Neonatal Dopamine Depletion Induces Changes in ...

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BRUCE LADENHEIM", KEVIN G. BECKER', JEAN LUD CADET" AND CHRISTINE R HOHMANN*''. "Molecular Neiiropsychiatry Branch, National Imlitute on ...
Neurotoxicity Research. 2007, VOL 11(2). pp. 107-130

EP. Graham Publishing Co.

Neonatal Dopamine Depletion Induces Changes in Morphogenesis and Gene Expression in the Developing Cortex IRINA N.

KRASNOVA'.

ELIZABETH S. BETTS'^ ABIOLA

DADA" AKILAH

JEFFERSON^,

BRUCE LADENHEIM", KEVIN G. BECKER', JEAN LUD CADET" AND CHRISTINE R HOHMANN*'' "Molecular Neiiropsychiatry Branch, National Imlitute on Drug Abuse, NIH/DHHS: Department of Biology. Morgan Stale University: ^Gene Expression and Genomies Unit. National Institute on Aging. NIH/DHHS, Baltimore, MD 21251 USA. [email protected] (Submitted 5 May 2006: Revised 18 August 2006: In final form 12 September 2006)

The mesocorticolimbic dopamine (DA) system is implicated in mental health disorders affecting attention, impulse inhibition and other cognitive functions. It has also been involved in the regulation of cortical morphogenesis. The present study uses focal injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle of BALB/c mice to examine morphological, behavioral and transcriptlonal responses to selective DA deficit in the fronto-parietal cortex. Mice that received injections of 6-OHDA on postnatal day 1 (PNDl) showed reduction in DA levels in their cortices at PND7. Histological analysis at PNDl20 revealed increased fronto-cortical width, but decreased width of somatosensory parietal cortex. Open Tield object recognition suggested impaired response inhibition in adult mice after 6-OHDA treatment. Transcriptional analyses using 17K mouse microarrays showed that such lesions caused up-regulation of 100 genes in the cortex at PND7. Notably, among these genes are Sema3A which plays a repulsive role in axonal guidance, RhoD which inhibits dendritic growth and tubulin p5 microtubule subunit. In contrast, 127 genes were down-regulated, including CCTe and CCT^ that play roles in actin and tubulin folding. Thus, neonatal DA

depletion affects transcripts involved in control of cytoskeletal formation and pathway finding, instrumental for normal differentiation and synaptogenesis. The observed gene expression changes are consistent with histological cortical and behavioral impairments in the adult mice treated with 6-OHDA on PNDl. Our results point towards specific molecular targets that might be involved in disease process mediated by altered developmental DA regulation. Keywords: Cerebral cortex; Dopamine; Gene expression; 6-Hydroxydopamine; Microarray; Morphology

INTRODUCTION Building a normal cerebral cortex requires coordinated generation, migration, and ditTcrentiation of neurons and glia along with the programmed cell death and formation of appropriate synaptic eonnections between neurons (Cohen-Cory, 2002; Guillemot et ai, 2006; Price et al, 2006). Well synehronized moleeular mechanisms and timely expression of speeifie genes are essential to the formation of cortical layers and the establishment of functional synapses that are eritical for normal eortical development (Ziv and Gamer, 2004; Guillemot

•Corresponding author; Tel.: 1 (443) 885-4002; FAX; 1 (443) 885-8285; E-mail: [email protected] ISSN 1029 8428 print/ ISSN 1476-3524 online. © 2007 FP Graham Pubhshing Co., www.NeurotoxicityResearch.com

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I.N. KRASNOVA et al.

