Proteomic Analysis of the Genotoxicant Methylazoxymethanol (MAM

Mar 29, 2006 - spectrometer MALDI-TOF-TOF (Auto Flex, Bruker Daltonics,. Fremont, CA) and a .... significant changes in several functional classes inc...
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Proteomic Analysis of the Genotoxicant Methylazoxymethanol (MAM)-Induced Changes in the Developing Cerebellum G. E. Kisby,† M. Standley,‡ T. Park,† A. Olivas,† S. Fei,‡ T. Jacob,‡ A. Reddy,‡ X. Lu,‡ P. Pattee,‡ and S. R. Nagalla*,‡ Center for Research on Occupational and Environmental Toxicology (CROET) and Center for Biomarker Discovery, Department of Pediatrics, Oregon Health & Science University, Portland, Oregon 97239 Received March 29, 2006

The genotoxicant methylazoxymethanol (MAM) is a widely used developmental neurotoxin, and its glucoside is an etiological factor for western Pacific amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS/PDC). Identification of global protein expression changes that occur in response to MAM in the developing cerebellum could provide valuable insight into the potential mechanisms involved in the neurodegeneration process. We have utilized fluorescence 2-dimensional differential gel electrophoresis (2D-DIGE), to determine the protein expression changes that occur during normal cerebellar development and in response to MAM. Three day-old postnatal C57BL/6 mice (PND3) received a single injection of MAM, and the cerebella of postnatal day 4 (PND4) and day 22 (PND22) were analyzed. Approximately, 1400 unique spots were matched and quantified in all samples. Comparison of PND4 and PND22 developing cerebellum showed that a significant fraction of the proteome (∼68%) changes at this stage. The immediate response of the developing cerebellum to MAM was minimal (∼10%). However, significant differences (27%) were noted 14 days after MAM exposure. In contrast, the transcriptome changes were more pronounced at 24 h compared to 14 days. MAM targeted several proteins networks including transport (e.g., R-synuclein), cytoskeletal (e.g., β-tubulin, vimentin), and mitochondrial (e.g., Atp5b) proteins. Immunochemistry confirmed several of the changes in protein expression (R-synuclein). Comparison with gene expression changes revealed that the temporal changes observed in the transcriptome and proteome are not correlative. These studies demonstrate for the first time the potential networks involved during neuronal development and neurodegenerative processes that are perturbed by MAM. Keywords: 2-D differential gel electrophoresis (DIGE) • western Pacific ALS/PDC • R-synuclein

Introduction Methylazoxymethanol (MAM) is a potent DNA alkylating agent (i.e., genotoxicant) and the active component of a glucoside (cycasin), which is found in the cycad plant. A characteristic feature of MAM (or cycasin) is the ability of this environmental genotoxicant to reproducibly induce brain maldevelopment.1,2 Postnatal exposure (days 1-4) leads to microencephaly (e.g., reduced folia and fissures) of the cerebellum and is characterized by specific targeting of glutaminergic and GABAergic precursor cells of the cerebellum (especially granule cells) resulting in misalignment of Purkinje cells and ectopic and multinucleated granule cells.1,3 Multinucleated and ectopic neurons have also been reported in the cerebellum and vestibular nuclei of subjects with western Pacific amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS/PDC),4 an observation suggesting that human exposure to MAM during * To whom correspondence should be addressed. Phone: (503) 494-1298. Fax: (503) 494-4821. E-mail: [email protected]. † Center for Research on Occupational and Environmental Toxicology (CROET). ‡ Center for Biomarker Discovery, Department of Pediatrics.

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early CNS development (up to the age of 1 year) may have arrested the mitotic and migratory developmental processes of neurons. Despite a wealth of information about the neurotoxic properties of MAM,5 the molecular mechanism by which this genotoxicant induces brain maldevelopment or chronic neurodegenerative disease is not clearly understood. Global gene expression profiling approaches have proven useful for revealing the complex molecular networks of normal brain development6-9 and the putative molecular mechanisms of the neurodegenerative disease process.10-13 Using a similar approach, our recent microarray studies indicate that MAM disrupts both developmentally regulated genes and genes that code for proteins with a potential role in the neurodegenerative disease process.14 The influence of MAM on these genes was also shown to be associated with a corresponding increase in DNA damage and cerebellar dysgenesis. Proteomic analysis using two-dimensional (2-D) gel electrophoresis is a well-established technique to separate and resolve hundreds of proteins. Flourescence 2-D differential gel electrophoresis (DIGE) provides enhanced sensitivity, reproducibility, and wide dynamic range for quantification of global 10.1021/pr060126g CCC: $33.50

 2006 American Chemical Society

MAM-Induced Changes in the Developing Cerebellum

proteomic changes through multiplex analysis of sample proteomes on the same gel reducing the gel-gel variation.15 Spot quantification of DIGE gels and identification of proteins using mass spectrometry (MS/MS) analysis could provide a more expansive list of cellular proteins that are regulated during CNS development or after toxin exposure. In the present study, we evaluate proteomic changes occurring in the developing cerebellum of mice at critical stages of CNS development and the key proteins that are influenced by the genotoxicant, MAM, during cerebellar development.

