Proteomic Analysis of Persistent Cortico-Striatal Synaptic NeuroAdaptations following Repeated Cocaine Exposure in Vervet monkeys.
Peter
1
Olausson ,
Dilja D.
1
Krueger
1Department
Christopher
of Psychiatry and
2
Colangelo ,
2Molecular
Kenneth R.
2
Williams ,
In order to assist the identification of potential mechanisms for cocaine-induced plasticity, we
attempted to provide a comprehensive analysis of protein alterations in the cortico-striatal circuitry
using an unbiased proteomics approach. We previously reported that repeated cocaine exposure to
Vervet monkeys (2 mg/kg/day for 14 days) was sufficient to produce concurrent and selective deficits
in reversal learning which is dependent on orbitofrontal cortex (OFC; see figure 1) and facilitation of
incentive aspects of motivation, supporting the notion that cocaine administration induces functionally
significant deficits in cortico-striatal functions (Olausson et al. 2007). The current study sought to
identify biochemical correlates of these behavioral effects in tissue taken from the same animals. Four
weeks after the last cocaine injection, monkeys were sacrificed, and tissue punches were taken from a
number of brain regions and subjected to multiplexed isobaric tagging technology (iTRAQ). A
number of cocaine-regulated proteins were identified that may be related to the behavioral effects
observed.
METHODS
Subjects and treatment: African green monkeys (Cercopithecus aethiops sabaeus) were trained to
perform food-rewarded object discriminations, and subsequently received daily injections of cocaine
(2 mg/kg, i.m.) or saline for 14 days (n = 8 per group). Following 14 days of withdrawal, monkeys
were tested on behavioral tasks, including attentional set-shifting, and sacrificed 4 weeks after the last
injection.
RESULTS
Prior Chronic Cocaine Exposure Produces Persistent
and Selective Deficits in Reversal Learning
Phase
140
Trials
120
Trial 1
Trial 2
to criterion
**
Saline
Simple
Cocaine
100
*
Complex
80
60
IDS
40
Reversal I
20
0
Simple Complex
IDS
Rev I
EDS
Rev II
EDS
Reversal II
Figure 1: Chronic Cocaine Exposure Persistently Impairs Reversal Learning. Prior
cocaine (2 mg/kg i.m.) exposure for 14 days produced profound and selective
deficits in reversal learning relative to saline-treated controls, but no effect on other
measures in the attentional set-shifting task.
Transcriptional Analyses of Cocaine-Induced Changes
in Synaptic Protein Expression
Persistent Cortico-Striatal Neuroadaptations at 1 Month Following Prior Chronic
Cocaine Exposure as Identified by iTRAQ
OFC (Area 11/12)
Dorsolateral PFC
Caudate
Protein ID
Fold regulation
Rho guanine nucleotide exchange factor (GEF) 17
-1.91
DJ179M20.1 (Adenosine deaminase)
-1.68
Putative lipid kinase
-1.65
Thymosin-like 4
-1.60
GDAP1-like1
-1.58
Claudin 11
-1.56
Seven transmembrane helix receptor
-1.56
DJ-1
-1.56
Endozepine (Fragment)
-1.55
Tropomyosin
-1.55
Mitochondrial NADP(+)-dependent malic enzyme 3
-1.54
Aquaporin 4
-1.51
Hypothetical protein DKFZp459K2229
-1.51
Neuronal pentraxin I precursor (NP-I)
-1.51
S100 calcium binding protein A1
-1.51
S100 protein, beta chain
-1.47
Fumarate hydratase
-1.47
GTP-binding protein G(I)/G(S)/G(O) gamma-3 subunit
-1.44
Carbonic Anhydrase II (Carbonate Dehydratase)
-1.44
Myelin Basic Protein (MBP)
-1.44
ACTA2 protein
-1.43
ELKS protein
-1.43
Hypothetical protein DKFZp468H1910
-1.43
Actin related protein 2/3 complex, subunit 4
-1.43
2'',3''-cyclic nucleotide 3'' phosphodiesterase
-1.42
Alpha-internexin (Neurofilament-66)
-1.41
Phosphoserine aminotransferase (PSAT)
-1.41
RAN protein
-1.40
Carbohydrate sulfotransferase 11
-1.40
Vacuolar protein sorting-associated protein 35
1.43
WD-repeat protein 37
1.43
Protein PM1
1.43
Cytochrome c oxidase subunit VIb isoform 1 (COX VIb-1)
1.43
NADH-ubiquinone oxidoreductase 30 kDa, mitochondrial
1.43
Kinesin-like protein KIF22
1.49
Hypothetical protein DKFZp459C1015
1.51
Succinyl-CoA ligase beta-chain, mitochondrial
1.52
Dipeptidylpeptidase VI isoform 1
1.53
MLL/SEPTIN6 fusion protein
1.55
Monoacyl glycerol lipase
1.57
Tubulin beta Class II