etal., 2006). The preeision required of these events renders the developing eortex extremely sensitive to disruptions by environmental influences as well as genetie predispositions implieated in a number of mental health disorders (Raedler et al., 1998; Riee and Barone, 2000; Johnston et al., 2001; Mick et ai., 2002; Durston, 2003). These insults might, in turn, eause persistent problems in eortieal ontogeny that later result in impaired eognition, memory and deeision making as well as in altered soeial behaviors in the exposed offspring (Riee and Barone, 2000; Miekero/., 2002). Dopaminergie afferent projections from the ventral tegmental area (VTA) to the cortex are the most frequently implieated pathways in neurodevelopmental disorders (Johnston et al., 1995; Berger-Sweeney and Hohmann, 1997; Benes et ai, 2000). Experimental neonatal depletion of dopamine (DA) is known to result in behavioral deficits which include changes in loeomotor activity (Miller ero/., 1981;Kalsbeeke/fl/., 1989a) and cognitive ftinetions (Raskin et al, 1983; Areher et al, 1988; Pappas et al, 1992; Luthman et al, 1997). Notably, developmental DA defieits are associated with decreased eortica! size and eurtailed dendritie growth (Kalsbeek et al, 1987; 1989b; Sherren and Pappas, 2005), in a fashion similar to the neuropathologic abnormalities reported in disorders thought to be secondary to altered DA homeostasis (Johnston et al, 1995; Shenton et al, 2001; Roth and Saykin, 2004). Thus, investigation of the moleeular and cellular bases for DA depletion-indueed impaired eortieal morphogenesis is of paramount importance because it might help to elarify the mechanisms underlying some human neurodevelopmental disorders. The use of 6-hydroxydopamine (6-OHDA)indueed lesions has been the most popular model for experimental DA depletion in the forebrain. However, some of the lesion studies published to date have several confounds. For example, intraeerebroventrieular (i.c.v.) injections of 6-OHDA, even if seleetive for DA, destroy projections not only to the eortex but also to the limbie system (Areher et al, 1988; Luthman et al, 1990). In addition, reactive serotoninergie hypertrophy into DA-denervated eortex has also been shown after i.e.v. applications of the toxin at birth (Blue and Molliver, 1987; Molina-Holgado et al, 1993; Breese et al, 2005). Similarly, thermal lesions to

the VTA are also nonseleetive, inducing reduetions in eortieal serotonin (5-HT) conecntrations (Kalsbeek e/t7/., 1987; 1989a). Altered 5-HT levels in the developing eortex also are known to affeet morphogenesis and behavior (Blue et al, 1991; Berger-Sweeney et al, 1998; Boylan et al, 2000; Hohmann et al, 2000). In addition, similarly treated rodents often have redueed brain and body weight (Berger-Sweeney and Hohmann, 1997; Bruno et al, 1998; Breese etal, 2005). Therefore, to isolate potentially deleterious influenees of DA depletion on eortieal development, we performed the present study using focal injeetions of 6-OHDA into the medial forebrain bundle (MFB) because it is a more selective animal model of deficient eortieal DA. We performed histologieal analysis in combination with large-seale mieroarray and RT-PCR approaehes in order to determine morphological and transcriptional responses speeifie to neonatal eortieal DA deafTerentation. Behavioral tests were eondueted to assess the possible cognitive consequences. We show perturbations in the transcriptional developmental programs in the mouse eortex that are consistent with the morphological changes. Our study identifies speeifie eellular and moleeular targets to further investigate dopaminergie morphogenetie influenees in eortieal development. MATERIALS AND METHODS Animals and 6-OHDA Treatment Male and female BALB/cByJ breeding stock was obtained from Jaekson Laboratory (Bar Harbor, ME). All animal proeedures were eondueted aeeording to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. BALB/cByJ mice, generated in our breeding eolony at Morgan State University, were removed from their mothers within the first 12-20 hours after birth, anesthetized on iee, placed into a speeially designed mouse pup holder, and positioned in a stereotaxic apparatus (Kopf, Tujunga, CA). We applied an injection protocol that we empirieally developed to perform maximal DA depletion in the eortex without major alterations in other afferent monoamine levels. 6-OHDA hydrobromide (Sigma, St.Louis, MO) was dissolved in 0.9% sterile saline containing 0.2% aseorbie aeid to a concentration 10 ^ig/^il. The solution was prepared fresh prior to injection and kept on iee in order to minimize oxidation.

