(I)/G(S)/G(T) BETA SUBUNIT 1 GAMMA-AMINOBUTYRIC ACID TYPE B RECEPTOR, SUBUNIT 2 PRECURSOR DOUBLE-STRANDED RNA-SPECIFIC EDITASE 1 CORTICOLIBERIN PRECURSOR CALRETICULIN PRECURSOR BRAIN-SPECIFIC POLYPEPTIDE PEP-19 BETA-SYNUCLEIN ATAXIN-2 BINDING PROTEIN ASPARTATE AMINOTRANSFERASE, MITOCHONDRIAL PRECURSOR ALPHA 2 GLOBIN
ENSG00000078369 ENSG00000136928 ENSG00000014442 ENSG00000147571 ENSG00000179218 ENSG00000183036 ENSG00000074317 ENSG00000078328 ENSG00000125166 ENSG00000130654
60 S RIBOSOMAL PROTEIN L11 Nucleophosmin B23.2 EST Neurofilament, light polypeptide 68 kDa
ENSG00000142676 ENSG00000182298 ENSG00000178038 NM_006158
likely to represent activation/deactivation of mechanisms of different nature. We briefly speculate about (i) the existence of yet unknown compensatory mechanisms is unraveled, (ii) the putative triggering of a developmental program in the mature brain in reaction to progressing degeneration and finally, (iii) the activation of mechanisms leading eventually to death in final stage.
To fully understand the present results, it is of paramount importance to remember that, in our progressive MPTP-lesioned macaque model, the kinetics of the decrease of DA neurons in the SNc follow a linear regression while that of striatal dopaminergic denervation follows an exponential regression (Bezard et al., 2001c). Thus, the D6 and the D12 animals, the extent of the SNc
Table 2 Down-regulated transcripts in D6 group AffyID
Description
Gene/transcript
209897_s_at 217864_s_at 212226_s_at 205094_at 205882_x_at 210949_s_at 210736_x_at 214334_x_at 203773_x_at 200027_at 212653_s_at 202615_at 213156_at 204073_s_at
SLIT HOMOLOG 2 PROTEIN PRECURSOR PROTEIN INHIBITOR OF ACTIVATED STAT PROTEIN 1 PHOSPHATIDIC ACID PHOSPHATASE TYPE 2B PEROXISOME ASSEMBLY PROTEIN 12 GAMMA ADDUCIN EUKARYOTIC TRANSLATION INITIATION FACTOR 3 SUBUNIT 8 DYSTROBREVIN ALPHA DAZ ASSOCIATED PROTEIN 2 BILIVERDIN REDUCTASE A PRECURSOR ASPARAGINYL-TRNA SYNTHETASE, CYTOPLASMIC KIAA0903 protein, partial cds Guanine nucleotide binding protein (G protein), q polypeptide EST Chromosome 11 open reading frame 9
ENSG00000145147 ENSG00000033800 ENSG00000162407 ENSG00000108733 ENSG00000148700 ENSG00000179927 ENSG00000134769 ENSG00000183283 ENSG00000106605 ENSG00000134440 ENSG00000115504 NM_002072 BG251521 ENSG00000124920
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Table 3 Up-regulated transcripts in D12 group AffyID
Article annotation
Gene/Transcript
208291_s_at 212309_at 201670_s_at 201627_s_at 205738_s_at 201435_s_at 212953_x_at 201128_s_at 200708_at 200010_at 210460_s_at 203895_at 200641_s_at 200638_s_at 219798_s_at 221750_at 212281_s_at 219549_s_at
TYROSINE 3-MONOOXYGENASE SIMILAR TO CLIP-ASSOCIATING PROTEIN 2 MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE INSULIN-INDUCED PROTEIN 1 FATTY ACID BINDING PROTEIN, HEART EUKARYOTIC TRANSLATION INITIATION FACTOR 4E CALRETICULIN PRECURSOR ATP-CITRATE SYNTHASE ASPARTATE AMINOTRANSFERASE, MITOCHONDRIAL PRECURSOR 60 S RIBOSOMAL PROTEIN L11 26 S PROTEASOME NON-ATPASE REGULATORY SUBUNIT 4 1-PHOSPHATIDYLINOSITOL-4,5-BISPHOSPHATE PHOSPHODIESTERASE BETA 4 14-3-3 PROTEIN ZETA/DELTA
