ARTICLE pubs.acs.org/jpr
Kainate Promotes Alterations in Neuronal RNA Splicing Machinery Michael Rohe,‡ Grit Nebrich,† Oliver Klein,† Lei Mao,† Claus Zabel,† Joachim Klose,† and Daniela Hartl*,† † ‡
Institute for Human Genetics, Charite-University Medicine, Berlin, Germany Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany
bS Supporting Information ABSTRACT: Kainate, a glutamate analogue, activates kainate and AMPA receptors inducing strong synaptic activation. Systemic kainate application to rodents results in seizures, neurodegeneration, and neuronal remodeling in the brain. It is therefore used to investigate molecular mechanisms responsible for these conditions. We analyzed proteome alterations in murine primary cortical neurons after 24 h of kainate treatment. Our 2-D gel based proteomics approach revealed 91 protein alterations, some already associated with kainate-induced pathology. In addition, we found a large number of proteins which have not previously been reported to be associated with kainate-induced pathology. Functional classification of altered proteins revealed that they predominantly participate in mRNA splicing and cytoskeleton remodeling. KEYWORDS: Kainic acid, kainate, epilepsy, primary neuron, proteome, BDNF, glutamate, alternative splicing, neurodegeneration
T
he glutamatergic system is by far the most abundant excitatory neurotransmitter system in the mammalian brain.1 Glutamate receptors are categorized into different subtypes that are defined by pharmacological agonists that were used in their characterization. Kainate receptors (KAR) are one subset of glutamate receptors with particular affinity to kainate (KA), a glutamate agonist, first isolated as toxic agent from seaweed more than 50 years ago. KARs consist of specific tetrameric combinations of glutamate receptor (GluR) subunits GluR5, GluR6, GluR7, KA1, and KA2.2 Cells from different brain regions express distinct subsets of KAR subunits, and even within one cell, subunit composition of KARs varies according to subcellular localization.2,3 KARs act as ligand-gated cation channels that can be activated by low concentrations of KA. These receptors display rapid activation and desensitization characteristics. The major physiological function of KARs is the regulation of network activity in many brain regions.2,4 KA also activates Ramino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR), a second type of glutamate receptors, which consist of tetrameric combinations of subunits GluR1-4 and possess an approximately 1000-fold lower affinity to KA than KARs. They are the most abundant receptors in the CNS and mediate fast synaptic transmission.4,5 Besides the endogenous functions of kainate as agonist for KARs and AMPARs, exogenous application of the glutamate analogue KA has been widely used to model temporal lobe epilepsy in rodents.4,6 Upon systemic or intracerebral administration, KA induces seizures originating in the hippocampus and spreading to other regions of the limbic system. KA receptors are particularly enriched in hippocampal CA3 pyramidal neurons. r 2011 American Chemical Society
This neuronal population is the first in the brain responding to even minimal KA doses.4,7 In addition to epileptic seizures, KA application was used to model neuronal excitotoxicity since high doses of KA can overactivate and damage neurons. After the induction of seizures and/or neuronal damage by KA, reorganization of neuronal architecture ensues.8 Despite using KA to model seizures, excitotoxicity, and neuronal remodeling, little is known about KA target proteins. Some studies have already analyzed proteome changes in total rat brains after KA administration, but as a result of brain complexity, interpretation of these results is difficult because of the limited spatial resolution power for protein changes when using total brain extracts.9-11 To increase our current