DOPAMINE & DEVELOPING CORTEX IN MICE

Injections were made into the MFB near the diagonal band of Broca, horizontal limb, similar to previously described (Hohmann el aL, 1988; Hohmann and Berger-Swceney, 1998). Coordinates for the injections were measured using the sinuses underlying the midline and frontonasal skull suture, and 0.6 microliters (= 6 ^ig) of 6-OHDA solution was injeeted into each hemisphere. On each side, a 27 gauge needle was lowered into the brain through skin and skull 2 mm anterior to the frontonasal suture and 1.5 mm lateral to midline, at an angle of 15° horizontally and 58° vertically. 6-OHDA was slowly administered in three injeetions (0.2 |il eaeh) at a depth of 4.0 mm (1st injection), 3.5 mm (2nd injection), or 3 mm (3d injection) along the posterior to anterior trajectory of the needle tract in the forcbrain. Litter mates, injected with vehiele, served as eontrols for all experiments. Each group contained mice from at least three different litters. After the injections, pups were re-warmed to normal body temperature on a heating pad and returned to their mothers. For HPLC and mieroarray analysis, male mice were saerificed by cervical dislocation on PND7, their brains were rapidly removed from the skulls and dissected on an iee-cooled plate. The samples were taken from dorsal fronto-parietal cortex to closely resemble the eortieal areas examined histologieally. In addition, striata were dissected out and used for HPLC analysis. Tissue samples were immediately frozen on dry ice, weighed, and processed for HPLC or RNA isolation as described below. For behavioral and histology experiments, male mice were weaned on PND30 and group housed with littcnnates until the start of behavioral testing. HPLC Analysis Cortical samples (A^=8 per group) were ultrasonieated in 0.1 M perehloric acid containing 10 ng/mg of internal standard dihydroxybenzilamine, then eentrifuged at 20,000^^ for 10 min. Concentrations of norepinephrine (NE), DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-HT, and 5-hydroxyindoleaeetie acid (5-HIAA) in eortieal and striatal tissue extracts of 6-OHDA and vehicle-treated miee were measured by HPLC with electrochemical detection as deseribed earlier (Krasnova el ai, 2001). Coneentrations of NE, DA, DOPAC, HVA, 5-HT, and 5-HlAA were ealeulated and expressed as pg/mg of tissue weight. Statistieal analysis was performed using analysis of varianee (ANOVA) followed by Fisher's proteeted least

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significant difference (PLSD) (StatView 4.02, SAS Institute, Cary, NC). Differences were eonsidered signifieant atp otic translation initiation factor 3 subunit 2 EIF3S2 BG078361 eukaryotic translation initiation factor 4 gamma 2 EIF4G2 BG063771 poly A binding protein, cytoplasmic 1 Pabpc] BG077146 seryl-aminoacyl-tRNA synthetase 1 Sarsl Protein Synthesis Up-regulated BQ552082 carbox>peptidase X 1 Cpxl AB030378 chondroitin 4-sulfotransferase 2 C4st2 BG072192 alpba-N-acetylglucosaminidase Naglu BG087134 D-dopachromc tautomerase Ddt BQ551115 geranylgeranyl diphosphate syntbase 1 Ggps 1 AW558740 mannosidase 1, beta Manlb BG076333 methylenetetraliydrofolate dehydrogenase Mtlifd2 BG084015 pbosphoserine aminotraiisferase 1 Psatl AU043387 proteasome 26S subunit. non-ATPase, 12 Psmdl2 BCOOl 982 proteasome subunit, alpha type 4 Psnia4 BG071790 protein phospliatase 1. gamma isoform Ppplcc BG074107 ribosomal protein L3 Rpl3 BC054388 ribosomal protein I37a Rpl37a BG084847 ribosomal protein L39 Rpl39 BG072802 ribosomal protein S16 Rpsl6 AW556256 ribosomal protein S6 Rps6 BG086349 ring linger protein 11 Rnfl 1 BQ5 52743 RNA binding protein gene Rbpms BG0706IO SUMO/serine specific protease 3 Snp3

±0.39 ±0.10 ±0.19 ±0.30 ±0.17 ±0.19 ±0.12 ±0.19 ±0.14 ±0.29 ±0.37 ±0.10 ±0.10

6-OHDA p values Means ± SE

1.23 ±0.10

0.76 ±0.21 0.70 ±0.12 0.73 ± 0.09 0.78 ±0.18 0.74 ±0.31 0.76 ±0.09 0.83 ±0.15 0.86 ±0.11 0.74 ±0.15 0.85 ±0.12 0.81 ±0.11 0.62 ±0.12 0.58 ±0.15 0.59 ±0.17

0.046 0.009 0.004 0.022 0.030 0.004 0.037 0.032 0.025 0.043 0.032 0.016 0.020 0.019