Experimental Procedures Animals. Neonatal C57BL/6J mice (postnatal day 3, PND3; n ) 3/timepoint) were administered a single mid-scapular injection of MAM (43 mg/kg, sc) as previously described.14 After 24 h (PND4) or 19 days (PND22), the cerebellum from each animal was dissected on ice, the meninges removed, the tissue placed in microcentrifuge tubes and immediately snap-frozen in liquid N2. Tissue was stored at -80 °C until analyzed. Isolation of Protein. The PND4 and PND22 cerebellum from saline- and MAM-treated mice was thawed, homogenized in extraction buffer (50 mM Tris, 2 mM EDTA, 0.02% NaN3, 0.1 M NaCl, pH 7.5, Complete Mini Protease Inhibitors (Roche Diagnostics Corp., Indianapolis, IN), 0.2 mM PMSF, and 1 mM DTT) and the homogenate sonicated for 3 × 10 s. The tissue homogenate was allowed to stand for 15 min before centrifugation at 20 000g for 1 h at 4 °C. The pellet was resuspended in buffer, an aliquot analyzed for protein concentration using the DC protein assay kit (Bio-Rad, Hercules, CA), and the remaining tissue homogenate stored at -80°C until analyzed. Fluorescence Two-Dimensional Differential In-Gel Electrophoresis (2D-DIGE). For each sample, 100 µg of sample was labeled with CyDye DIGE Fluor minimal dye (GE Healthcare Biosciences, Piscataway, NJ) at a concentration of 600 pmol of dye/100 µg of protein. Samples were labeled with Cy3 (MAM), Cy5 (saline-treated), or Cy2 (reference, MAM + saline-treated), and all three labeled samples were multiplexed and resolved in a single gel. Labeled proteins were purified by acetone precipitation and dissolved in isoelectric focusing (IEF) buffer containing 0.5% (v/v) ampholytes and rehydrated passively onto a 24 cm IPG strip (pH 4-7, GE) for 12 h at room temperature. After rehydration, the IPG strip was subjected to first dimension IEF for ∼10 h to attain a total of 65 kV/h (Ettan IPGphore, GE). Focused proteins in the IPG strip were first reduced by equilibrating with buffer containing 1% (w/v) dithiothreitol (DTT) for 15 min and then alkylated with buffer containing 2.5% (w/v) iodoacetamide (IAA) for 15 min. After reduction and alkylation, the IPG strip was loaded onto a gradient (8-16%) polyacrylamide gel (24 × 20 cm), and SDSPAGE was conducted at 80-90 V for 18 h to resolve proteins in the second dimension (Ettan DALT System, GE). Following electrophoresis, gels were scanned in a Typhoon 9400 scanner (GE) using appropriate lasers and filters with the PMT biased at 550 V. Images in different channels (saline- and MAMtreated) were overlaid using selected colors, and differences were visualized using Image Quant software (Amersham Biosciences). Differential protein analysis was performed using Phoretix (Nonlinear Dynamics) software. To evaluate protein spots showing differential expression, 2-D gel images were analyzed using Phoretix 2D Evolution (v.2005, Nonlinear USA, Inc., Durham, NC). A fixed area was selected from every gel, and cross-stain analysis was performed. Background subtraction was done using the ‘mode of non spot’

research articles method, and images were wrapped to maximize spot matching. A ratiometric normalization algorithm was applied to account for any possible concentration differences in the dyes occurring during protein labeling. To generate ratios of relative expression for individual protein spots, normalized spots in the Cy3 and Cy5 channels were compared to the corresponding spot in the Cy2 channel (internal standard) for each multiplex group. The statistical significance of differences in intensity between spots was determined by t-tests on averaged gels for each group. Protein spots with a relative ratio >1.5 with a t-test value of 10.6) and OpenSea (significance score >100) scoring algorithms18 were chosen. In total, 234 unique proteins were identified using the criteria of proteins with more than 2 peptide hits and/or higher scores. A majority of the spots showed a single protein in the high confidence range with multiple peptides. Multiple spots showed similar protein ID’s. Proteins listed in Tables 2 and 3 were chosen from spots with unique protein ID’s and/or the most abundant protein represented in the spot. Journal of Proteome Research • Vol. 5, No. 10, 2006 2657