1.60
Regulator of G-protein signaling 6 (RGS6)
1.62
Antigen MLAA-34
1.64
Flotillin 1
1.67
KIAA0851 protein (Fragment)
1.74
Macrophage migration inhibitory factor
1.74
Ras homolog gene family, member T1
1.74
Rap1 GTPase-GDP dissociation stimulator 1
1.76
Protein phosphatase 2A, regulatory subunit B'
1.77
Transketolase
1.86
Rho-associated protein kinase 1
1.95
PKA catalytic subunit beta, isoform 2
2.06
Microsomal glutathione S-transferase 3
2.17
Ubiquinol-cytochrome c reductase complex 7.2kDa
2.28
Protein ID
Fold regulation
Hypothetical protein DKFZp459A0327
-1.59
WD-repeat protein 1 (Actin interacting protein 1)
-1.45
Hypothetical protein DKFZp459D219
-1.44
Crystallin, alpha B
1.41
Carbonyl reductase 1
1.41
Sirtuin 2 (NAD-dependent deacetylase)
1.48
Myelin-oligodendrocyte glycoprotein precursor
1.48
Versican V2 core protein precursor
1.49
ATP-binding cassette, sub-family C, member 1 isoform 7
1.56
Dorsal neural-tube nuclear protein
1.61
2'',3''-cyclic nucleotide 3'' phosphodiesterase
1.88
Myelin basic protein (MBP)
1.93
Myelin proteolipid protein (PLP)
1.93
Cytochrome c oxidase subunit Va (Fragment)
2.00
Myelin-associated glycoprotein precursor (Siglec-4a)
2.14
Rho/rac-interacting citron kinase
2.35
CD9 antigen
2.60
Protein ID
Nitric-oxide synthase, endothelial
Septin-5
Rac/Cdc42 guanine nucleotide exchange factor 6
ETHE1 protein, mitochondrial precursor
Synapsin-1
Mitochondrial carrier homolog 2
NADP-dependent malic enzyme, mitochondrial precursor
4F2 cell-surface antigen heavy chain (CD98 antigen)
Zinc transporter 3 (ZnT-3)
Trypsin precursor
Myelin basic protein (MBP
NADH dehydrogenase iron-sulfur protein 6, mitochondrial
Protein DJ-1 (Parkinson disease protein 7 homolog)
Riboflavin synthase beta chain
Acyl-CoA-binding protein (ACBP)
CD81 partner 3
Tubulin alpha-2 chain
Voltage-dependent anion-selective channel protein 2
NADH dehydrogenase 1 beta subcomplex subunit 4
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Ubiquinol-cytochrome c reductase complex 11 kDa
Actin, cytoskeletal 2
Pyruvate dehydrogenase protein X component
Septin-11
Receptor-type tyrosine-protein phosphatase zeta
Nucleoside diphosphate kinase A
Hexokinase-1
Myelin-oligodendrocyte glycoprotein precursor
Coiled-coil-helix-coiled-coil-helix domain protein 3
Heat-shock protein 105 kDa
Neural cell adhesion molecule L1 precursor (N-CAM L1)
Brain acid soluble protein 1 (BASP1 protein)
Cytochrome c oxidase subunit 5B, mitochondrial
Cytochrome c oxidase subunit 4 isoform 1
Tyrosine phosphatase, non-receptor type substrate 1
ATP synthase lipid-binding protein, mitochondrial
Metallothionein-3 (MT-3)
Glutamine synthetase
OFC (Area 13/14)
Protein ID
Fold regulation
Ras-related protein Rab-2A
-1.84
Guanine nucleotide binding protein (G protein), beta polypeptide
-1.70
2
Cytochrome c-1
-1.62
protein phosphatase 2A, regulatory subunit B
-1.53
Hexokinase 1 isoform HKI-R
-1.52
Heat shock protein 12A
-1.50
Myosin heavy chain
-1.45
14-3-3 protein eta
-1.43
Opioid-binding protein/cell adhesion molecule
-1.41
Insulin-like growth factor II precursor
1.45
Hippocalcin-1
1.49
Myelin Basic Protein (MBP)
1.54
ACTA2 protein
1.54
RAB3C
1.74
Attentional set-shifting: The effects of prior repeated cocaine exposure on cognitive processing was
examined using the attentional set-shifting task, a primate version of the Wisconsin Card Sorting Test
that is sensitive to neuroanatomically and neurochemically dissociable functions of the prefrontal
cortex. Specifically, monkeys were required to respond at either of two objects that differed based on
two distinct perceptual dimensions (i.e. shape and color/pattern). The task consisted of six distinct
phases within each session (1. Simple discrimination, 2. Complex discrimination, 3. Intra-dimensional
shift, 4. Reversal I, 5. Extra-dimensional shift, and 6. Reversal II). Following successful acquisition of
the response criterion (6 consecutive correct responses) the next test phase was initiated. Monkeys
were also tested on extinction learning and measures of motivated responding prior to secrifice (not
shown).