understanding of molecular mechanisms contributing to KA administration in neurons, we analyzed the proteome of mouse KAtreated primary cortical neurons using a large gel 2-D electrophoresis-based proteomics approach (2-DE). We compared our data set of changed proteins to a newly established reference data set that comprises a large fraction of the brain proteins identified on our 2-DE gels. This data set is important to perform a meaningful enrichment analysis for the KAinduced protein changes and to determine overrepresented pathways. Interestingly, we found primarily proteins associated with mRNA splicing to be altered after KA treatment of primary cortical neurons, a process important in neurons to maintain excitability. Received: July 28, 2010 Published: January 25, 2011 1459
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Table 1. Proteins Altered after KA Treatment of Primary Cortical Neurons direction of protein name
gene regulation (treated p- ratio (treated/ SwissProt name vs control) value control) accession
pI
MW
score
identified sequence peptides coverage (%)
Acetyl-CoA acetyltransferase, mitochondrial
Acat1
down
0.027
0.719
Q8QZT1
8.71
44787 248
5
12
Vacuolar ATP synthase subunit F
Atp6v1f
up
0.002
1.213
Q9D1K2
5.52
13362 131
8
84
T-complex protein 1 subunit γ (TCP-1-γ)
Cct3
down
0.001
0.764
P80318
6.28
60591 172
4
5
Charged multivesicular body protein 2b
Chmp2b
up
0.043
1.384
Q8BJF9
8.82
23919
51
1
5
Creatine kinase U-type, mitochondrial
Ckmt1
down
0.014
0.867
P30275
8.39
46974 560
9
25
Dihydropyrimidinase-related protein 1 Dihydropyrimidinase-related protein 1
Crmp1 Crmp1
down down
0.015 0.023
0.839 0.859
P97427 P97427
6.63 6.63
62129 135 62129 59
15 7
13 13
Dihydropyrimidinase-related protein 1
Crmp1
down
0.041
0.851
P97427
6.63
62129
59
7
13
Dynactin subunit 2
Dctn2
up
0.049
1.217
Q99KJ8
5.14
44090 134
2
7
Dihydropyrimidinase-related protein 2
Dpysl2
up
0.023
1.244
O08553
5.95
62132
54
5
13
Destrin (Actin-depolymerizing factor) (ADF)
Dstn
up
0.015
1.309
Q9R0P5
8.14
18509 382
6
31
Fumarate hydratase 1
Fh1
down
0.044
0.827
P97807
9.12
54336 200
3
10
Fascin (Singed-like protein)
Fscn1
down
0.034
0.796
Q61553
6.21
54371
75
2
5
Ferritin light chain 1 Glial fibrillary acidic protein, astrocyte (GFAP)
Ftl1 Gfap
down up
0.040 0.002
0.801 1.105
P29391 P03995
5.66 5.36
20790 248 49878 683
4 9
20 22
Glial fibrillary acidic protein, astrocyte
Gfap
up
0.008
1.152
P03995
5.36
49878 299
5
12
Growth factor receptor-bound protein 2
Grb2
up
0.012
1.214
Q60631
5.89
25222 184
5
21
Hepatoma-derived growth factor-related protein 3 Hdgfrp3
up
0.008
1.199
Q9JMG7
8.39
22417
96
1
12
Heterogeneous nuclear ribonucleoprotein D0
Hnrnpd
down
0.003
0.871
Q60668
7.62
38330 231
4
14
Heterogeneous nuclear ribonucleoprotein L
Hnrnpl
up
0.022
1.120
Q8R081
8.33
63923 224
4
8
Heterogeneous nuclear ribonucleoprotein A1 Heterogeneous nuclear ribonucleoprotein K
Hnrnpa1 Hnrnpk
up down
0.031 0.043
1.163 0.884
P49312 P61979
9.27 5.39
34175 269 50944 226
5 5
22 11
Heterogeneous nuclear ribonucleoprotein K
Hnrnpk
up
0.031
1.279
P61979
5.39
50944 366
7
13
Homer protein homologue 1
Homer1
up
0.018
1.510
Q9Z2Y3
5.39
41388 586
9
32
Stress-70 protein, mitochondrial precursor
Hspa9
down
0.007
0.901
P38647
5.91
73483 657
8
15
Insulin-like growth factor 2 mRNA-binding
Igf2bp3
up
0.013
1.354
Q9CPN8
8.99
63535 104
2
5
(Adapter protein GRB2)
protein 3 LIM and SH3 domain protein 1
Lasp1
down
0.030
0.861
Q543N3
5.11
23087 444
7
31
Microtubule-associated protein 1B Trans-2-enoyl-CoA reductase, mitochondrial
Mtap1b Mecr
up up
0.017 0.007
1.445 1.826
P14873 Q9DCS3
4.76 270245 178 9.17 40317 703
5 5
2 15
NADH dehydrogenase [ubiquinone]
Ndufb10
up
0.042
1.210
Q9DCS9
8.19
21010 168
3
21
Nhp2l1
down
0.001
0.773
Q9D0T1
8.72
14165
70
2
9
Programmed cell death 6-interacting protein
Pdcd6ip
down
0.0387
0.592
Q9WU78
6.15
95950
83
2
2
Astrocytic phosphoprotein PEA-15
Pea15a
up
0.007
1.602
Q62048
4.94