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Gene Microarray Assays. In total, 27 648 sequence-verified mouse cDNA clones were used to examine gene expression changes in the cerebellum of saline- or MAM-treated PND4 and PND22 mice as previously described.14 In brief, total RNA (10 µg) extracted from cerebellar tissue was labeled with Cy5 by an amino-allyl labeling protocol. Arrays were scanned using SA 5000 fluorescent scanner (Perkin-Elmer) and data analyzed with QuantArray software (Perkin-Elmer). Detailed microarray protocols and full data sets are available on our Web site at http:/medir.ohsu.edu/∼geneview/. Western Blot. Protein extracts (25 µg) from the PND4 and PND22 cerebellum of saline- or MAM-treated mice were resolved on a 12% SDS-PAGE discontinuous gel, the samples transferred onto a nitrocellulose membrane (Bio-Rad), and the membrane stained with Ponceau S to confirm equal protein loading. The membrane was blocked in 5% nonfat milk in PBS for 1 h, incubated with monoclonal anti-R-synuclein (1:2000; BD Transduction Labs) or anti-γ-actin (1:2000; Chemicon) in PBS-Tween 20 (0.1%) for 1 h, and the bands were visualized by probing with goat anti-mouse IRDye 800 (1:5000; Rockland) for 30 min. Membranes were scanned on an Odyssey fluorescence imager (LiCor), and each band was quantified using Molecular Analyst software (Bio-Rad, Inc.) with background subtraction. Values are expressed as optical density (OD) units ( SEM. Immunohistochemistry. Saline- or MAM-treated mice were perfused with 4% buffered paraformaldehyde, the brains cryoprotected in sucrose, and the tissues rapidly frozen and sectioned as previously described.14 Sagittal brain tissue sections (20 µm) were washed in 1% SDS for 5 min, blocked with M.O.M. reagent (Vector Labs) for 1 h, and then incubated with monoclonal anti-R-synuclein (1:250; BD Transduction Labs) for 30 min followed by anti-mouse Alexa 488 (1:500; Invitrogen) for 10 min. Tissue sections were counterstained with DAPI and examined by epifluorescence microscopy using a Zeiss Axioskop 2.

Results and Discussion Distinct Protein Profiles of the Normal Developing Cerebellum. To identify proteins that are targeted by MAM during cerebellar development, initial experiments focused on determining the proteins that are expressed at specific stages of cerebellar development such as granule cell proliferation (PND4) and maturation (PND22).19 Global gene expression analysis of the corresponding stages of cerebellar development showed that a significant portion of the transcriptome is regulated during maturation of the cerebellum.14 In this study, we examined the global changes in the proteome of the developing cerebellum and compared that with matching gene expression data.14 Protein extracts from the PND4 and PND22 cerebellum of saline-treated mice (controls) were labeled with Cy5 or Cy3 (respectively), the differentially labeled protein extracts were resolved by 2-D DIGE (see Figure 1), and protein identification was confirmed using tandem MS/MS. Spots that are predominantly red (Cy5) were preferentially expressed on PND4, while those that are predominantly green (Cy3) were preferentially expressed on PND22. Those that are yellow were comparably expressed at both ages. To quantitatively compare the differences between PND4 and PND22 samples, spot quantification and statistical analysis were performed using Phoretix (Nonlinear Dynamics, Inc.) software. The detection protocol identified 1024 spots (Table 1). After background subtraction and ratiometeric normalization, matched spots 2658

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Figure 1. Protein expression in the developing cerebellum of neonatal mice after treatment with MAM. Protein extracts (100 µg) prepared from the cerebellum of neonatal mice treated with saline or MAM at PND3 were combined and examined for differentially expressed proteins by 2D-DIGE. Representative gels from the following samples: (A) the cerebellum of saline-treated mice covalently labeled with Cy5 (PND4) or Cy3 (PND22), (B) the PND4 cerebellum of saline- and MAM-treated mice covalently labeled with Cy5 (saline) or Cy3 (MAM), and (C) the PND22 cerebellum of saline- and MAM-treated mice labeled with Cy5 (saline) or Cy3 (MAM).

were used for statistical analysis. Using the criteria of differential abundance of >1.5-fold change and p-value < 0.05, 742 (73%) spots were determined to be differentially expressed during development of the murine cerebellum (Table 1). Proteins identified from the differentially expressed spots and the corresponding mRNA expression14 are listed in Table 2. This is consistent with our previous work which showed that 63% of the genome is regulated during development of the neonatal mouse cerebellum.14 However, only ∼50% of the differentially expressed proteins exhibited a comparable change in gene expression.14 The discordance between proteomic and mRNA data has been noted before in several different systems.20-27 Although the transcriptome and proteome of the developing

MAM-Induced Changes in the Developing Cerebellum Table 1. Global Protein Expression Changes in the Cerebellum of Neonatal Micea comparison (vs saline)

no. spots matched

no. differentially expressed spots

% matched spots differentially expressed

PND4 vs PND22 PND4 MAM PND22 MAM

1024 1327 1239

742 394 401

72.46 29.69 32.36

a Spots were determined to be differentially expressed if the fold change was greater than 1.5.