1.32 t 0.15 1.28±0.15 1.30 ±0.12

0.65 ±0.21 0.59±0.17 0.61 ±0.19

0.011 0.026 0.036

0.83 ± 0.06 0.83 ± 0.09 0.85 ± 0.05 0.88 ± 0.07 1.29 ±0.13

2.27 1.30 1.70 2.36 0.69

±0.59 ±0.14 ±0.26 ± 0.69 ±0.14

0.003 0.022 0.004 0.030 0.046

1.33 ±0.16

0.89 0.76 0.69 0.79 0.75 0.88 0.80 0.67

±0.12 ±0.10 ±0.13 ± 0.12 d: 0.18 ±0.11 ±0.08 ±0.13

0.044 0.023 0.017 0.047 0.042 0.033 0.003 0.025

1.91 ± 0.45 1.28 ±0.18 1.38±0.l8 1.39 ± 0.24 1.38 ±0.21 2.18 ±0.67 1.30 i 0.20 1.69 ±0.47 1.56 ±0.32 1.85 ±0.35 i.27±0.17 1.62 ±0.29 1.39 ±0.21 1.86 ±0.37 1.54 ±0.28 1.33 ±0.18

0.047 0.024 0.027 0.048 0.026 0.014 0.027 0.024 0.030 0.008 0.034 0.027 0.047 0.007 0.036 0.046

1.45 ±0.22 1.41 ±0.20 1.45 + 0.26

0.041 0.024 0.033

i.n±o.o7 1.27 ±0.19 1.34±0.18 1.30 ±0.23 1.52±0.2l 1.46 ±0.14 1.20 ±0.15 0.94 ± 0.13 0.73 ±0.12 0.92 ± 0.07 0.85 ±0.10 0.84 ±0.11 0.63 ±0.13 0.81 ± 0.09 0.78 ±0.10 0.79 ±0.18 0.79*0.10 0.88 ± 0.07 0.87 ±0.11 0.85 ±0.09 0.81 ±0.13 0.86 ± 0.07 0.91 ±0.05 0.88 ±0.15 0.88 ±0.11 0.89 ±0.10

DOPAMINE & DEVELOPING CORTEX IN MICE

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Supplementary Table I. Functional classification of transcripts whose expression was changed after 6-OHDA treatment GenBanktf

Symbol

Gene Name

Control Means ± S£

Protein Synthesis Down-regulated 1.37 ±0.20 Aloxl2 BCiO69982 arachidonate 12-lipoxygenase 1.22 ±0.08 Siat7b siillyltransferase 7B BQ55I716 2.25 ± 0.94 Usp39 ubiquitin specific protease 39 BG076775 1.38 + 0.21 Slc3a2 solute carrier family 3 (aiiiino acid transport) 2 BG064714 1.69 ±0.30 Scnp6 SUMO/scntHn specific protease 6 BG077813 1.33 ±0.13 THm6 tripartite motif protein 6 1JGO7()343 1.40 ±0.23 Usp21 ubiquitin specific protease 21 BGO650O3 1.54 ± 0.23 ubiquitin specific protease 3 Usp3 BG069818 1.37 ±0.20 1,4- galactosyltransferase polypeptide I B4galtl BG075068 1.63 ±0.33 component of oligomeric golgi complex 8 Cog8 BG067882 I,27±0.15 DnaJ (Hsp40) bomolog subfamily C member 7 I)najc7 BG077676 1.77 ±0.34 EL0VL6 elongation of long chain fatty acids EIovl6 BG069686 1.34 ±0.14 enolase 1 alpha non-neuron Enol BG0784I0 3-hydroxy-3metliylglutaryl-Coenzyme A synthasc HMG-CoA s>Titliase 1.60 ±0.30 AU020524 1.76 ± 0.33 heterogeneous nucleiir ribonucleoprotein D Hnrpd BG078653 1.49 ±0.41 inter-alpha tr>psin inhibitor hea\7 chain 1 llihl BG066944 1.26 ±0.15 low-density lipoprotein receptor- related protein 10 LrplO BGO64451 1.40 ±0.18 EroU BG077757 EROl-like 1.33 ±0.14 Facl5 BG084205 fatty acid Cocnzyme A ligase long chain 5 1.48 ±0.18 Ppap2b BG080374 phosphatidic acid phosphatase type 2B 1.44 ±0.21 Yifl AW539280 Yipl interacting factor homolog 1.29 ±0.17 Gnpi BG064771 glucosaniine-6-phosphate deaminasc 1.57 ±0.31 Gmpr2 AW 556794 guanosine monophosphate reductiLse 2 1.30 ±0.12 Pon3 BG074714 paraoxonasc 3 1.20 ±0.10 Tkt BG075312 Traiisketolase 1.24 ±0.11 Modi malic enzyme BG064680 1.43 ±0.23 Mctap2 methionine aminopcptidase 2 BG065235 1.53 ±0.29 Mrpll mitochondrial ribosomal protein 1 BG073306 1.41 ±0.21 Ndufafl NADU dehydrogenase assembly factor I BG063606 1.70 ±0.41 Pnini 1 phosphomannomutase 1 BG077408 1.37 t 0.20 PPP2Ab protein phosphatasc 2a beta 075970 1.15±O.I1 PTP4A1 protein tyrosine phosphatase 4a 1 BG068173 Rpl5 Rpl7 RpI8 Rps6ka2 Spi6 Signal transduction