15045
86
2
22
Phosphoglycerate kinase 1 Polyglutamine-binding protein 1
Pgk1 Pqbp1
down up
0.046 0.014
0.896 1.509
P09411 Q91VJ5
7.53 5.86
44508 178 30579 120
4 4
10 13 18
1 β subcomplex subunit 10 NHP2-like protein 1 (High mobility group-like nuclear protein 2 homologue 1)
Peptidyl-tRNA hydrolase 2, mitochondrial OS
Ptrh2
up
0.027
1.467
Q8R2Y8
6.96
19514 195
2
RNA-binding protein 4B
Rbm4b
up
0.007
1.141
Q8VE92
6.28
39966
83
1
4
Oxygen-regulated protein 1
Rp1
down
0.019
0.791
P56716
7.4
234241
27
1
0
Stromal cell-derived factor 2 precursor (SDF-2)
Sdf2
up
0.001
1.246
Q9DCT5
6.83
23144
27
1
6
Septin-7
Sept7
up
0.017
1.094
O55131
8.73
50518 568
9
23
Septin-7
Sept7
up
0.048
1.226
O55131
8.73
50518 515
9
20
Plasminogen activator inhibitor 1 RNA-binding protein
Serbp1
up
0.031
1.148
Q9CY58
8.6
44687 277
4
12
Plasminogen activator inhibitor 1
Serbp1
up
0.003
1.194
Q9CY58
8.6
44687 225
4
16
RNA-binding protein Splicing factor 1
Sf1
up
0.003
1.339
Q64213
8.98
70363
86
2
2
Splicing factor, arginine/serine-rich 1
Sfrs1
down
0.023
0.754
Q6PDM2 10.37
27728
45
1
4
Syntaxin-binding protein 1
Stxbp1
up
0.045
1.425
O08599
67526 183
2
2
1460
6.49
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Table 1. Continued direction of gene regulation (treated p- ratio (treated/ SwissProt protein name
name
vs control)
value
control)
identified
accession
pI
MW
score
sequence
peptides coverage (%)
Synapsin-1
Syn1
up
0.013
1.327
O88935
9.81
74052 137
4
Transaldolase
Taldo1
down
0.030
0.711
Q93092
6.57
37363 117
3
6 8
Transaldolase
Taldo1
down
0.013
0.742
Q93092
6.57
37363 194
19
41
Translationally controlled tumor protein
Tpt1
down
0.050
0.824
P63028
4.76
19450 157
3
21
Tubulin R-1 chain (R-tubulin 1)
Tuba1a
down
0.035
0.724
P68369
4.94
50104
46
1
3
Tubulin R-1 chain (R-tubulin 1)
Tuba1a
down
0.003
0.776
P68369
4.94
50104
69
1
3
Tubulin R-1 chain (R-tubulin 1) Elongation factor Tu, mitochondrial precursor
Tuba1a Tufm
down down
0.024 0.022
0.818 0.869
P68369 Q8BFR5
4.94 7.23
50104 49477
84 94
1 2
4 5
Ubiquitin carboxyl-terminal hydrolase isozyme L5 Uchl5
down
0.046
0.713
Q9WU7
5.24
37593
62
2
7
Ubiquinol-cytochrome-c reductase complex core
up
0.042
1.114
Q9CZ13
5.75
52735 290
4
10
Uqcrc1
protein 1, mitochondrial precursor
’ MATERIALS AND METHODS Preparation and Treatment of Primary Neurons
Primary cortical neurons were prepared from newborn Balb/c mice at postnatal day 1. Cortical cells were dissociated using papain (1 h at 37 °C) and cultured on poly-D-lysine/collagencoated culture dishes. Neurons were cultured for 5 days in Neurobasal-A medium (Gibco) containing B27 supplement (Sigma) and GlutaMAX (Invitrogen). The neurons were then treated with 50 μM KA (Sigma) or medium only (control) for 24 h by replacing half of the culture medium with fresh medium. After treatment, neurons were still viable and no differences in death rate were observed between treated and untreated cells. The cells were harvested in ice-cold PBS, and cell pellets were frozen immediately in liquid nitrogen. Six individual samples of treated and control neurons were collected (n = 6). Protein Extraction and 2-DE
Protein extracts were prepared from frozen cell pellets according to our updated protein extraction protocol.12,13 Briefly, sample buffer (50 mM TRIZMA Base (Sigma-Aldrich), 50 mM KCl, 4.5% CHAPS, and 20% w/v glycerol at pH 7.5) and a proteinase inhibitor cocktail (Complete, Roche Diagnostics) were added to the samples. They were ground to fine powder in a mortar cooled by liquid nitrogen and subsequently sonicated on ice. Afterward, DNase and urea/thiourea were added to the samples. The protein extracts were then supplied with 70 mM dithiothreitol (Biorad), 2% v/w of ampholyte mixture (Servalyte pH 2-4, Serva) and stored at -80 °C until use. Protein samples were separated by the large-gel 2-DE developed in our laboratory as described previously.12,14-19 The gel format was 40 cm (isoelectric focusing) 30 cm (SDS-PAGE) 0.9 mm (gel width). Two-dimensional protein patterns were obtained after silver staining.12 Spot Evaluation Procedure