ENSG00000180176 ENSG00000163539 ENSG00000155130 ENSG00000186480 ENSG00000121769 ENSG00000151247 ENSG00000179218 ENSG00000131473 ENSG00000125166 ENSG00000142676 ENSG00000159352 ENSG00000101333 ENSG00000164924
Hypothetical protein FLJ20257, mRNA 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (soluble) EST Reticulon 3 (RTN3)
ENSG00000146834 BG035985 ENSG00000109084 ENSG00000180519
lesion reaches 20 and 40%, respectively. In the D25 group, degeneration is 85% in the SNc (Bezard et al., 2001c). This is pivotal in so much as a stable transcription level at D12 for instance, should actually correspond to an up-regulation, since the number of neurons has diminished. One caveat of the study, however, is that we cannot completely dissect SNc from SNr neurons. Thus, a change of expression might occur in the GABAergic cells of the SNr as well or in glial cells. The SN has however been dissected in order to study the dorsal part, i.e. the area that contains the DA cells. The ratio of DA neurons versus GABA neurons is in favor of the DA neurons. Among the regulated transcripts, three are hallmarks of PD. Those transcripts are DAT, VMAT2 and TH. As expected, these three transcripts are strongly down-regulated in the D25 group (Table 6). Such a dramatic down-regulation most probably indicates the disappearance of SNc neurons (Bezard et al., 2001c). However, it is interesting to observe that TH is upregulated in the D12 group (Fig. 3), suggesting that surviving DA neurons undergo functional changes aimed at preserving DA release in the striatum (Zigmond et al., 1990). Interestingly, while such an adaptive mechanism was thought to be unlikely from studies using standard techniques (Bezard et al., 2001c, 2003), we here demonstrate that subtle changes occur before symptom appearance, but not very early in the process since D6 animals do not display comparable modifications. In keeping with this upregulation of TH at day 12 is the regulation of nurr-1, for which a
linkage with a familial form of PD has been shown (Le et al., 2003). Although its expression is down-regulated in the D25 group (Fig. 3), no up-regulation is observed at day 12 as for TH. Interestingly, nurr-1 has been shown to directly trans-activate the TH promoter (Kim et al., 2003). As evoked above, stable expression in a system where fewer neurons are able to express a marker would actually correspond to a relative increase in expression of that marker. Thus, the increase in TH expression may, at least in part, be triggered by the relative increase in nurr-1 transcription. Although quite speculative since no in situ evidence is yet available (but real-time PCR confirmation has been obtained for all discussed transcripts), we would like to propose that a sequence of events takes place in the course of neurodegeneration (Fig. 4). Such events could be classified according to the potential functional outcome of their regulation. For instance, other transcripts might participate to compensation during the presymptomatic period. Among those, the Fatty Acid Binding Protein-3 (FABP3) is a good candidate since it is known to be able to promote D2 receptor internalization (Takeuchi and Fukunaga, 2003). Interestingly, FABP3 expression is maximal at day 12 (Table 3, Fig. 3), at a time point where D2 binding is decreased in the striatum of monkeys treated according to the same MPTP regimen (Bezard et al., 2001c). Decreased auto-inhibition of DA neurons could thus be mediated by increased expression of FABP3. When considering these results altogether, i.e. the increase in TH