Tissue preparation: Four weeks after the last cocaine injection, monkeys were anesthetized with
ketamine, and brains were removed for biochemical analysis. During this process, monkeys were
intracardially perfused with ice-cold saline containing 25 mM sodium fluoride and 1 mM sodium
orthovanadate to minimize protein degradation and loss of post-translational modifications. Brains
were then cut into 5 mm thick slices using a primate brain matrix, and tissue punches were taken from
20 brain regions of interest using a large gauge tissue punch. Immediately, synaptoneurosomes were
isolated from the brain tissue using a modified version of Hollingsworth protocol (Hollingsworth,
1985) in a HEPES buffer and frozen in liquid nitrogen until use.
Sample preparation for iTRAQ analysis: iTRAQ analysis and mass spectrometric identification of
proteins was carried out by the Yale/NIDA Neuroproteomics Center on samples from four brain
regions: Nucleus accumbens, caudate, orbitofrontal cortex and medial prefrontal cortex. For each
brain region, 100 µg total protein per animal from each treatment group were pooled and resuspended
in iTRAQ buffer. Following Amino Acid Analysis, samples from saline- and cocaine-treated monkeys
were digested using trypsin and labeled with iTRAQ reagents 114 or 116 respectively. Pairs of
differentially labeled samples were pooled, subjected to cation exchange fractionation and on average
20 fractions analyzed using reverse-phase LC/MS/MS. Subsequent identification of peptides and
quantification of protein expression was conducted and searched using the Celera Primate database
with Protein Pilot 2.0.
and Jane R.
1
Taylor
Biophysics and Biochemistry, Yale University, New Haven CT.
INTRODUCTION
Cocaine addiction is known to involve long-lasting or persistent behavioral and neurochemical
alterations. Of particular importance may be the drug-induced changes in synaptic connections that
occur within cortico-striatal brain circuits that normally mediate reward, motivation and inhibitory
control. It has been previously shown that chronic administration of cocaine alters the density of
dendritic spines in the nucleus accumbens and prefrontal cortex (e.g. Robinson and Kolb 1999),
structures that have been related to cocaine-induced behavioral alterations. In addition, a number of
molecular substrates have been identified that are altered following cocaine administration that
influences synaptic function. However, the exact mechanisms underlying these cocaine-induced
structural and functional rearrangements remain to be identified.
Angus C.
1
Nairn
Figure 2: Bioinformatic transcriptional analysis of alterations in protein expression
within the OFC and the striatal regions using GeneGo MetaCore (see tables) suggest
that the transcription factors HNF4alpha and SP1 are highly integrated in the
proteomic map. Red circles identify up-regulated proteins, blue circles downregulated proteins.
Medial PFC
Protein ID
Kinase insert domain rec (type III receptor tyr kinase)
SNAP gamma
CD81 antigen
Mitotic checkpoint protein
Sirtuin type 2 (NAD-dependent deacetylase)
Tropomyosin
Dynamin-1
NAD-dependent aldehyde dehydrogenases
Neuronal calcium sensor 1 (NCS-1)
NCK-associated protein 1
Neurocan core protein precursor
Programmed cell death 8 isoform 2
Peroxiredoxin 6
Hypothetical protein DKFZp459G2127
BASP 1
TPA: Ras-related small GTPase
Flotillin 1
Fold regulation
-2.03
-1.81
-1.70
-1.62
-1.62
-1.61
-1.59
-1.58
-1.41
-1.41
1.40
1.41
1.43
1.44
1.48
1.55
2.09
Putamen
Nucleus Accumbens
Protein ID
Nucleoside diphosphate kinase B
Myelin proteolipid protein (PLP)
NADH-cytochrome b5 reductase
NADH dehydrogenase iron-sulfur protein 2
Heat shock 70 kDa protein II (HSP70 II)
Myelin-oligodendrocyte glycoprotein precursor
Tubulin polymerization-promoting protein (TPPP)
Vacuolar ATP synthase catalytic subunit A
Plexin-A1 precursor (Semaphorin receptor NOV)
Homer1
Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial
NAD-dependent deacetylase sirtuin-2
CD9 antigen
Myelin basic protein (MBP)
Ubiquitin-conjugating enzyme E2 N
Glyceraldehyde-3-phosphate dehydrogenase 4
UDP-glucuronosyltransferase 2B5 precursor
Electrogenic sodium bicarbonate cotransporter 1
Carbonic anhydrase 2
Growth factor receptor-bound protein 2 (GRB2)
Macrophage migration inhibitory factor (MIF)
Hippocalcin-like protein 1
Tropomyosin 1 alpha chain
Aquaporin-4
Malate dehydrogenase, mitochondrial precursor
Multidrug resistance-like ATP-binding protein mdlB
Wiskott-Aldrich syndrome protein 1 (WAVE-1)
3-hydroxyisobutyrate dehydrogenase, mitochondrial
DBP5 activity protein 2
Secretogranin-2 precursor (Secretogranin II)
Glutamate decarboxylase 2
Probable oxidoreductase KIAA1576
Acyl-CoA-binding domain-containing protein 7
Cystatin-B (Stefin-B)
Fold regulation
-2.58
-1.88
-1.87
-1.71
-1.68
-1.56
-1.52
-1.50
-1.46
-1.46
-1.45
-1.44
-1.44
-1.41
-1.41
-1.41
-1.41
1.41
1.41
1.42
1.42
1.46
1.48
1.49
1.54
1.56
1.63
1.63
1.64
1.64
1.69
1.76
1.92
2.16
2.97
3.01
3.18
3.59
Fold regulation
-1.72
-1.64
-1.60
-1.58
-1.56
-1.55
-1.53
-1.52
-1.51
-1.51
-1.50
-1.49
-1.43
-1.43
-1.41
-1.41
1.41
1.41
1.42
1.43
1.43
1.44
1.44
1.47
1.47
1.48
1.51
1.52
1.58
1.59
1.80
1.85
1.88
2.12
Protein ID
S100-A8
NADH dehydrogenase
Histidine triad nucleotide-binding protein 1
NADH dehydrogenase iron-sulfur protein 4, mitochondrial
Thioredoxin
Limbic system-associated membrane protein
AP-2 complex subunit alpha-1
Protein phosphatase PP2A 55 kDa regulatory subunit
Tubulin alpha-13 chain
cAMP and cAMP-inhibited cGMP 3'',5''-cyclic PDE10A
Myelin proteolipid protein (PLP)
GTP-binding protein G(I)/G(S)/G(O) gamma-3 subunit
Aconitate hydratase, mitochondrial precursor
Ran-specific GTPase-activating protein
Myelin basic protein (MBP)
Neurogranin
Pyruvate dehydrogenase protein X component, mitochondrial
Sarcoplasmic/endoplasmic reticulum calcium ATPase 1
D-beta-hydroxybutyrate dehydrogenase, mitochondrial
Centrosome-associated protein CEP250
Myelin-associated glycoprotein precursor
Thioredoxin-dependent peroxide reductase, mitochondrial
Annexin A7
Phosphoglucomutase-1
Hydroxyethylthiazole kinase 2
2'',3''-cyclic-nucleotide 3''-phosphodiesterase
Adenylate kinase isoenzyme 4, mitochondrial
Claudin-11
Inclusion membrane protein D
Ferritin light chain
Enolase
Undecaprenyl-diphosphatase 1
Phospholemman precursor
Cell division control protein 42 homolog
Glial fibrillary acidic protein, astrocyte (GFAP)
Fold regulation
-5.22
-2.18
-2.00
-1.80
-1.67
-1.59
-1.57
-1.55
-1.54
-1.54
-1.52
-1.52
-1.43
-1.43
-1.43
-1.42
-1.41
-1.41
1.41
1.42
1.42
1.42
1.48
1.48
1.49
1.52
1.54
1.55
1.59
1.62
1.69
1.82
2.00
2.03
2.85
Figure 3: Tables of synaptic proteins identified as regulated following chronic cocaine administration. Numbers represent the
fold regulation in cocaine-administered animals, based on the ratio between iTRAQ ligand 116 (cocaine) and 114 (saline).
Current experiments are conducting secondary confirmations of selected target proteins using Western Blot and quantitative
real-time PCR.
CONCLUSIONS
• Chronic exposure to cocaine results in regulation of a large number of metabolic enzymes, suggesting an changes in energy demand possibly due to alterations in
metabolically active spines and synapses.
• In addition, a number of proteins related to intracellular signaling (including small GTPases, kinases, phosphatases and calcium-binding proteins), protein
turnover and cytoskeletal rearrangement were identified, consistent with the hypothesis that cocaine-induced changes include both structural and functional
alterations of synaptic function.
•Transcriptional analysis of cocaine-induced regulation of synaptic proteins using bioinformatics suggest the involvement of the transcription factors HNF4alpha
and SP1.
Supported by Yale/NIDA Neuroproteomics Research Center (1 P30 DA018343-0), NIDA grants DA11717 (JRT) and DA10044 (ACN).