Protein spot patterns were evaluated by Delta2D imaging software (version 4.0, Decodon). Briefly, protein patterns were matched using “exact” mode of Delta2D. Subsequently, a fusion image was generated employing “union” mode, creating a protein pattern containing all spots from all 2-D gels within the project. Digital spot detection was carried out on the fusion image, followed by manual removal of detected entities that could not be assigned to a spot. A spot pattern containing 2182 protein
spots was then transferred from the fusion image to all other images in the project. Percent spot volume was used for quantitative analysis of protein expression. The spot volume of each spot is the product of its size and intensity. Normalized values (after background subtraction) were exported from Delta2D to spreadsheet format for statistical analysis. After confirming normal distribution, data sets were analyzed using a paired Student’s t test.20 Treated and control samples were always handled in pairs starting at protein extraction until after completion of 2-D gel runs (n = 6). Mass Spectrometry
For protein identification by mass spectrometry, 1200 μg of protein extract was separated by 2-DE and stained using a MScompatible silver staining protocol.12 Protein spots of interest were excised from 2-D gels and subjected to in-gel tryptic digestion. Peptides were analyzed by an ESI-MS/MS on a LCQ Deca XP ion trap instrument (Thermo Finnigan, Waltham, MA). Alternatively, a Reflex 4 MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen Germany) was used for identification, as described previously.14,15,20-22 Mass spectra were analyzed using our in-house MASCOT software package (version 2.1) automatically searching NCBI databases (SwissProt, version 51.8). MS/MS ion search was performed using this set of parameters: (i) taxonomy, Mus musculus; (ii) proteolytic enzyme, trypsin; (iii) maximum of accepted missed cleavages, 1; (iv) mass value, monoisotopic; (v) peptide mass tolerance, 0.8 Da; (vi) fragment mass tolerance, 0.8 Da; and (vii) variable modifications, oxidation of methionine and acrylamide adducts (propionamide) for cysteine. Furthermore, the molecular weight and pI of each protein identified by database search was compared to values obtained from our 2-DE patterns. All significantly altered proteins identified by mass spectrometry are listed in Table 1. Functional Annotation and Enrichment Analysis
Gene symbols representing identified proteins were used to determine GO and KEGG categories using an online tool, Webgestalt, version 2 (http://bioinfo.vanderbilt.edu/webgestalt/index.php).23 In addition, enrichment of cellular pathways as compared to proteins identified on a 2-D gel was determined using a Hypergeometric test with more than 2 proteins per group and p e 0.05. Multiple test adjustment (Benjamini and 1461
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Figure 1. Protein spot alterations after KA treatment of primary cortical neurons. Protein spot changes are highlighted in blue on a representative 2-D gel of primary mouse cortical neurons. Changes of the same protein found in more than one spot are highlighted in red along with the corresponding gene name. Results were obtained by comparing KA-treated (t = 24 h) to untreated primary cortical neurons.
Figure 2. (A) Immunoblot analysis of Sf1 and SF2/ASF after KA treatment. In neurons treated with KA (50 μM; for 24 h) the splicing factors Sf1 and SF2/ASF were significantly altered as determined by Western blot analysis. (B) Densitometric quantification of replicate experiments (n = 4) revealed significant (p e 0.05) down-regulation of SF2/ASF and up-regulation of Sf1 in KA-treated as compared to nontreated (Ctrl) neurons. Tubulin served as loading control.