CNS has not been compared, a recent study revealed that 409 genes (23%) out of 1758 genes in the adult mouse brain were significantly correlated with their gene products.28 These studies demonstrate that there are varying levels of expression between mRNAs and proteins in both neural and nonneural tissue emphasizing the importance of potential post-transcriptional regulatory mechanisms in CNS development that can be revealed only through integrated analysis of both proteins and mRNAs.29 The differentially expressed cerebellar proteins revealed significant changes in several functional classes including cytoskeletal, cell cycle and differentiation, ion binding and transport, chaperone, and metabolic proteins (Table 2). The high level expression of cytoskeletal and cell cycle and differentiation proteins during early stages of cerebellar development (i.e., PND4; Figure 1) indicates that these protein networks play an essential role in the proliferation and migration of neurons30 and that exposure to genotoxicants (e.g., MAM) at this early age may explain the pronounced influence of these agents on neonatal brain development.1,2 Ion channels, transporters, and cell signaling pathways are required to maintain an ionic gradient across the membrane, and these classes of proteins were especially upregulated in the late postnatal period of cerebellar development (i.e., PND22), a time when neurons require an ionic gradient for neurotransmisson. The results presented here indicate that the complex protein signaling networks of the mature cerebellum are attained through the temporal regulation of distinct classes of proteins. Previous studies have shown that genes that code for these different classes of proteins are especially enriched during development of the mouse cerebellum9,31 (see also cerebellar developmental transcriptome database, http://www.cdtdb.brain.riken.jp) and the maturation of cultured cerebellar granule cells.32 MAM Targets Distinct Proteins during Cerebellar Development. The neonatal cerebellum is a prime target for a number of environmental chemicals including the genotoxicant MAM.33-35 As a consequence, neurons within the neonatal cerebellum lose their ability to migrate appropriately to their final destination resulting in cellular dysmorphology and laminar disorganization, pathological features that often occur in many neurodevelopmental disorders (e.g., schizophrenia, epilepsy).36-38 We have recently demonstrated that DNA damage and altered gene expression precede MAM-induced cerebellar dysgenesis,14 suggesting that this genotoxicant induces neurodevelopmental changes by targeting genes that control the proliferation, migration, and maturation of neurons during critical periods of CNS development. Proteomic analysis of PND4 and PND22 cerebellum of MAMtreated mice by 2D-DIGE (Figure 1) and spot quantification revealed temporal changes induced by MAM with a minimal (10%) response at 24 h and robust changes at 14 days (27%)

research articles following treatment (Table 1). MAM altered the expression of several structural proteins (Table 3) including those that contribute to the maturation of neurons (i.e., Tubb2) and glia (i.e., GFAP, vimentin). These results are consistent with the pronounced influence of MAM on the in vitro assembly of microtubules and the axonal outgrowth of developing neurons39 and the corresponding disruption of granule cell migration and overexpression of GFAP and vimentin in the cerebellum of MAM-treated neonatal rats.40 Microtubules are polar, filamentous fibers that are dynamic structures in neurons, and they play an important role in mitosis, cell migration, neuronal differentiation, and the transport of cargo. The increased expression of the intermediate filament vimentin suggests that this genotoxicant disrupts neuronal migration by distorting the structural proteins of scaffolding cells (e.g., Bergman glia) such that granule cells ‘lose their way’ as they move into the internal granule cell layer. In the cerebellum, Bergman glia are generated before neurogenesis, and they play a critical role in guiding neuronal migration. However, the pronounced influence of MAM on other classes of proteins such as calcium homeostasis (e.g., calretinin, parvalbumin), neurofilaments (e.g., R-internexin), and the processing of mRNA (e.g., Tcea1) indicates that this genotoxicant disrupted multiple protein networks that may have contributed to the cerebellar developmental changes. The influence of MAM on several calcium binding proteins could have altered the intrinsic neuronal excitability of cerebellar granule cells41 and thus disturbed the cellular pathways that maintain the neuronal circuitry within the cerebellum. R-Internexin is the predominant neurofilament protein in immature granule cells,42 and mice that overexpress this protein (2-3fold) have pronounced deficits in motor coordination and balance.43 The overexpression of this protein could have contributed to the motor deficits observed in MAM-treated mice. MAM also influences RNA synthesis by inhibiting the activity of RNA polymerase II44 and by disturbing the processing and transport of RNA.45 The persistent downregulation of the transcription elongation factor (Tcea1 or S-II), a protein that reactivates RNA poly II after DNA damage,46 and RNA processing (e.g, Sfrs3) and transport proteins (e.g., Rbm8a) indicates that MAM severely disrupts the processing of RNA within the developing CNS, possibly by a DNA damage mechanism.47 The targeting of this important cellular process by MAM may have contributed to the neurodevelopmental and neurobehavioral changes. This global proteomic profiling approach has not only detected proteins that are known to be targeted by MAM (i.e., vimentin, GFAP), but has also revealed disturbances in other novel protein networks (i.e, calcium homeostasis, neurofilaments, and RNA processing) that could play a significant role in the neurodevelopmental changes triggered by this genotoxicant. MAM Targets Key Proteins Involved in Neurodegenerative Disease. Protein identification of differentially expressed spots revealed (Table 3) that MAM perturbs the expression of several different classes of developmentally regulated proteins (e.g., structural, transport, calcium homeostasis) and supports the hypothesis that the influence of MAM on CNS development is most likely due to the targeting of multiple protein networks and not due to specific disruption of a single pathway. The glucoside of MAM (i.e., cycasin) is considered as an etiological candidate for the prototypical neurodegenerative disorder western Pacific ALS/PDC,48,49 which is characterized by neurofibrillary tangles and R-synuclein aggregates in multiple brain regions (including cerebellum).50-52 The presence of mitochonJournal of Proteome Research • Vol. 5, No. 10, 2006 2659