Table 4 Down-regulated transcripts in D12 group AffyID
Description
Gene/Transcript
209897_s_at 217864_s_at 211696_x_at 217232_x_at 213515_x_at 210736_x_at 217414_x_at 221476_s_at 204073_s_at
SLIT HOMOLOG 2 PROTEIN PRECURSOR PROTEIN INHIBITOR OF ACTIVATED STAT PROTEIN 1 HEMOGLOBIN GAMMA-A AND GAMMA-G CHAINS
ENSG00000145147 ENSG00000033800 ENSG00000161280
DYSTROBREVIN ALPHA ALPHA 2 GLOBIN 60 S RIBOSOMAL PROTEIN L15 Chromosome 11open reading frame 9
ENSG00000134769 ENSG00000130654 ENSG00000174748 ENSG00000124920
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Table 5 Up-regulated transcripts in D25 group AffyID
Article annotation
Gene/Transcript
218495_at 204367_at 201698_s_at 202457_s_at 210039_s_at 206552_s_at 213791_at 209233_at 209631_s_at 204081_at 215707_s_at 207157_s_at 220359_s_at 202742_s_at 221217_s_at 211699_x_at 211745_x_at 210517_s_at 200010_at 200639_s_at 221691_x_at 220122_at 219449_s_at 204155_s_at 217819_at
UXT PROTEIN TRANSCRIPTION FACTOR SP2 SPLICING FACTOR, ARGININE/SERINE-RICH 9 SERINE/THREONINE PROTEIN PHOSPHATASE 2B CATALYTIC SUBUNIT, ALPHA PROTEIN KINASE C, THETA TYPE PROTACHYKININ 1 PRECURSOR PROENKEPHALIN A PRECURSOR PROBABLE RIBOSOME BIOGENESIS PROTEIN NEP1 PROBABLE G PROTEIN-COUPLED RECEPTOR GPR37 PRECURSOR NEUROGRANIN MAJOR PRION PROTEIN PRECURSOR GUANINE NUCLEOTIDE-BINDING PROTEIN G(I)/G(S)/G(O) GAMMA-5 SUBUNIT cAMP-REGULATED PHOSPHOPROTEIN 21 cAMP-DEPENDENT PROTEIN KINASE, BETA-CATALYTIC SUBUNIT ATAXIN-2 BINDING PROTEIN
ENSG00000126756 ENSG00000167182 ENSG00000111786 ENSG00000138814 ENSG00000065675 ENSG00000006128 ENSG00000181195 ENSG00000126749 ENSG00000170775 ENSG00000154146 ENSG00000171867 ENSG00000174021 ENSG00000172995 ENSG00000142875 ENSG00000078328
ALPHA 2 GLOBIN A-KINASE ANCHOR PROTEIN 12 60 S RIBOSOMAL PROTEIN L11 14-3-3 PROTEIN ZETA/DELTA Nucleophosmin B23.2 Hypothetical protein FLJ22344, mRNA Hypothetical protein FLJ20533, mRNA EST HSPC041 protein (LOC51125), mRNA
ENSG00000130654 ENSG00000131016 ENSG00000142676 ENSG00000164924 ENSG00000182298 ENSG00000175471 ENSG00000175606 NM_025164 ENSG00000147533
Table 6 Down-regulated transcripts in D25 group AffyID
Description
Gene/Transcript
208738_x_at 208291_s_at 204141_at 209372_x_at 212083_at 205857_at 206836_at 209897_s_at 204337_at 202361_at 204622_x_at 216248_s_at 216963_s_at 221011_s_at 218118_s_at 204454_at 200697_at 214434_at 205110_s_at 210949_s_at 218100_s_at 217838_s_at 203408_s_at 209560_s_at 200613_at 205294_at 212224_at 205378_s_at 221796_at 212148_at 212151_at 213242_x_at
UBIQUITIN-LIKE PROTEIN SMT3B TYROSINE 3-MONOOXYGENASE TUBULIN BETA-2 CHAIN
ENSG00000180283 ENSG00000180176 ENSG00000137267