Hochberg) was also carried out as provided by Webgestalt. However, it is important to mention that the changes in protein levels observed may not be independent because of coexpression of proteins as modules and networks, though hypergeometricbased tests will assume that they are. This could limit the performance of our statistical test based on hypergeometric distribution. In addition to Webgestalt, we evaluated our data using the online tool DAVID (http://david.abcc.ncifcrf.gov/).24,25 Using this tool, gene functional classification applying GOannotation was performed. Enrichment scores were calculated for functional groups. A score of 1.3 is equivalent to non-log
scale 0.05; scores higher than 1.3 indicate more significant enrichment. Immunoblotting
Protein concentration was determined using the Roti-Nanoquant assay (Carl Roth). Neuronal protein extracts were separated using 12% SDS-PAGE gels, blotted to PVDF membranes, and probed with antibodies directed against Splicing factor, arginine/serine-rich 1 (Sfrs1; SF2/ASF) or Splicing factor 1 (Sf1) (Abcam) according to standard immunoblotting procedures. Detection of signals was carried out using a Fujifilm 1462
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Table 2. Proteins Altered in This Study Previously Associated with KA Administration expression protein (gene name)
changea
role in KA treatment outcome
v
Interacts with GluR5 by targeting mGluR5 to dendritic synaptic sites and/or axons, regulated by neuronal
Homer 1
activity;56,57 up-regulation observed after KA administration;11,28 changes in alternative splice variant distribution induced by seizures.58 Grb2
v
Electroconvulsive seizure and KA treatment induce up-regulation of Grb2, an important mediator of surface receptor signaling.11,29,59
Gfap
v
KA induces up-regulation of Gfap.29,60
Hnrnpa1 Stxbp1 (Munc-18-1)
v v
Shows increased binding to RNA after depolarization in neurons.47 Munc-18-1 is crucial to calcium-dependent neurotransmitter release, facilitates synaptic vesicle docking and fusion.61
Syn1
v
Synapse-specific protein, up-regulation was observed in synapses of mice following KA administration.11
Sept7
v
Up-regulation of mRNA observed in KA-treated rats.28
Sfrs1 (SF2/ASF)
V
Regulates GluR2 alternative flip/flop splicing.51
Tufm
V
Down-regulated in the brain of KA-treated rats.10
Tpt1
V
Altered in mouse synapses after KA administration.11 Exposure of cortical neurons to KA increases dendrite growth mediated by cytoskeletal reorganization.27
Proteins associated with cytoskeletal organization: Tuba1a, Lasp1, Fscn1, Crmp1
V
Mtap1b, Hdgfrp3
v
Fscn1 is important for filopodial formation.62 Crmp1 is involved into neurotrophin-induced neurite extension.63 Mtap1b is highly expressed in neuronal growth cones.64 Hdgfrp3 promotes neurite outgrowth by interaction with microtubules.65
a
v = up-regulation, V = down-regulation.
LAS-1000 device and densitometric scans of replicate experiments were performed with Advanced Image Data Analyzer (AIDA) software (Raytest) (Student’s t test p e 0.05, n = 4).