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Table 2. Comparison of Gene and Protein Expression Changes of Developmentally Regulated Proteins in the Murine Cerebellum (PND4 vs PND22) Fold Change (P22/P4)

protein IPI ID

gene accession ID

00227299 00230394

BG073184 BG077403

Vimentin Lamin B1

00110851 00117352 00180675 00338039

AI836926 AI894247 AI840757 AI845505

Actin, beta, cytoplasmic Tubulin, beta 5 Tubulin, alpha 6 Tubulin, beta 2

00162945 00316959 00230766

AI844696 BG068687 AI846671

Internexin, alpha Centromere autoantigen F Parvalbumin

00116775

AI835962

RAB18, member RAS oncogene family

00115157 00119346

AI844674 AI836013

Synuclein, alpha Calretinin/Calbindin 2

00399510 00121209 00131614

BG077941 AI848203 AI836590

PDZ and LIM domain 5 Apolipoprotein A-I Synuclein, beta

Pdlim5 Apoa1 Sncb

36.51 29.75 20.81

1.06 -1.32 1.70

00132314 00131695 00331066

AI838705 BG079989 BG076015

Nucleobindin 1 Albumin 1 Calbindin-28K

Nucb1 Alb1 Calb1

12.00 9.83 2.98

-1.62 1.41 1.25

00399449 00416577

AI528529 AI528719

Syntaxin binding protein 3 Guanosine diphosphate (GDP) dissociation inhibitor 3

Stxbp3 Gdi3

-2.79 -1.63

1.01 -1.88

00468481

AI836589

General Cellular Function Atp5b 20.19

1.24

catalytic subunit in mitochondrial ATP synthesis

00407692

BG081377

3.13

1.72

00420349 00402788

AI845631 BG076306

ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit ATPase, H+ transporting, V1 subunit A, isoform 1 Dihydropyrimidinase-like 2 Beta-transducin repeat containing protein

Dpysl2 Btrc

-2.30 -2.16

-2.38 -1.33

Catalytic subunit of mitochondrial complex VI hydrolase activity in mitochondrion promotes degradation of phosphorylated proteins

00308885

BG073067

Other Hspd1

36.51

1.28

00318810 00230108 00128446

AI848309 AU042964 AI854369

-11.42 -3.24 -3.20

1.08 -1.09 -1.03

00406762 00223253

BG082990 BG084957

Cdc42bpb Hnrpk

3.17 -2.27

1.11 1.10

00136703 00319992

AI839796 AI843553

Cd80 Hspa5

2.05 -1.66

1.49 1.35

00153103

AI841681

1.90

1.32

name

symbol

Structural Components Vim -9.14 Lmnb1 6.52 Actb -5.42 Tubb5 4.77 Tuba6 -3.31 Tubb2 -1.94 Cell Cycle and Differentiation Ina 36.51 Cenpf -3.08 Pvalb 2.67

1.26 1.18 -1.15 -1.12 -2.88 -1.31 -1.04 1.53 -1.08 -1.03

Ion Binding and Transport Snca 54.56 Calb2 48.65

-1.39 -1.18

60 kDa heat shock protein, mitochondrial precursor GH regulated TBC protein 1 Coenzyme Q4 homologue (yeast) Cholinergic receptor, nicotinic, alpha polypeptide 4 Cdc42 binding protein kinase beta Heterogeneous nuclear ribonucleoprotein K CD80 antigen Heat shock 70kD protein 5 (glucose-regulated protein) JTV1 gene

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gene expression

-2.06

Rab18

Atp6v1a1

Grtp1 Coq4 Chrna4

Jtv1

drial proteins within these neuropathological hallmarks of neurodegenerative disease53 suggests that disturbances in energy metabolism may contribute to their accumulation. The proteins that were influenced by MAM (Table 3) demonstrate for the first time that this genotoxicant disrupts the processing of synuclein (i.e., R-synuclein, β-synuclein) and key proteins that maintain mitochondrial function (i.e., energy metabolism). The targeting of these two classes of proteins may help explain how this genotoxicant induces long-term effects on the CNS. R-Synuclein is a 140 amino acid (21 kDa), natively unfolded protein that acts as a chaperone,54 aids in vesicular transport,55 2660

protein expression

function

intracellular transport in Bergmann glia maintenance of nuclear structural integrity growth of dendritic spines microtubule-based process microtubule-based process microtubule-based process neuronal morphogenesis cell differentiation/ development calcium binding protein in stellate, basket, and Purkinje cells small GTPase mediated signal transduction synaptic vesicle transport calcium binding in granule cells and their parallel fibers links protein kinase C to Ca2+ channels cholesterol transport negative regulator of R-synuclein aggregation calcium homeostasis transport calcium regulation and signaling in Purkinje cells intracellular protein traffic protein transport

facilitates protein folding of mitochondrial proteins hormone activity unknown opens ACh receptor ion-channel for synaptic transmission diacylglycerol binding a pre-mRNA-binding protein T lymphocyte activation facilitates the assembly of multimeric protein complexes in the ER unknown

and contributes to synaptic plasticity. R-Synuclein inclusions are elevated in various neurodegenerative disorders including Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple system atrophy (MSA), and western Pacific ALS/PDC52,54,56 and in various animal models of neurodegenerative disease (e.g., paraquat, MPTP).57,58 Lewy bodies in the brain of patients with PD have been found to contain aggregates of R-synuclein. Studies have also shown that the non-Aβ component (NAC) of amyloid plaques that form in the brains of AD patients is the central hydrophobic region of R-synuclein.59 We show here that MAM had a pronounced effect on R-synuclein expression by