TESTIS EXPRESSED GENE 261 SYNAPTIC VESICLE AMINE TRANSPORTER SODIUM-DEPENDENT DOPAMINE TRANSPORTER SLIT HOMOLOG 2 PROTEIN PRECURSOR REGULATOR OF G-PROTEIN SIGNALING 4 PROTEIN TRANSPORT PROTEIN SEC24C ORPHAN NUCLEAR RECEPTOR NURR-1
ENSG00000144043 ENSG00000165646 ENSG00000142319 ENSG00000145147 ENSG00000117152 ENSG00000148571 ENSG00000153234
NEUROMODULIN MSTP014 MITOCHONDRIAL IMPORT INNER MEMBRANE TRANSLOCASE SUBUNIT TIM23 LDOC1 PROTEIN HEXOKINASE, TYPE I HEAT SHOCK 70 kDa PROTEIN 12A FIBROBLAST GROWTH FACTOR-13 EUKARYOTIC TRANSLATION INITIATION FACTOR 3 SUBUNIT 8 ESTROGEN-RELATED RECEPTOR BETA LIKE 1 ENA/VASODILATOR STIMULATED PHOSPHOPROTEIN-LIKE PROTEIN DNA-BINDING PROTEIN SATB1 DELTA-LIKE PROTEIN PRECURSOR CLATHRIN COAT ASSEMBLY PROTEIN AP50 BAI1-ASSOCIATED PROTEIN 2 ISOFORM 3 ALDEHYDE DEHYDROGENASE 1A1 ACETYLCHOLINESTERASE PRECURSOR EST EST EST KIAA0284 protein, partial cds
ENSG00000172020 ENSG00000185082 ENSG00000138297 ENSG00000182195 ENSG00000156515 ENSG00000165868 ENSG00000129682 ENSG00000179927 ENSG00000114446 ENSG00000089465 ENSG00000182568 ENSG00000185559 ENSG00000161203 ENSG00000175866 ENSG00000165092 ENSG00000087085 AA707199 AL049381 BF967998 ENSG00000099814
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Fig. 1. Hierarchical clustering of ANOVA positive transcripts. Maximal (red) and minimal (green) values of z scores for the heat map are respectively 2 and
and FABP3 expression and the relative increase in nurr-1 expression at day 12, it suggests that DA neurons undergo compensatory changes leading to increased functional DA activity that has not yet been demonstrated. Certain factors involved in maintenance of function, survival or promotion of differentiation of DA neurons are regulated during the presymptomatic period. Together with Pitx3 or Lmx1b, nurr-1 is a key player in SNc neuron differentiation (Smidt et al., 2003) and maintenance of function (Le et al., 1999). Interestingly, diminishing nurr-1 transcriptional expression leads to increased sensitivity to MPTP intoxication (Le et al., 1999). Thus, relative increase in nurr-1 level per surviving neuron would have two roles, i.e. promoting TH activity and bprotectingQ from MPTP intoxication, resulting in a delay of symptom appearance. Surprisingly, the injured SNc seems to react in the first place by going back to a non-mature state (Fig. 4), if not a developmental state, as suggested by the regulation of nurr-1 and Tcf7l2, a member of the Wnt family proteins that play a critical role in development and oncogenesis and have been shown to affect proliferation and differentiation of rat ventral midbrain dopaminergic neurons (Castelo-Branco et al., 2003). This is further supported by the profile 5-prototypical transcript SLIT2 that belongs to a family of secreted chemorepellents and contributes to the maintenance of dorsal position by prevention of axonal growth into ventral regions, the prevention of axonal extension toward and across the midline and the channeling of axons toward particular regions (Bagri et al., 2002). Dramatic decrease in SLIT2, especially in the presymptomatic period, i.e. at day 6 and day 12, would remove this inhibitory tone. SN neurons are thus in a position to be (re)-innervated by new afferents.
2.