’ RESULTS Proteins Altered after KA Treatment
Mouse primary cortical neurons were cultivated and treated with 50 μM kainic acid (KA) or medium (control) for 24 h. Six individual samples of treated and control neurons were collected, and proteins were extracted and separated by 2-DE. After quantitative evaluation of the 2-D gels using Delta2D, we applied a statistical analysis (paired Student’s t test) to identify protein spots significantly altered (p e 0.05) between treated and control neurons. We found that 91 protein spots (47 up- and 44 down-regulated) were altered significantly when treated neurons were compared to controls (Figure 1). Identification and Functional Analysis of Altered Proteins
We next identified significantly altered protein spots by mass spectrometry. We determined the identity of 56 (62%) of all altered protein spots (Table 1). This corresponds to 47 nonredundant proteins. Seven proteins were identified in more than one protein spot representing different protein isoforms (protein splice variants or modified forms of the same protein; see Figure 1). Only one protein, Heterogeneous nuclear ribonucleoprotein K (Hnrpk), showed differential regulation in protein isoforms. Of a total of two, one isoform was up- and one downregulated. All other proteins identified with more than one isospot showed always the same direction of expression alteration. To see whether observed alterations can be reproduced using another method we analyzed the expression levels of two proteins, Splicing factor, arginine/serine-rich 1 (Sfrs1; SF2/ASF) and Splicing factor 1 (Sf1), through Western blot analysis. Immunoblots of both proteins showed significant (p e 0.05)
quantitative changes between KA-treated and control neurons (a representative blot is shown in Figure 2). The direction of changes was the same as previously observed on 2-D gels (upregulation of Sf1 and down-regulation of SF2/ASF in KA-treated neurons, respectively). We next determined if some of the altered proteins were already reported to be regulated in response to KA treatment. Seven proteins have been described previously to be altered after KA treatment in rodents. Furthermore, 8 proteins are indirectly associated to KA as they participate in processes that have been linked to change upon KA treatment. These proteins and a summary of their relationship to KA treatment are summarized in Table 2. We next characterized major functional groups within our data set of altered proteins. Careful literature-based functional classification of altered proteins suggested that the majority of altered proteins were associated with mRNA splicing. In addition, cytoskeleton remodeling, protein sorting and metabolism are also associated with the proteins altered after KA treatment. We next used the software tool Webgestalt (http://bioinfo.vanderbilt.edu/ webgestalt/) to characterize our data set. We decided to employ Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) based terms to analyze our data set. KEGG annotation of altered proteins yielded the following pathways: Spliceosome, Oxidative phosphorylation, MAPK signaling, Neurodegenerative Disorders, Fatty acid metabolism, Amino acid metabolism, Glycolysis, Citrate cycle, Axon guidance and Insulin signaling/JakSTAT signaling/Focal adhesion. However, only 13 of 47 nonredundant proteins could be annotated with KEGG terms. In contrast, all proteins could be annotated with GO terms. We therefore decided that KEGG analysis was not suitable for our data set, and our further analysis was conducted using GO annotation. Enrichment Analysis of Altered Proteins
In order to determine overrepresentation of any pathways in our data set, we created a reference data set using a large number 1463
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Table 3. Enriched GO Terms with Associated Proteins Altered after KA Treatment enrichment,a compared to mouse GO term
2-D reference data set
genome
proteins (gene names)
Biological Process mRNA metabolic process
X
X
Hnrnpd, Sf1, Hnrnpl, Hnrnpk, Rbm4b, Sfrs1, Nhp2l1, Hnrnpa1
X
X
Hnrnpd, Igf2bp3. Stxbp1
RNA splicing
X
X
Hnrnpk, Rbm4b, Nhp2l1, Hnrnpa1 Sfrs1, Sf1
Generation of precursor metabolites and energy
X
Pgk1, Ndufb10, Fh1, Atp6v1f, Uqcrc1
Microtubule-based process
X
Dctn2, Tuba1a, Mtap1b
RNA binding
X
Posttranscriptional
regulation
of
gene
expression
Molecular Function Cytoskeletal protein binding
X
Sf1, Nhp2l1, Rbm4b, Hnrpa1, Hnrpl, Sfrs1, Hnrnpk, Igf2bp3, Serbp1, Hnrnpd
X
Dstn, Lasp1, Fscn1, Mtap1b, Syn1
Cellular Component Spliceosomal complex
X
X
Ribonucleoprotein complex
X
X
Hnrnpa1, Hnrnpk, Hnrpl, Hnrpd Sf1, Nhp2l1, Sfrs1
Microtubule associated complex
X
X
Pea15a, Dctn2, Mtap1b
X
Ndufb10, Fh1, Stxbp1, Dpysl2, Acat1, Tufm, Mecr, Ckmt1, Ptrh2, Hspa9, Uqcrc1
Mitochondrion
Sf1, Nhp2l1, Hnrnpa1, Hnrnpk, Sfrs1
a
Enrichment of GO-terms among proteins altered after KA treatment of neurons as compared to a reference data set comprising over 60% of all proteins that can be found on large 2-D gels for neurons or the mouse genome, respectively.