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MAM-Induced Changes in the Developing Cerebellum

Table 3. Comparison of Gene and Protein Expression Fold Changes MAM-Responsive Proteins in the Mouse Cerebelluma Fold Change (MAM/Saline) protein IPI ID

gene accession ID

p4 name

symbol

protein

Structural Components Gfap n/a

-1.47

20.61

Tubb2 Vim

1.40 1.74

1.55 -1.12

4.99 2.56

Tuba1 Lmnb1

1.25 2.55

1.27 -1.01

1.73 -1.36

Capping protein (actin Capza2 -2.01 -2.79 filament) muscle Z-line, alpha 2 Cell Cycle and Differentiation Internexin, alpha Ina 1.46 1.05 Parvalbumin Pvalb n/a -1.80

-1.12

-2.27 1.10

-1.82 -1.34

Glial fibrillary acidic protein, astrocyte 00338039 AI845505 Tubulin, beta 2 00227299 BG073184 Vimentin 00110753 AI840757 Tubulin, alpha 1 00230394 BG077403 Lamin B1 00111265 AI528740

00162945 AI844696 00230766 AI846671

00406163 AI843939 00129685 AI840032

Adenylate kinase 1 Translationally controlled tumor protein

00115157 AI844674 00119346 AI836013

Synuclein, alpha Calretinin/Calbindin 2

00226872 AI847883

EF-hand domain-containing protein 2 Synuclein, beta

00131614 AI836590

p22 mRNA

00117042 AI836096

Ak1 Tpt1

protein

n/a -2.29

2.40 -2.04

Ion Binding and Transport Snca -1.19 -1.11 -16.03 Calb2 1.00 1.22 -5.72 Efhd2

-1.18

-1.32

-2.42

Sncb

-1.20

1.51

-2.27

00131695 BG079989 Albumin 1 00227392 BG065012 Tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein, eta polypeptide 00227585 AI845489 Fatty acid-binding protein 7, brain

Alb1 Ywhah

2.05 1.08

1.06 1.17

-1.89 1.79

Fabp7

1.21

2.19

-1.79

00121209 AI848203 Apolipoprotein A-I 00331066 BG076015 Calbindin-28K

Apoa1 Calb1

-1.46 1.68

-1.57 -1.38

-1.61 1.59

mRNA

1.37 intermediate filaments in astrocytes 1.00 microtubule-based process 1.10 intracellular transport in Bergmann glia 1.22 microtubule-based process -1.03 maintenance of nuclear structural integrity -1.24 binds to fast growing ends of actin filaments -1.04 neuronal morphogenesis 1.03 calcium binding protein in stellate, basket, and Purkinje cells; absent in ALS spinal neurons -1.17 cell cycle control -1.12 apoptosis control -1.01 synaptic vesicle transport 1.08 calcium binding in granule cells and their parallel fibers -1.02 calcium binding -1.10 negative regulator of R-synuclein aggregation -1.03 transport -1.05 intracellular protein transport 1.41 growth of radial glial fibers in developing brain 1.35 cholesterol transport -1.21 calcium regulation and signaling in Purkinje cells

General Cellular Function Atp5b 1.32

n/a

Rbm8a

-1.10

n/a

-2.83 n/a

Cox5b

-1.08

1.27

-2.53

-1.43

00229510 BG073920 Lactate dehydrogenase 2, B chain 00404870 AI528616 Splicing factor, arginine/serine-rich 3

Ldh2 Sfrs3

-1.44 -4.47

-1.06 -2.04

2.26 -1.99

1.01 1.63

00228674 BG081444 Transcription elongation factor A

Tcea1

-1.93

-1.01

-1.96

-1.12

Enolase 2, gamma neuronal Eno2 NADH-ubiquinone oxidoreductase Ndufv2 24 kDa subunit, mitochondrial precursor 00230507 BG082389 ATP synthase, proton transporting, Atp5l mitochondrial F0 complex, subunit d Other 00230108 BG087515 Protein disulfide-isomerase A3 Pdia3

1.67 -2.67

1.44 1.35

-1.83 1.23

-1.10 1.01

-2.02

1.25

-1.10

1.13

-1.02

1.17

-1.74

00475138 AI840344

Stathmin

Stmn3

1.08

n/a

-1.65

00319992 AI843553

Heat shock 70kD protein 5 (glucose-regulated protein)

Hspa5

-1.62

n/a

1.32

00468481 AI836589

ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit 00410937 AK009953 RNA-binding protein 8A

00116154 AI851168

Cytochrome c oxidase, subunit Vb

00331704 AI839881 00109167 AI847609

a

9.86

function

-1.08 catalytic subunit in complex V surveillance and nuclear export of spliced mRNAs terminal oxidase in mitochondrial electron transport glucose metabolism RNA processing during cellular proliferation and/or maturation cleaves transcript at stalled RNA pol sites to allow resumption of elongation glucose metabolism component of mitochondrial complex I catalytic subunit in complex V

1.24 catalyzes rearrangement of disulfide bonds -1.16 regulates microtubules by destabilizing -1.00 facilitates the assembly of multimeric protein complexes in the ER

Names in bold are developmentally regulated proteins.