The abovementioned genes are all involved in putative positive responses of SNc neurons aimed at either compensating functionally for the loss of other DA neurons or delaying their own neuronal death at different time points. However, the same neurons might also trigger pathological mechanisms leading eventually to their death, though at later time points in the process. For example, we report an overexpression of GPR37, also known as Pael receptor, in D25 animals (Table 5). Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations of the parkin gene and Pael-R, a parkin-binding protein (Imai et al., 2001) highly expressed in SNc neurons, accumulates in the brains of AR-JP patients (Murakami et al., 2004). This, associated with the present discovery of an increased Pael-R expression, strongly suggests that accumulation of unfolded Pael-R may lead to selective death of DA neurons in PD and that the later event in MPTP-induced neuronal death is an ER stress. Recently, Heat shock 70 kDa protein (Hsp70) and a co-chaperone have been identified as novel parkin-binding partners and shown to enhance the ability of parkin to inhibit cell death induced by Pael-R (Imai et al., 2003). Interestingly, we here show that Hsp70 expression is down-regulated in D25 animals (Table 6, Fig. 4), further supporting the possibility of a pivotal role for Pael-R accumulation in the late stage of MPTPinduced neuronal death. In keeping with the ER stress hypothesis raised by Pael-R overexpression is the discovery that ataxin-2 binding protein (A2BP) expression is increased in day 6 and day 25 animals (Tables 1 and 5). A2BP is a protein known to be involved in type 2 spinocerebellar ataxia (SCA2), a neurodegenerative condition caused by an unstable trinucleotide
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slightly below 1.6 fold. One could however note that this transcript meets all the other criteria necessary for ANOVA selection. This only shows that one should not be too strict when deciding on a cut-off (see http://www.neurophy.u-bordeaux2.fr/ sn _ microarray for raw data). The transcript-by-transcript approach can probably stand looser parameters. The present results are thus supportive of the generally held belief that model systems recapitulate human disease, albeit in a com-
Fig. 2. Profiles of hierarchically identified clusters. Those profiles are organized from top to bottom in the same order as the clusters of Fig. 1.
repeat (polyQ) in the protein (Klockgether and Evert, 1998), and recently identified as a minor cause of familial parkinsonism (Lu et al., 2004). The present results suggest a link between PD and SCA2 and raise the possibility of a relationship between A2BP up-regulation, ER stress and SNc neuronal death. Of course, other transcripts known to play a role in PD are present on the microarrays and may be regulated, but absent from our list. In fact, alpha synuclein (204466_s_at) is not in the list generated by the automated process. The reason is that its up-regulation is
Fig. 3. Examples of real-time PCR confirmation of transcriptional regulation observed with the microarray experiment. Values are log ratios of the different time points compared to the control group.
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Fig. 4. Schematic diagram illustrating the posited sequential transcriptional activation of different programs in relation with the gradual process of nigral degeneration in the course of the experimentally induced parkinsonism (adapted from Bezard et al., 2001c). The cell counting maps show the typical patterns of degeneration in the SNc. Tyrosine hydroxylase (TH) positive neurons (i.e. DA neurons) are marked in red whereas the blue symbols represent the Nissl-stained cells which were not TH-positive. The horizontal line above/below each map indicates the mean percentage of TH-positive cells. The colored arrows indicate the time point from which a specific transcript is up- or down-regulated, the color referring to a family of mechanisms. The originality of the present study is to suggest that, besides classic functional compensation, the SNc neurons undergo plastic changes during the presymptomatic period to delay neuronal death by going back to a developmental state. However, once degeneration is extreme, mechanisms enhancing neuronal death are promoted. Changes in expression of all these transcripts have been confirmed by real-time PCR.