BG065196 BG063883 BG077480 BG063083 BG077478

ribosomal protein L5 ribosomal protein L7 ribosomal protein L8 ribosomal protein S6 kinasc polypeptide 2 serine protease inhihitor 6

Up-regiilated BQ552I59 BQ552281 BQ551016 BG087137 BQ551164 BQ551079 BG080I66 BQ552129

ankyrin repeat and SOCS box-protein 6 annexin A6 homer, neuronal immediate early gene kit oncogeiie PAS serine/threonine kinase protein phosphatase 2 subunit B RAB, member of R.\S oncogenc family-tike 3 wingless-related MMI'V integration site 5B

Asb6 Anxa6 Homer3 Kit PASK PPP2r2a Rab!3 Wnt5b

p values Means ± SB

0.73 ±0.12 0.72 ±0.15 0.80 ± 0.09 0.76 ±0.11 0.76 ± 0.08 0.85 ±0.06 0.77 ±0.14 0.78 ±0.11 0.67 ±0.21 0.73 ±0.14 0.78 ±0.10 0.90 ±0.17 0.72 ± 0.11 0.82 + 0.15 0.90 ± 0.09 0.65 ±0.16 0.74 ± 0.09 0.79 ±0.12 0.68 ± 0.15 0.62 ±0.11 0.72 ±0.15 0.71 ±0.12 0.79 ± 0.11 0.58 ±0.16 0.79 ± 0.09 0.86 + 0.06 0.77 ±0.15 0.74 ±0.16 0.84 ±0.12 0.82 ±0.12 0.83 ±0.11 0.73 ±0.14

0.034 0.024 0.029

±0.44 ±0.57 ±0.17 ±0.16 ±0.30

0.79 ± 0.14 0.90 ±0.13 0.73 ± 0.11 0.87 + 0.07 0.76 ±0.15

0.019 0.039 0.018 0.006 O.OiO

0.83 ± 0.07 0.86 ± 0.11 0.82 ±0.10 0.72 ±0.16 0.86 ±0.11 0.89 ±0.07 0.85 ±0.18 0.91 ± 0.09

l,24±0.11 1.64 ±0.35 1.33 ±0.16 1.76 ± 0.57 2.34 ±0.53 1.86 ±0.40 2.12 ±0.57 1.25 ±0.11

0.006 0.046 0.014 0.031 0.016 0.020 0.039 0.048

1.75 1.99 1.29 1.41 1.59

0.021 0.002 0.003

0.021 0.005 0.023 0.008 0.020 0.042 0.007 0.047 0,018 0.037

0.014 0.021 0.007 0.000 0.025 0.022 O.OIl 0.017 0.026 0.015 0.024 0.031

0.041 0.046 0.049 0.027

I.N. KRASNOVAe/a/.