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Figure 2. R-Synuclein expression in the cerebellum of MAM-treated neonatal mice. The cerebellum of MAM-treated mice was examined for R-synuclein levels by both Western blotting and immunohistochemistry. (A) Western blot analysis of R-synuclein levels in the PND4 and PND22 cerebellum of saline- or MAM-treated mice (n ) 3). Protein extracts from a similar set of MAM-treated mice (n ) 3) were also immunoprobed for γ-actin levels. For comparison, the distribution of R-synuclein was examined by immunoprobing parasagittal sections (10 µm) of the cerebellum from PND22 mice that had been treated with saline (B) or MAM (43 mg/kg, sc) (C) with a monoclonal antibody to R-synuclein (Chemicon). Nuclei were identified by counterstaining tissues sections with DAPI [right panels in B and C]. Note folia (f) were significantly smaller in the cerebellum of MAM-treated mice, and heavy staining was localized over granule cells (GC, arrows) within certain folia. Magnification, ×10 (Insets: ×40). S ) saline.

causing a 16-fold downregulation of the protein in the cerebellum of PND22 mice (Table 3). This was confirmed by examining the PND22 cerebellum of several MAM-treated mice for R-synuclein levels (Figure 2A). A concomitant reduction in β-synuclein levels, a protein that inhibits the formation of R-synuclein inclusions or aggregates,60 was also observed in the PND22 cerebellum of MAM-treated mice. In contrast, R-synuclein levels were comparable in the PND4 cerebellum indicating that the influence of MAM on this protein was 2662

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delayed even though levels of this cerebellar protein do not vary much during neonatal mouse61 or human development.62 Since the two ages chosen reflect key stages of granule cell development, additional studies were conducted to determine if MAM also significantly reduced the expression of R-synuclein within the granule cell layer of the PND22 cerebellum (Figure 2). In contrast to the Western blotting data, immunohistochemistry (IHC) revealed heavy deposits of R-synuclein over granule cells within several cerebellar folia (i.e., IX & X) of MAM-

research articles

MAM-Induced Changes in the Developing Cerebellum

treated mice when compared with the corresponding cerebellum of saline-treated mice (compare Figure 2, panels B and C). The lack of staining of these deposits in adjacent sections probed without the antibody (data not shown) suggests that these immunoreactive deposits may be R-synuclein inclusions much like that produced in the brain of paraquat or MPTPtreated mice.58,63 One possible explanation for the differences noted is that the soluble form of R-synuclein was identified by Western blotting and DIGE because the supernatant was used for these experiments, while the IHC studies identified the insoluble form. Further studies will help determine if the observed heavy deposits within the cerebellum of MAM-treated mice are inclusions of R-synuclein. Of note, R-synuclein aggregates are also found in the cerebellum of ALS/PDC subjects, suggesting that these deposits may have been triggered by early exposure to cycad medicinal or food products. Mitochondrial dysfunction and free radical-induced oxidative damage have been implicated in the pathogenesis of neurodegenerative diseases such as Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD).64 Proteomics has recently provided insight into the disturbances of several mitochondrial and glycolytic proteins in the brains of subjects with ALS, PD, and AD.65-67 In this study, we observed that a number of mitochondrial respiratory chain complexes [e.g., complex I (Ndufv2), complex IV (cytochrome c oxidase), complex V (ATP synthase)] and glycolytic proteins (e.g., Ldh2, Eno2) were perturbed within the cerebellum of MAM-treated mice. MAM disturbed the expression of multiple complexes in the mitochondrial oxidative phosphorylation pathway, the primary cellular pathway for synthesizing ATP. MAM reduced the expression of one of the subunits of complex 1 (Ndufv2), a subunit that exhibits polymorphisms, which is a risk factor for PD68 and bipolar disorders.60 A subunit of cytochrome oxidase (i.e., Cox 5b), 1 of the 10 subunits of the mitochondrial complex IV that is encoded by a nuclear gene, was persistently downregulated by MAM. Two subunits of complex V (i.e., Atp5b, Atp5l), a motor enzyme that is essential for mitochondrial cristae formation,69 were also perturbed by MAM. Interestingly, cytochrome oxidase activity and ATP synthase are reportedly reduced in the brain and peripheral tissues of subjects with neurodegenerative disease.53,70-73 These findings suggest that MAM may induce its long-term effects on the CNS by disrupting mitochondrial function.