pressed time frame, since our studies are able to detect gene expression changes consistent with existing literature from the study of human PD. Surprisingly, a limited number of studies have used the MPTP mouse model of PD to investigate dysregulation of gene expression in the SN (e.g. Grunblatt et al., 2001; Miller et al., 2004). A single study can be compared to the present work in that special care was paid to only dissect the nigra and that early versus late phase of MPTP intoxication was investigated (Miller et al., 2004). Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity and cell cycle and apoptosis (Miller et al., 2004), thereby confirming our data. However, these authors investigated mostly the very early MPTP-induced changes since 7 days after the last MPTP injection, the number of TH-positive neurons was only decreased by 35%. MPTP-induced neuronal death taking 5 days to be complete in the mouse model (JacksonLewis et al., 1995) suggests that the changes they measured correspond to a period anterior to our D6 group. Still, besides the abovementioned global changes, a significant number of genes involved in cell proliferation and developmental processes were found to be dysregulated at this early stage (Miller et al., 2004). We posit, at the light of our results, that SN neurons might also positively respond to MPTP injury by triggering proliferation or developmental processes. Further studies, such as double in situ hybridization, are now needed to confirm the changes of expression of the identified targets in DA or GABA neurons. Confirmation of those later,
either as compensatory or delaying/enhancing neuronal death mechanisms in a clinically relevant experimental design, should help to develop new therapeutic approaches either aimed at enhancing existing compensatory mechanisms (Bezard et al., 2003) or at neuroprotection of remaining DA neurons (Meissner et al., 2004). References Bagri, A., Marin, O., Plump, A.S., Mak, J., Pleasure, S.J., Rubenstein, J.L., Tessier-Lavigne, M., 2002. Slit proteins prevent midline crossing and determine the dorsoventral position of major axonal pathways in the mammalian forebrain. Neuron 33, 233 – 248. Berg, D., Gerlach, M., Youdim, MB., Double, KL., Zecca, L., Riederer, P., Becker, G., 2001. Brain iron pathways and their relevance to Parkinson’s disease. J. Neurochem. 79, 225 – 236. Bernheimer, H., Birkmayer, W., Hornykiewicz, O., Jellinger, K., Seitelberger, F., 1973. Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J. Neurol. Sci. 20, 415 – 455. Bezard, E., Imbert, C., Deloire, X., Bioulac, B., Gross, C.E., 1997. A chronic MPTP model reproducing the slow evolution of Parkinson’s disease: evolution of motor symptoms in the monkey. Brain Res. 766, 107 – 112. Bezard, E., Boraud, T., Bioulac, B., Gross, C.E., 1999. Involvement of the subthalamic nucleus in glutamatergic compensatory mechanisms. Eur. J. Neurosci. 11, 2167 – 2170. Bezard, E., Crossman, A.R., Gross, C.E., Brotchie, J.M., 2001a. Structures outside the basal ganglia may compensate for dopamine
F. Bassilana et al. / Neurobiology of Disease 20 (2005) 93–103 loss in the presymptomatic stages of Parkinson’s disease. FASEB J. 15, 1092 – 1094. Bezard, E., Ravenscroft, P., Gross, C.E., Crossman, A.R., Brotchie, J.M., 2001b. Upregulation of striatal preproenkephalin gene expression occurs before the appearance of parkinsonian signs in 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine monkeys. Neurobiol. Dis. 8, 343 – 350. Bezard, E., Dovero, S., Prunier, C., Ravenscroft, P., Chalon, S., Guilloteau, D., Crossman, A.R., Bioulac, B., Brotchie, J.M., Gross, C.E., 2001c. Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine-lesioned macaque model of Parkinson’s disease. J. Neurosci. 21, 6853 – 6861. Bezard, E., Gross, C.E., Brotchie, J.M., 2003. Presymptomatic compensation in Parkinson’s disease is not dopamine-mediated. Trends Neurosci. 26, 215 – 221. Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A.L., Sadoul, R., Verna, J.M., 2001. Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease. Prog. Neurobiol. 65, 135 – 172. Castelo-Branco, G., Wagner, J., Rodriguez, F.J., Kele, J., Sousa, K., Rawal, N., Pasolli, H.A., Fuchs, E., Kitajewski, J., Arenas, E., 2003. Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a. Proc. Natl. Acad. Sci. U. S. A. 100, 12747 – 12752. Dawson, T.M., Dawson, V.L., 2003a. Molecular pathways of neurodegeneration in Parkinson’s disease. Science 302, 819 – 822. Dawson, T.M., Dawson, V.L., 2003b. Rare genetic mutations shed light on the pathogenesis of Parkinson disease. J. Clin. Invest. 111, 145 – 151. Giasson, B.I., Lee, V.M., 2001. Parkin and the molecular pathways of Parkinson’s disease. Neuron 31, 885 – 888. Grunblatt, E., Mandel, S., Maor, G., Youdim, M.B., 2001. Gene expression analysis in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mice model of Parkinson’s disease using cDNA microarray: effect of R-apomorphine. J. Neurochem. 78, 1 – 12. Hassler, R., 1938. Zur Pathologie der Paralysis Agitans und des postenzephalitischen Parkinsonismus. J. Psychol. Neurol. 48, 387 – 476. Hoehn, M.M., Yahr, M.D., 1967. Parkinsonism: onset, progression and mortality. Neurology 17, 427 – 442. Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y., Takahashi, R., 2001. An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell 105, 891 – 902. Imai, Y., Soda, M., Murakami, T., Shoji, M., Abe, K., Takahashi, R., 2003. A product of the human gene adjacent to parkin is a component of Lewy bodies and suppresses pael receptor-induced cell death. J. Biol. Chem. 278, 51901 – 51910. Electronic publication 52003 Oct 51907. Jackson-Lewis, V., Jakowec, M., Burke, R.E., Przedborski, S., 1995. Time course and morphology of dopaminergic neuronal death caused by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neurodegeneration 4, 257 – 269.