of proteins that were identified on our 2-D gels without bias to disorder, brain region, and age. We used data from a large number of studies employing 2-D gels that investigated mouse brain and neuronal cells. Since the data set contained proteins identified in mouse brain and we analyzed neurons in this study, we also checked that all protein spots identified in brain tissue were also present on 2-D gels of primary neurons. Experimental data from different studies were combined using the gene names obtained from identified proteins. This approach was chosen because different protein names but only one gene name exists for many proteins. This proteome reference data set consisted of 1022 nonredundant proteins (Supplementary Table 1). This number represents about 60% of the protein diversity which can be resolved on our large 2-D gels as protein spots. This was estimated by counting the total number of protein spots observed on the gels and comparing it to the number of proteins included in our reference data set. We then analyzed overrepresentation of protein categories within our reference data set as compared to the whole mouse genome. We found significant enrichment of GO terms Oxidation reduction, Cellular carbohydrate catabolic process, carboxic acid metabolic process and generation of precursor metabolites and energy, Coenzyme binding, RNA binding, Purine nucleotide binding, Oxidoreductase activity, Cytoplasm, Organelle and Mitochondrion. We next compared our GO-annotated subset of proteins altered in primary cortical neurons after KA treatment to our reference proteome data set and to the mouse genome, respectively. We found enrichment of proteins associated to mRNA metabolism and splicing. Proteins associated to the cytoskeleton were also enriched. This was in line with our previous observation that the majority of proteins altered after KA treatment in neurons were associated to mRNA splicing, followed by cytoskeleton. A detailed summary of all enriched categories is shown in Table 3. When employing correction for multiple testing, only GO-terms enriched in comparison to the mouse genome
remained significant. However, correction for multiple testing is usually used for evaluation of microarray data sets that are much larger than our proteome data set. Therefore, this test method might be too stringent in our case. To make sure that enrichment was not biased by the analysis tool (Webgestalt), we next analyzed enrichment of functional groups within our data set of proteins altered after KA treatment using another tool, DAVID. The analysis approved our previous results concerning RNA splicing. In detail, gene functional classification revealed two enriched clusters, one comprising proteins associated to RNA splicing (enrichment score 3.5) and one comprising proteins associated to cellular stress response (enrichment score 1.82). We included significantly altered proteins with very small changes in expression in our data set. Next, we investigated if the observed enrichment of mRNA metabolism/splicing and actin cytoskeleton was also observed when only robust changes (ratios over 20%) were considered. We observed enrichment of the terms RNA splicing, Spliceosomal complex, and Microtubule associated complex as compared to the reference data set using Webgestalt. This approved our previous results. Importantly, a comparison of our enrichment data to other data sets generated in our group revealed that the observed enrichment of protein groups was specific for KA-treated neurons. For instance, we compared the data set to data of primary cortical neurons treated with BDNF and soluble APP and found no enrichment of the functional groups enriched in KA-treated neurons (unpublished, data not shown). In addition, when analyzing enrichment of functional protein groups among proteins altered in the brains of mouse models for neurodegenerative diseases (Alzheimer, Parkinson, and Huntington diseases), we also did not observe enrichment of mRNA splicing.26
’ DISCUSSION In the study presented here, we analyzed proteome alterations after prolonged KA administration to primary cortical neurons. 1464
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Journal of Proteome Research So far, the effects of KA have mostly been analyzed in the brains of KA-injected rodents. Due to the complexity of the brain, interpretation of these results is difficult.9,10 Only one study focused on synapse-specific proteome changes following KA administration to mice.11 These studies provided a number of altered proteins some of which were also found in our study. We analyzed proteome changes in primary cortical neurons of mice representing a model with high In Vivo relevance. In addition, analysis of primary neurons helps to distinguish the effects of KA on neuronal versus glial cells. We used a KA concentration (50 μM) that was already described to increase dendrite growth without affecting survival of primary neurons.27 In agreement with this observation, we identified alterations in cytoskeletal proteins that facilitate dendrite growth (Table 2) but no significant differences in survival of neurons after KA treatment. After 24 h of KA administration, 91 protein spots were significantly altered in expression. Interestingly, many of these proteins were already associated with expression changes after KA treatment. Up-regulation of Homer1, Gfap, Hsp9a, and Grb2 was observed in the hippocampus of KA-treated rats.28,29 These proteins were also identified to be