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Supplementary Table I. Functional classification of transcripts whose expression was changed after 6-OHDA treatment GenBank;#

Gene Name

Symbol

Control Means ± SE

p values 6-OHDA Means ± SE

Signal tramduction Down-regulated BG088471 B-ceil receptor-associated protein 29 BC054805 calniodulin 1 AW554667 constitutive photomorphogen 9 subunit 5 BG064928 disabled honiolog 2 (Drosophila) BGO65213 growtli factor receptor bound protein 10 BG078399 guanine nucleotide binding protein beta 2 BG064826 HSI binding protein BG081243 inositol 1 A5-triphosphate receptor 1 BG063736 nemo like kuiase EJG071651 RABl la, member I^\S oncogene family BG080845 related RAS viral (r-ras) oncogene 2 BG077776 seHneihreonine kinaiic 10 BQ551075 SH3-domain binding protein 1 BG079034 sphingosine kinase 2 BG070360 suppressor of cytokine signaling 4 L33417 very low density lipoprotein receptor

Bcap29 Calml Cops5 Dab2 GrblO Gnb2 Hslbpl Itprl Nik Rablla Rras2 StklO Sh3bpl Sphk2 Socs4 Vldlr

1.24 1.45 1.27 1.36 1.27 1.40 1.40 1.56 1.38 2.01 1.48 1.45 0.89 1.60 1.24 1.52

±0.10 ±0.19 ±0.12 ±0.29 ±0.14 ±0.19 ± 0.28 ±0.37 ±0.2! ± 0.56 ±0.22 ±0.24 ±0.07 ± 0.24 ±0.13 ±0.29

0.80 ±0.09 0 91 ±0.12 0 64 ±0.17 0.78 ±0.11 0.87 ±0.11 0.73 ±0.15 0.78 ±0.14 0.80 ± 0.20 0.85 ±0.11 0.68 ±0,19 0.68 ±0.17 0.74 ±0.10 0.58 ±0.26 0.67 ±0.17 0.73 ±0.13 0.71 ±0.15

0.007 0.039 0.028 0.038 0.041 0.022 0.043 0.045 0.049 O.OIO 0.008 0.013 0.015 0.005 0.018 0.029

0.72 0.80 0.85 0.86 0.86 0.79 0.82 0.82 0,87 0.77 0.86 0.94 0.84 0.87 0.85

± 0.09 ±0.15 ± 0.08 + 0.08 ± 0.11 ±0.10 ±0.11 ±0.09 ±0.12 ± 0.08 ± 0.08 ±0.12 ±0.10 ±0.15 ±0.12

149 ± 0 35 1 53 ±0.34 1.61 ±0.24 1.45 ±0.22 1.67 ±0.37 1.71 ±0.29 1.17 ±0.08 1.30 ±0.19 1.39 ±0.19 1.59 ±0.27 1.55 ±0.22 r28±0.11 2.13 ±0.65 1.47 ±0.19 1,26 ±0.11

0.030 0.044 0.007 0.018 0.032 0.0 U 0.046 0.047 0.044 0.009 0.009 0.048 0.036 0.018 0.034

1.42 ± 0.15 1.36 ±0.16 1.43 ±0.22 1.35 ±0.21 1.33 ±0.16 1.24±0.!2 1.17±0.05 1.20 ±0.08 0.72 ± 0.09 1.31 ±0.19 1.43 ±0.27 1.33 ±0.12 1.15 ±0.09 1.31 ±0.10 1.43 ±0.21 1.78 ±0.36 1.76 ± 0.43

0.74 ± 0.1 0.75 ±0.12 0.88 ±0.10 0.78 ±0.12 0.83 ±0.13 0.75 ±0.16 0.59 ±0.13 0.75 ± 0.09 1.38 ±0.21 0.80 ±0.15 0.79 ±0.10 0.80 ±0.14 0.64±0.17 0.62 ±0.15 0.85±0.18 0.92 ±0.22 0.93 ±0.15

0.006 0.018 0.048 0.034 0.042 0.029 0.046 0.008 0.007 0,038 0,038 0,015 0.040 0.028 0.028 0.049 0.045

Intracellular transport Up-regulated AUO4239O BQ552336 BG085675 BG087256 BG072229 BG086010 BG085334 BG083967 BG087217 BG084323 BQ552559 BG088027 BG071195 BQ551106 BC047281