Summary A number of aspects of the developing nervous system are poorly understood. This includes its mechanisms of signaling, its cellular interactions, its complex protein networks, and the impact of environmental agents on these processes. However, technical developments in the field of proteomics have advanced our understanding of protein expression, function, and organization in signaling processes and regulatory networks, thus, providing deeper insight into how cellular protein networks are regulated in the developing nervous system. The present studies used the recently developed 2D-DIGE technique to identify the key protein networks that change during neonatal development of the cerebellum. As expected, cerebellar development was accompanied by changes in both neuronal and glial proteins, and this was shown by specific changes in structural, cell cycle and differentiation, ion binding and transport proteins, and metabolic enzymes. The pronounced targeting of many of these developmentally regulated proteins by MAM indicates that the neonatal CNS is especially vulner-

able to environmental genotoxicants. Perturbations in specific structural (e.g., GFAP, vimentin) and differentiation (e.g., internexin) proteins are consistent with the established neurodevelopmental changes (i.e., cerebellar dysgenesis) and motor deficits (e.g., ataxia) that develop in neonatal animals exposed to MAM. However, perturbations in several other classes of proteins (e.g., calcium homeostasis, RNA processing) were also noted, suggesting that the action of MAM on the developing CNS is more complex than previously thought and may involve the targeting of multiple protein networks. Along the same lines, we observed novel changes in synuclein (i.e., R-synuclein, β-synuclein) and mitochondrial proteins, indicating that this genotoxicant may induce long-term effects on the CNS by promoting inclusion formation or disrupting mitochondrial function. Further investigation of these latter cellular processes may provide important insight into how this genotoxicant acts as a ‘slow toxin’ to trigger neurodegenerative disease.74 Comparison of gene and protein expression patterns has also revealed differences in temporal expression, suggesting that the transcriptional response to an insult may not translate into measurable protein expression changes.

Acknowledgment. Supported by NIH Grant 5P42ES10338-02 [NIEHS Toxicogenomics Consortium] (S. R.N,) and, in part, by DOD grant DAMD 17-98-1-8625 (G.E.K.) and a NIEHS training grant ES007060 (T.P.). References (1) Ferguson, S. A. Neuroatnatomical and functional alterations resulting from early postnatal cerebellar insults in rodents. Pharmacol. Biochem. Behav. 1996, 55, 663-671. (2) Colacitti, C.; Sancini, G.; DeBiasi, S.; Franceschetti, S.; Caputi, A.; Frassoni, C.; Cattabeni, F.; Avanzini, G.; Spreafico, R.; Di Luca, M.; Battaglia, G. Prenatal methylazoxymethanol treatment in rats produces brain abnormalities with morphological similarities to human developmental brain dysgeneses. J. Neuropathol. Exp. Neurol. 1999, 58 (1), 92-106. (3) Sullivan-Jones, P.; Gouch, A. B.; Holson, R. R. Postnatal methylazoxymethanol: sensitive periods and regional selectivity of effects. Neurotoxicol. Teratol. 1994, 16, 631-637. (4) Shiraki, H.; Yase, Y. Amyotrophic lateral sclerosis in Japan. In Handbook of Clinical Neurology Vol. 22, System Disorders and Atrophy, Part 2; Vinken, P. J., Bruyn, G. W., Eds; American Elsevier: New York, 1975; pp 353-419. (5) Spencer, P. S.; Kisby, G. E.; Palmer, V. S.; Obendorf, P. Cycasin, methylazoxymethanol, and related compounds. In Experimental and Clinical Neurotoxicology, 2nd ed.; Spencer, P. S., Schaumburg, H. H., Eds.; Oxford University Press: New York, 1999; pp 436-447. (6) Mody, M.; Cao, Y.; Cui, Z.; Tay, K. Y.; Shyong, A.; Shimizu, E.; Pham, K.; Schultz, P.; Welsh, D.; Tsien, J. Z. Genome-wide gene expression profiles of the developing mouse hippocampus. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (15), 8862-8867. (7) Matsuki, T.; Hori, G.; Furuichi, T. Gene expression profiling during the embryonic development of mouse brain using an oligonucleotide-based microarray system. Brain Res. Mol. Brain Res. 2005, 136 (1-2), 231-254. (8) Kagami, Y.; Furuichi, T. Investigation of differentially expressed genes during the development of mouse cerebellum. Brain Res. Gene Expression Patterns 2001, 1 (1), 39-59. (9) Lim, C. R.; Fukakusa, A.; Matsubara, K. Gene expression profiling of mouse postnatal cerebellar development using cDNA microarrays. Gene 2004, 333, 3-13. (10) Katsel, P. L.; Davis, K. L.; Haroutunian, V. Large-scale microarray studies of gene expression in multiple regions of the brain in schizophrenia and Alzheimer’s disease. Int. Rev. Neurobiol. 2005, 63, 41-82. (11) Baranzini, S. E. Gene expression profiling in neurological disorders: toward a systems-level understanding of the brain. Neuromol. Med. 2004, 6 (1), 31-52. (12) Pasinetti, G. M. Use of cDNA microarray in the search for molecular markers involved in the onset of Alzheimer’s disease dementia. J. Neurosci. Res. 2001, 65 (6), 471-476.

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