up-regulated in our study, demonstrating the validity of our findings and a high In Vivo relevance of our data. The growth factor receptor bound 2 (Grb2) was up-regulated after KA administration. It is an important adaptor molecule in several growth-factor signaling cascades as it couples cell surface receptor signaling to downstream targets.30-34 Because KA is known to induce an increase in expression and signaling of brain derived neurotrophic factor (BDNF), up-regulation of Grb2 might be important for BDNF signaling.35,36 KA also induced up-regulation of Igf3bp3 (IMP3). IMP3 binds to mRNA of Insulin-like growth factor 2 (Igf2) and regulates its expression.37 Interestingly, alterations in Igf2 were also observed following systemic KA administration.38,39 According to our literature-based analysis of altered proteins, KA treatment of primary cortical neurons mainly induced alterations of proteins involved in mRNA splicing and actin cytoskeleton remodeling. Interestingly, enrichment analysis of our data revealed a predominant enrichment of proteins associated to mRNA splicing via spliceosome. Splicing is essential for the expression of proteins from genes. One well-documented effect of KA administration to neurons is up-regulation of BDNF expression, which has opposing effects depending on its subcellular site of action. Only one out of several BDNF splice variants analyzed was reported to be up-regulated in hippocampal dendrites after KA administration. This effect was specific for KA as administration of another epileptogenic agent, pilocarpine, led to the up-regulation of two other splice-variants of BDNF in dendrites.40 Growing evidence shows that various biological stimuli, such as cell excitation, stress, and cell cycle activation, induce rapid changes in alternative splicing.41,42 Alternative mRNA splicing in neurons is important for a number of processes such as neuronal development, axon guidance, synaptogenesis, and neuronal excitability.43 Interestingly, alternative splicing was shown to regulate membrane delivery of different KAR subunits displaying different kinetics.44-46 Furthermore, cell excitation with KCl-induced membrane depolarization induced skipping of several exons, including exons of the N-methyl D-aspartate (NMDA) glutamate receptor NR1 subunit. These receptors also undergo excitation-dependent alternative splicing in primary neurons.47,48 In addition, activitydependent changes in alternative splicing were demonstrated in hippocampal slices treated with pilocarpine.49
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The examples clearly demonstrate the importance of alternative splicing as a result of KA administration. Splicing decisions are carried out by processes associated with spliceosomes that assemble on pre-mRNA. Thereby, RNA binding proteins from serine/arginine-rich (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) families play key roles in recognizing splicing enhancer or silencer sequences on the RNA.50 One of these proteins, SF2/ASF (Sfrs1), a member of the SR protein family, was down-regulated after KA treatment of neurons (Figure 2, Table 2). Interestingly, the importance of this protein for excitation-related alternative splicing was already demonstrated. It was shown that SF2/ASF regulates alternative flip/ flop splicing of GluR2.51 Flip/flop splicing of GluR2 is developmentally regulated with the flip isoform being more abundant during early development. Besides developmental regulation, flip and flop isoforms also display different kinetics. SF2/ASF was shown to induce flop splicing, which is more abundant in adult tissue and decreases sensitivity faster compared to the flip isoform.51 However, the Heterogeneous nuclear ribonucleoprotein A1 (Hnrnpa1, hnRNP A1), a member of the hnRNP family, is known to directly counteract the effects of SF2/ASF with SF2/ASF favoring the proximal and HnRNP A1 promoting the distal choice for competing 50 splice sites.52 Intruguingly, HnRNP A1 was, in contrast to SF2/ASF, up-regulated after KA administration in our data set (Table 2) and it was also previously shown to mediate excitation-induced alternative splicing in primary neurons.47 However, there are also other proteins altered after KA treatment in our study that have functions in alternative splicing. For example, the RNA-binding motif protein 4B (Rbm4b) modulates alternative pre-mRNA splicing and regulates alternative splicing of microtubule-binding protein tau.53-55 Together, our results add to growing evidence that neurons adapt to an overload or lack of neurotransmitter by mechanisms that include alternative splicing in order to stabilize excitability.43
’ CONCLUSION In summary, our data clearly suggest the importance of altered mRNA splicing as a consequence of KA administration to neurons. In addition, we provide a large number of new candidate proteins that might be related to KA-induced processes. These may help elucidating mechanisms such as maintenance of neuronal excitability via alternative splicing in further studies. ’ ASSOCIATED CONTENT
bS
Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Ph: þ49 30 450566258. Fax: þ49 30 450566904. E-mail:
[email protected].
’ ACKNOWLEDGMENT We thank Yvonne Kl€are and Sabine Groebert for excellent technical assistance. This study was funded by Deutsche Forschungsgemeinschaft (DFG), project HA6155/1-1. 1465
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Journal of Proteome Research
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