ATPase, Ca++ transporting, slow twitch 2 Atp2a2 ATPase, Cu++ transporting, alpha polypeptide Atp7a ATPase, H+ transporting, lysosomal Atp6gl ATP-binding cassette, subfamily E member 1 Abce 1 caIbindin-28K Calbl dynein, cyloplasmic. intermediate chain 2 Dncic2 SARU gene liomolog Saral siderollexin 1 Sfxnl sideronexin3 Sfxn3 solute carrier family 34 Slc34a2 solute carrier family 6 Slc6a6 striatin, calmodulin binding protein 3 Stm3 Vitellifomi macular dystrophy 2 Vmd2 T>T3/Trp5-monooxygenase activation protein Ywhab l-acylglycerol-3-phosphateO-acyltransferase 1 Agpat4 Intracellular transport Down-regulated BG064835 a disintegrin-like and metalloprotease AdamtslO BG078400 adaptor protein complex AP-1 mu 2 Aplm2 BG077736 aldehyde dehydrogenase 9 subfamily AI Aldh9al BG076762 ATPase, Na+/K+transporting beta 1 Atplbl BG064795 ferritin light chain I Ftll BG085818 hemoglobin alpba adult chain 1 Hba-al BCK)85146 lactotransferrin Ltf BG088166 lysosomal apyrase-like 1 Lysall BQ551346 MucoHpin 3 Mcoln3 BG064735 peroxiredoxin 5 Prdx5 BG069106 potassium voltage-gated channel H member 1 Kcnhl BG064853 monocarbox>late transporter 4 MCT4 BG072114 solute carrier 21 prostaglandin transporter 2 S!c21a2 BG085151 solute carrier 27 fatly acid transporter 4 Slc27a4 BG075243 solute carrier 30 zinc transporter 3 Slc30a3 BG080898 transient receptor potential cation channel M7 Trpm7 BG069221 T-cell immunoglobulin and mucin domain 2 Tim-2

DOPAMrNE & DEVELOPING CORTEX IN MICE

129

Supplementary Table I. Functional classification of transcripts whose expression was changed after 6-OIIDA trealmcnl GenBank#

Symbol

Gene Name

p values

Control Means ± SE

Means ± SE

0.92 ± 0.06 0.92 ± 0.05 0.90 ± 0.10 0.85 ± 0.06 0.83 ±0.10

1.38 2.08 1.99 1.69 1.26

±0.19 ± 0.62 t 0.51 ±0.46 ±0.16

0.031 0.037 0.035 0.036 0.029

1.47 ±0.22 1.25*0.19 1.65 ±0.30 1.14 i 0.09 1.53 ± 0.29 1.31 ±0.14 1,4] I. 0.21

0.88 ±0.10 0.73 ±0.19 0.83 ± 0.08 0.75 ±0.16 0.74 ± 0.16 0.68 ±0.19 0.84 ±0.12

0.031 0.035 0.013 0.041 0.031 0.025 0.041

0.76 0.75 0.85 0.78 0.79 0.75

±0.12 ±0.12 ±0.08 ± 0.20 ±0.12 ±0.11

1.28 ±0.17 1.60 10.43 1.44±0.19 1.74 ±0.50 L96 tO.5I 1.92 ±0.52

0.018 0.045 0.013 0.044 0.022 0.046

1.21 ±0.13 1.87 ±0.46 1.36 ±0.18 L38±0.18

0.81 ±0.10 0.65 ±0.14 0.84 ±0.13 0.90 ±0.11

0.038 0.012 0.042 0.046

0.80 0.89 0.86 0.89 0.75

L42±0.31 I.25±ai4 1.81 ±0.49 2.37 ± 0.79 1.25±0.I5

0.048 0.044 0.048 0.034 0.039

0.008 0.042 0.039

Mitochondria Up-regulated riG073437 BG085306 BG087498 BQ552525 iiC024673

ATP synthase, H+ transporting mitochondrial beta cytochrome c oxidase, subunit Vic electron transferring flavoprotein, alpha MitochondHal rihosoniiil protein L45 NADH dehydrogenase (ubiquinone) 1 alpha 8