103
Kasprzyk, A., Keefe, D., Smedley, D., London, D., Spooner, W., Melsopp, C., Hammond, M., Rocca-Serra, P., Cox, T., Birney, E., 2004. EnsMart: a generic system for fast and flexible access to biological data. Genome Res. 14, 160 – 169. Kim, K.S., Kim, C.H., Hwang, D.Y., Seo, H., Chung, S., Hong, S.J., Lim, J.K., Anderson, T., Isacson, O., 2003. Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell-specific manner. J. Neurochem. 85, 622 – 634. Klockgether, T., Evert, B., 1998. Genes involved in hereditary ataxias. Trends Neurosci. 21, 413 – 418. Le, W., Conneely, O.M., He, Y., Jankovic, J., Appel, S.H., 1999. Reduced Nurr1 expression increases the vulnerability of mesencephalic dopamine neurons to MPTP-induced injury. J. Neurochem. 73, 2218 – 2221. Le, W.D., Xu, P., Jankovic, J., Jiang, H., Appel, S.H., Smith, R.G., Vassilatis, D.K., 2003. Mutations in NR4A2 associated with familial Parkinson disease. Nat. Genet. 33, 85 – 89. Lu, C.S.W.C.Y., Kuo, P.C., Chang, H.C., Weng, Y.H., 2004. The parkinsonian phenotype of spinocerebellar ataxia type 2. Arch. Neurol. 61, 35 – 38. Martin, R.F., Bowden, D.M., 1996. A stereotaxic template atlas of the macaque brain for digital imaging and quantitative neuroanatomy. NeuroImage 4, 119 – 150. Marvanova, M., Menager, J., Bezard, E., Bontrop, R.E., Pradier, L., Wong, G., 2003. Microarray analysis of nonhuman primates: validation of experimental models in neurological disorders. FASEB J. 17, 929 – 931. Meissner, W., Hill, M.P., Tison, F., Gross, C.E., Bezard, E., 2004. Neuroprotective strategies for Parkinson’s disease: conceptual limits of clinical trials and animal models. Trends Pharmacol. Sci. 25, 249 – 253. Miller, R.M., Callahan, L.M., Casaceli, C., Chen, L., Kiser, G.L., Chui, B., Kaysser-Kranich, T.M., Sendera, T.J., Palaniappan, C., Federoff, H.J., 2004. Dysregulation of gene expression in the 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-lesioned mouse substantia nigra. J. Neurosci. 24, 7445 – 7454. Murakami, T., Shoji, M., Imai, Y., Inoue, H., Kawarabayashi, T., Matsubara, E., Harigaya, Y., Sasaki, A., Takahashi, R., Abe, K., 2004. Pael-R is accumulated in Lewy bodies of Parkinson’s disease. Ann. Neurol. 55, 439 – 442. Smidt, M.P., Smits, S.M., Burbach, J.P., 2003. Molecular mechanisms underlying midbrain dopamine neuron development and function. Eur. J. Pharmacol. 480, 75 – 88. Szabo, J., Cowan, W.M., 1984. A stereotaxic atlas of the brain of the cynomolgus monkey (Macaca fascicularis). J. Comp. Neurol. 222, 265 – 300. Takeuchi, Y., Fukunaga, K., 2003. Differential subcellular localization of two dopamine D2 receptor isoforms in transfected NG108-15 cells. J. Neurochem. 85, 1064 – 1074. Zigmond, M.J., Abercrombie, E.D., Berger, T.W., Grace, A.A., Stricker, E.M., 1990. Compensations after lesions of central dopaminergic neurons: some clinical and basic implications. Trends Neurosci. 13, 290 – 296.