Atp5b COX6C Etfa MrpI45 Ndufa8

Down-regulated Atp5al BG078689 ATP synthase H+ transporting niito alpha 1 Atp6vlcl AW537398 ATPase, H+ transporting. V lCl Bzrp BG076653 benzodiazepine reccplor. peripheral COX8A BG076988 cytochrome c oxidase, subunit Villa Mrpll BG073306 mitochondrial ribosomal protein LI NdulalO BQ550897 NADU dehydrogenase (ubiquinone) 1 alpha 10 Ndufaf] BG()63606 NADH debydrogenase (ubiquinone) 1 alpha Cell Adhesion Up-regulated CD81 BG075757 CD 81 antigen Co!6a] BG075779 procollagen. type VI alpha 1 lipin 3/Eniilin3 BG076147 clastin microfibril interfacer 3 Col 5a 1 BQ552186 procollagen. type V alpha 1 Nisch BG073805 nischarin Selel BCiO88919 selectin, endothelial cell, ligand Cell Adhesion Down-regulated Cspg6 BG085432 chondroitin sulfate proteoglycan 6 Cfdp BG06986I craniofacial development protein 1 Loxl3 BQ551553 lysyl oxidase-Iike 3 Vcl BG063694 vincuUn Cell cycle Up-regulated Cdc7Il BQ55I224 cell division cycle 71 homolog like 1 Ccne2 BQ55O528 cyclin E2 Ccnll BG083664 cyclin LI Gsptl BG086478 Gl to phase transition 1 Tepl BG075056 telomerase associated protein 1

tO.lO ±0.10 ± 0.09 ± 0.07 ±0.14

Cell cycle Down-regulated BG063118 inner centromere protein FJG064865 mini chromosome maintenance deficient 5 BG066368 checkpoijU kinase 1 homolog Up-regulated BQ551034 Bone morphogenetic protein 8b BG088057 Glia maturation factor beta AU040433 Translonning growth ("aL-tor alpha Down-regulated BC052677 Growth differentiation factor 9

Incenp Mcm5 Chekl Growth factors

1.55 ±0.23 L46±0.27 1.62 ±0.31

0.79 ±0.09 0.80±0.19 0.72 ±0.13

BMP8b GMFB TGFa

0.83 ±0.08 0.95±0.07 0.79 ±0.09

1.42 ±0.21 0.021 1.31 ±0.15 0.046 1.73 ±0.33 0.010

GDF9

1.45 ±0.34

0.73 ±0.13

0.032

I.N. KRASNOVA et al.

130

Supplementary Table L Functional classification of transcripts whose expression was changed after 6-OHDA treatment GenBank#

Gene Name

Symbol

DNA damage / Stress response proteins Up-regulated BG071480 DNA polymerase epsilon suhunit 2 Pole2 BG076183 Structural maintenance of chromosomes 1 SMCl BG077269 Structural maintenance of cliromosomes 4 SMC4 BG071892 RAD21 homolog RAD21 BQ552179 REV3-Iike DNA polymerase zela Rev31 BG085381 Degenerative spermatocyte homolog Degs RC030444 Glutathione S-transferase, mu 4 Gstni4 BG074109 fleat shock protein, 86 kDa 1 Hsp86-1 BG087426 Heat sbock 70kD protein 8 Hspa8 BG072457 Myotropbin Mtpn Down-regidated BC034517 excision repair cross-complementing group 2 Ercc2 BG088755 DnaJ (Hsp40) homolog, subfamily A member 1 Dnajal BG065483 DnaJ (Hsp40) bomolog, subfamily B memher 1 Dnajbl Unclassified/Other Up-regulated BG071424 integral membrane protein 2C Itm2C Down-regii laled Ewsh BG064201 Ewing sarcoma bomolog Fbxo32 C86521 f-box only protein 32 BQ551727 unc-84 homolog A Unc84a

Control Means ± SE

6-OHDA p values Means ± SE

0.87 ± 0.06 0.78 ±0.10 0.7710.16 0.83 ±0.13 0.83 ±0.10 0.83 ± 0.07 0.85 ± 0 1 2 0.86 ±0.07 0.87 ±0.08 0.89 ±0.10

1.46 ±0.25 L54±0.18 2.48 ±0.61 1.61 ±0.27 1.33 ±0.16 1.45 ±0.23 1.77 ±0.38 1.56 ±0.42 1.42 ±0.17 1.56 ±0.23

0.038 0.041 0.019 0.019

1.42 ±0.20 1.20 ±0.10 1,63 ± 0.27

0.69 ± 0.09 0.78 ±0.11 0.86 ±0.14

0.002 0.049 0.031

0.78 ±0.12

1.28 ±0.20

0.035

1.51 ±0.31 1.34±0.17 1.2I±0.15

0.76 ±0.12 0.77 ±0.13 0.60 ±0.18

0.026 0.026 0.014

0.011 0.005 0.028 0.021 0.025

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